Since early electron microscopy studies, microtubules have been invariably described as being surrounded by a "clear zone" which gives the impression of a "halo" around them when they are viewed in cross section. A 5-10 nanometer distance from the surface of MT is free of cytoplasmic ground substance or any other material normally seen elsewhere throughout the cell. These clear spaces were initially thought to be electron microscopic artifacts, or layerings of less dense filamentous proteins on the surface of the microtubules. However, newer fixation and staining techniques and freeze etching have confirmed that the space immediately surrounding microtubules are seldom encroached upon by other organelles or cytoplasmic ground substance. Stebbings and Hunt (1982) have studied the "clear zone" and point out that the surface of microtubules is strongly "anionic" since tubulin is an acidic protein due to its high content of acidic amino acids such as glutamate and aspartate. These amino acids give up positively charged hydrogen ions to solution, leaving MT with excess electrons. Stebbings and Hunt propose that anionic, or electronegative fields at MT surfaces can explain the clear zones as well as the staining of MT by positively charged dyes, binding to MT of positively charged proteins, cations such as calcium, metals and other compounds. Electronegative fields surrounding MT may act as excitable ionic charge layers ("Debye layers") which are also thought to occur immediately adjacent to cell membranes (Green and Triffet, 1985). Excitable "clear zone" charge layers next to MT could facilitate collective communicative mechanisms within the cytoskeleton (Figure 6 and 8).

The question of the hollow core within MT is even more mysterious; it too appears devoid of ground substance. It is unknown whether the interiors of MT are also electronegative zones, or perhaps positive ones which would create voltage gradients across MT walls. Del Giudice and colleagues (1986) at the University of Milan have even suggested electromagnetic focusing and "superconductivity" within microtubule cores (Chapters 6 and 8). Insulated from "aqueous" surroundings and held together by water-excluding hydrophobic forces, MT and the rest of the cytoskeleton comprise a "solid state" network within living cells.

What do microtubules do? For openers they are the cytoskeleton, being the most rigid structures in most cells. To establish the pattern of the cytoskeleton and the form and function of living cells, MT assemble from subunits at the proper time, place, and direction. They are often anchored and guided by MT organizing centers (MTOC) containing centrioles. Once in place, they participate in movement of cytoplasm, organelles and materials, growth, reproduction, synaptic plasticity, and nearly all examples of dynamic cytoplasmic activity. The mechanisms for MT organization are unknown, but several theories of MT based information processing have been proposed and will be described in Chapter 8.

Many MT functions involve control and signaling of the activities of attached proteins including those which interconnect MT in assemblies like centrioles, cilia and flagella. Active sliding by motile bridges attached along MT are involved in cell shape determination, and extension of cytoplasmic projections like neuron growth cones, dendritic spines and amoeba lamellipodia. These extensions contain actin filament bundles without MT, however MT generally establish the architecture which orients these extensions, provide their raw materials, anchor them and integrate their functions with the cell. In cytoplasmic transport, components moving along parallel arrays of cytoplasmic microtubules deliver cellular materials wherever they are needed. In lengthy asymmetrical cytoplasmic processes like nerve axons, specialized mechanisms of axoplasmic transport have evolved to transport cell constituents at rates up to five thousand nanometers per second! MT orchestrate the motile force supplied by "dynein" mechanical arms to move organelles which include chromosomes, nuclei, mitochondria, neurotransmitter synaptic vesicles, liposomes, phagosomes, granules, ribosomes, and other proteins. MT assembly determines the form and function of biological systems.

MT also participate in sensory perception of the cell's external environments. Many sensory receptors are modified cilia, assemblies of microtubules similar in structure to centrioles. Insect mechanoreceptors and sensory cilia in our ears appear to utilize MT to transduce mechanical force to the base of each cilium and into the nervous system. Atema (1973) has proposed that propagation of conformational changes along sensory cilia convey information to the cell as a whole. Moran and Varela (1971) have suggested that sensory cilia MT act as engines driven backwards. When an external force moves the microtubules of the mechanoreceptor cilium, they suggest that the MT release ions like calcium which can regulate cellular activities. Sensory transduction, guidance and alignment are "intelligent" MT activities which are vital to biological growth, embryological development, secretion, synapse formation and many other important biological functions. Temporal and spatial control of MT deployment are achieved by two mechanisms of pattern formation: directed assembly from MT organizing centers (MTOC) and self assembly of multitubular arrays by means of intertubule linkers (Figure 5.6). MT are the scaffolding, conveyor belts, and computers of living cells.

Figure 5.6: Microtubule arrays interconnected by MAP bridges. Hollow circles are MT arrays in cross section. By Paul Jablonka.

5.2.2      Microtubule Assembly and the Generation of Form

As changeable cellular superstructures, MT can assemble into rigid tubular rods when and where they are needed, and then disassemble into subunits which may be transported away by other MT. Like many viruses, MT self assemble from their subunits into orderly polymers. The large increase in order, or negative entropy associated with MT assembly would appear to flow upstream against the second law of thermodynamics which, in general, states that order tends towards disorder. The increase in order observed during MT assembly can be related to the dispersal (disordering) of tightly bound structured water from the subunits as they polymerize, consistent with the concept that MT subunits associate by hydrophobic interactions (Chapter 6).

Figure 5.7: Microtubule mitotic spindles (wispy filaments separating chromosomes (heavy rods) establishing daughter cell polarity during cell division. Centrioles and MTOC are at nidus of each array. With permission from Marc DeBrabander (1982).

MT vary markedly in stability and function. Structurally similar MT may be highly stable, or fleetingly transient. This variability can result from differences in tubulin chemistry, from effects of secondary proteins attached at specific points on MT surface lattices (microtubule associated proteins: MAPs), local cytoplasmic influences, membrane interactions and many other factors. Highly stable MT are found in cilia and flagella in which they are assembled into complex patterns and carry out their functions without disassembly. The other extreme are MT within mitotic spindles which separate chromosomes and establish daughter cell shape in cell division ("mitosis"-Figure 5.7). These "labile" MT not only assemble before mitosis and disassemble afterwards, but apparently undergo localized disassembly/reassembly during mitosis ("dynamic instability"). In some animal cells and during plant cell mitosis, assembly of MT subunits into MT appears to take place in the cytoplasm without any relation to a particular organelle. In other cells, structures including centrioles, basal bodies, centrosomes, and kinetochores facilitate assembly of microtubules at a specific site and orientation. These structures are called microtubule organizing centers (MTOC), and they consist of a pair of centrioles and a dense granular material. In general, negatively charged ends of MT attach to the MTOC, and positively charged ends grow distal to the MTOC, establishing cell polarity, shape and orientation.

The MT assembly process may be tracked by parameters that crudely reflect the polymer level such as turbidity or viscosity. Generally, there is first a lag phase when no microtubules form, then a phase of exponential growth and finally a stable plateau. At low concentrations, no microtubules are formed. Above a certain critical concentration (Cc) of tubulin, microtubules increase in relation to the total tubulin concentration. However, MT assembly is more complex than being simply at equilibrium with a pool of unassembled subunits.

Spontaneous assembly of tubulin dimers depends on physiological conditions which alter the critical concentration (Cc) of subunits required. Spontaneous MT assembly thus depends on many cofactors: calcium ion concentration, temperature, presence of microtubule inhibitors or stabilizers (Figure 5.8), microtubule associated proteins (Figure 5.12), the presence of microtubule organizing centers (MTOC-Figure 5.9) and the availability of GTP. Like ATP (adenosine triphosphate-required for actin assembly), GTP (guanosine triphosphate) is a source of biochemical energy. As will be described in Chapter 6, ATP and GTP can each donate phosphate bond energy by being "hydrolyzed" to their "diphosphates" ADP and GDP, respectively. In the case of assembly of both MT and actin filaments, the presence of GTP or ATP without being hydrolyzed is an important requirement for assembly. Nonhydrolyzable analogs of GTP are equally effective in promoting assembly of MT; hydrolysis and energy input take place after incorporation of a dimer into a tubule. MT formed with nonhydrolyzable analogs are more stable than those with GTP, so hydrolysis energy may be related to disassembly. In actin filaments, hydrolysis also occurs after the formation of the polymer. Therefore a paradox exists in that GTP binding is required for microtubule assembly, but GTP hydrolysis occurs later and is not required for assembly. An identical situation occurs with actin and ATP. Depletion of ATP (for actin) and GTP (for MT) strongly inhibits disassembly but does not hamper assembly per se. However, in energy depleted cells, random as8embly prevails instead of organized assembly. The energy from phosphate bond hydrolysis, the main energy currency in all biological systems, is unaccounted for within the cytoskeleton, the dynamic organizer of cell function. Perhaps the energy is used to generate communicative lattice vibrations, coherent excitations, or "solitons" in MT and the cytoskeleton in general (Chapters 6 and 8).

Figure 5.8: Effects of MT disassembly drug nocodazole in PtK2 cells. MT visualized with peroxidase-antiperoxidase (PAP) method. Upper left: organized MT radiate from dense MTOC region near nucleus. Upper right: cells have been treated with nocodazole (2 x 10^-5 M, 4 hours) and MT depolymerized. Lower left: cells washed after nocodazole, 5 minutes later MT growing from MTOCs. Lower right: cells 20 minutes after wash have reorganized MT system. With permission from DeBrabander, Geuens, Nuydens, Willebrords and DeMey (1981).

MT assembly also requires the presence of magnesium ion and a low concentration of calcium ion. Calcium is an important messenger system within many forms of cytoplasm. Waves of calcium can be caused by membrane and cytoskeletal activities, and can induce conformational changes in proteins and dissolve cytoplasmic gel, converting it to a more aqueous state: "sol." Tubulin dimers loosely bind 16 calcium ions per dimer (Hayashi and Matsumara 1975); an excess of calcium, however, causes MT disassembly. In the presence of abundant zinc ions, tubulin polymerizes into flat expansive sheets rather than cylinders.

The dimer subunits alpha/beta tubulin are all arranged in the same direction. The beta subunit protrudes at the "plus" end and contains the exchangeable GTP binding site. "Cc" for assembly is lower at the plus end than it is at the minus end. At steady state in the presence of hydrolyzable GTP, a net incorporation of dimer subunits is seen at the plus end, and a net loss of dimer subunits is seen at the minus end. Subunits thus move from the plus end to the minus end in MT-a phenomenon called "treadmilling." In MT anchored at one end to organizing centers, treadmilling is a net movement of subunits within the MT lattice, and may correspond to the slow 1 millimeter per day component of axoplasmic transport. MT thus appear to be continually growing, perhaps twisting, at a rate of about 10 nanometers per second. Labile MT can polymerize their way through the cytoplasm, adding GTP-tubulin at the beta plus end, and dumping GDP tubulin at the alpha minus end. These types of MT can thus behave like mobile tractors, cytoskeletal caterpillars (Figure 5.10).

Disassembly or separation of tubulin dimer subunits from MT is induced when terminal dimers bind GDP. When GTP is hydrolyzed, a phosphate group is lost and guanosine triphosphate (GTP) becomes guanosine diphosphate (GDP). GTP bound dimers at open ends of MT (GTP "caps") are stable: they stay assembled. However, when open ends of MT contain subunits binding GDP, they tend to release and shorten the MT cylinder. MT whose distal ends have exposed GDP tubulin, and are not anchored by structures like MTOC, shrink and depolymerize. Generally, free MT polymers (those not anchored by MTOC) are "chimeric" with stretches of unhydrolyzed GTP at the ends (GTP caps) and GDP in the interior. Growing polymers have "protective" GTP subunit caps at their end; shrinking polymers lose their GTP caps and expose GDP subunits at the end, causing disassembly.

Microtubules thus appear to exist in two populations: the majority growing at an appreciable rate and the minority shrinking very rapidly and providing new subunits for growth. This concept has been called "dynamic instability" with the rapid shrinkage of MT referred to as "microtubule catastrophes" (Kirschner and Mitchison, 1986). The faster the growth rate, the larger the GTP cap and the lower the probability of the cap disappearing and the microtubule depolymerizing. When the cap disappears and GDP subunits are exposed, the polymer enters a rapid depolymerizing ("catastrophic") phase. MT contain thirteen parallel protofilaments and it is unclear how many exposed GTP or GDP containing subunits at MT terminals are required for stability or instability. An essential factor is the rate of GTP hydrolysis which results in GDP tubulin and induces disassembly. The utilization of GTP hydrolysis energy by MT and the cytoskeleton is a significant portion of biological energy consumption, yet remains unappreciated. Theories of protein conformational state regulation such as solitons and coherent excitations which could account for the useful consumption of hydrolysis energy will be described in Chapter 6. Solitons, coherent lattice vibrations, and cooperative resonance present possibilities for collective, and possibly intelligent effects within cells.

Japanese investigators Horio and Hotani (1986) have directly observed growing and shrinking MT using dark field microscopic video. They observed that both ends of a microtubule can exist in either the growing or the shortening phase and alternate quite frequently between the two phases in an apparently random manner. Further, growing and shortening ends can coexist on a single microtubule: treadmilling. One end may continue to grow simultaneously with shortening at the other end. The two ends of any given microtubule have remarkably different characteristics. One "active" end (presumably the beta, plus end) grows faster, alternates in phase between growing and shrinking more frequently, and fluctuates in length to a greater extent than the inactive end. Microtubule associated proteins ("MAPs") suppress the phase conversion and stabilize microtubules in the growing phase.

There are many apparent mechanisms for stabilizing polymerized microtubules: they may be capped or bound to various structures. Binding at the proximal end to MTOCs stabilizes MT and prevents their depolymerizing at the opposite end. MT need to be stabilized at merely one end to predominate within cells because, even if they depolymerize, another will reform in the same place and orientation (DeBrabander, 1985). Thus MTOC provide cells with a means of selective retention of a subclass of specifically oriented MT.

MTOC oriented in a specific direction can determine cell polarity, including extension of cells in embryological growth, formation of probing appendages called filopodia in locomotory cells and growth of nerve processes. Signals at the cell periphery apparently cause an asymmetry of the microtubule cytoskeleton and overall cell polarity. How can a peripheral clue lead to reorganization deep in the cell? One possible explanation is that a signal is relayed through the cytoskeleton to the MTOC, leading to a change in its orientation and directed nucleation of microtubules. Another is that a signal at the periphery affects the MT distribution directly. Since the entire cytoskeletal array is dynamic, it might only be necessary to transiently stabilize a particular subset of microtubules for the cellular cytoskeletal array to rapidly transform. Kirschner and Mitchison (1986) have proposed that the dynamic microtubule array probes many regions at random. By stabilizing certain MT configurations as they arise, they believe the cell can arrive at a structure that is not precisely defined by genetic information but one that adapts to fulfill a particular functional role.

Dynamic structural rearrangement of the MT cytoskeleton appears to require intelligence. Like the brain as a whole, cytoskeletal intelligence has features of connectionism, parallelism, distributedness, and hierarchy. The apparent cytoskeletal commanders are MTOC, of which the critical structures are the organelles which may have hijacked evolution: centrioles.

5.2.3      Microtubule Organizing Centers (MTOC) and Centrioles

MTOC and their chief components, centrioles, are the specific apparatus within living cells which trigger and guide reorganization of cytoplasm such as occurs during growth, generation of form and function ("differentiation") and cell movement. The enigmatic MTOC determine where, when, and how these functions occur (Figure 5.11).

MTOC (or "centrosomes") contain centrioles and "pericentriolar substance" which facilitates tubulin assembly by somehow lowering Cc. Centrioles are the common structure in all of these cellular control centers. Centrioles are composed of two similar cylinders; their diameters are 0.2 microns or 200 nanometers. Each cylinder possesses a 9 fold radial symmetry and is constructed essentially of 9 triplets of microtubules fused longitudinally. "Satellites," electron dense proteins, appear to orbit the centrioles. A cartwheel filamentous structure ("pinwheel") connects all the microtubules within each centriole at one end. One centriole begets another by replication perpendicular to the cylindrical surface of the centriole. The first step in cell division is maturation of the "mother" cell, followed by separation of pairs of centrioles and migration to establish architecture of "daughter" cells. Centriole mechanisms of perpendicular replication, orientation and guidance are unknown.

Figure 5.9: Microtubules in mitotic PtK2 cell labeled with tyrosine tubulin immunogold. The spindle pole region (MTOC) at left is a focus for spindle MT which radiate toward chromosomes (dark material). Insert upper right: kinetochore MT attaching chromosome. With permission from Geuens, Gundersen, Nuydens, Cornellisen, Bulinski, DeBrabander (1986).

Between cell division cycles ("interphase"), the MT system is relatively quiescent and "radial." Most cell MT are anchored at one end to the MTOC, although they don't appear to contact any structures. Rather, MT minus ends are somehow stabilized by the "pericentriolar material." This favors MT assembly and protects against disassembly by binding and capping the minus end of MT. Most tubulin is polymerized during interphase, however free MT not radiating from the MTOC occur near the periphery of the cell. Except for free MT, the network is rather stable with low turnover rates and minimal treadmilling and dynamic instability.

During "prophase", the cytoskeleton prepares for the separation of duplicated chromosomes so the cell can make a copy of itself. DeBrabander (1985):

It appears that nature selected a near fail safe microtubule based mechanism to allow the eukaryotic cell to handle more and more complex genetic libraries.

Figure 5.10: DeBrabander's model of MTOC action. Upper left: A centriole is surrounded by a dense material that lowers Cc, the critical concentration of tubulin required for microtubule assembly. At the beginning of mitosis, new MT are preferentially nucleated near the MTOC. Upper right: Kinetochore plates associated with chromosomes also have the Cc lowering material but anchor plus ends; new MT form. Middle: MT minus ends are stabilized by the pericentriolar dense material and plus ends grow outward. Free MT are unstabilized and remain short due to consumption of tubulin subunits by MTOC stabilized MT. Bottom: stabilized MT slide along one another to pull chromosomes apart. With permission from Marc DeBrabander (1982).

Figure 5.11: Microtubules in PtK2 cells illustrated by tubulin antibody at two different magnifications. Nucleus (left) is devoid of MT, which densely emanate from nearby MTOC. With permission from Geuens, Gundersen, Nuydens, Cornellisen, Bulinski, and DeBrabander (1986).

As the chromosomes condense in prophase, the MT system changes its display and turnover rate. Interphase MT are gradually shortened while new MT start to grow from the centrosome. At this point, the system is extremely sensitive to polymerization inhibitors. Drugs which are used against cancer are often mitotic inhibitors which prevent the rapid polymerization of prophase MT from MTOC. Kinetochores are mobile MTOC which attach chromosomes and bind MT at their plus end (Figure 5.10). MT assemble between centrosomes (which bind their minus end) and kinetochores (which bind their plus end) and separate the genetic material towards the daughter cell poles in the mitotic cycle. The delicate array of the two centrioles, connected MT spindles and "star-like astral projections" (MT which "overshoot" the centrioles) have suggested to many observers some type of electromagnetic field because of the resemblance to a magnetic field pattern. The contractile ring formed perpendicular to the axis of mitosis by the microtrabecular lattice establishes a cleavage furrow which separates the daughter cells. The chromosome material decondenses while a new nuclear membrane is zipped onto it, and cellular elements assume again an organized aspect. Later, the cells flatten and gradually reassume their normal interphase shape.

Figure 5.12: Schematic representation of a microtubule associated protein (MAP dark lines) facilitating assembly of a microtubule (MT). The longitudinal binding retards dissociation of subunits and stabilizes the MT structure. With permission from DeBrabander, DeMey, Geuens, Nuydens, Aerts, Willebrords and Moerman (1985).

MTOC/centrioles are involved in orientation, shape, timing of division and growth. With MT and other cytoskeletal structures, MTOC/centrioles determine what cells do, when and how they do it, and what kind of cells they are! Are MTOC the command centers of the cytoskeleton? Chapter 3 described the significant position of centrioles and MT in evolution, and Chapter 8 will describe theories which explain centrioles' enigmatic capabilities. These include consideration (Bornens, 1979) that centrioles rotationally oscillate with a gyroscopic inertia which senses and establishes cell orientation to neighbor cells, tissue, environment, chemical gradient fields, gravity, and perhaps other factors. Centrioles seem to be a relatively immobile anchor around which other cytoskeletal elements move and are organized. Bornens suggests that centrioles communicate with actin filament networks by propagating conformational effects, and thus regulate cellular activities. Another centriole theory has been lodged by Northwestern University's Guenter Albrecht-Buehler (1977, 1985), who proposes that centrioles detect signals (such as infra-red, ultrasound, microwave) propagating linearly throughout the cytoplasm. Albrecht-Buehler has considered the design principles for a nanoscale directional signal detector and finds that centrioles are appropriate. Centrioles, or their future hybrids, may have interesting technological applications (Chapter 10).

5.2.4      Microtubule Associated Proteins (MAPs)

The full range of MT functions is achieved by the actions of various proteins which bind in precise fashions at specific tubulin dimers in MT lattices. These microtubule associated proteins (MAPs) include electromechanical enzymes which generate force and movement, communicative crossbridges to other cytoskeletal filaments and organelles, MAPs which enhance MT assembly (Figure 5.12) and a class of MAPs within neurons whose functions are not understood.

In many cases, the attachment patterns of MAPs to MT lattice walls have a precise geometrical configuration which appears related to the function of the MAP-MT complex (Figures 5.13 thru 5.16). For example Allen (1979, 1985) and colleagues showed that smooth passage of vesicles in axoplasmic transport requires contractile MAPs spaced between 15 and 25 nanometers apart along MT lengths. Many other MAP binding patterns are spirals which wind around MT cylinders in super-helices, and several theories suggest how these sites can be determined. These include linear sequencing of varying tubulin isozymes along protofilaments (Kim, 1986), crystal symmetry describing each MAP terrain (Figure 5.17, Koruga, 1986) or by the lattice steps necessary to get from one MAP site to its nearest neighbor MAP. For example, electron micrographs of MAP-MT binding by Burns (1978) clearly show MAP binding which may be described as "over 3, up 1." That is, if the tubulin subunit to which the MAP is bound is the starting point, moving 3 monomers along the leftward row helix, and then up 1 monomer along the protofilament will indicate the next attachment site. A number of geometrical super-helical patterns have been observed which match other "chess-like" movement rules. These are shown in Figures 5.12--5.15. In other instances such as brain MAPs, attachment patterns are irregular and heterogeneous, and thus are capable of representing information.

Figure 5.13: Patterns of MAP attachment to microtubules observed by electron microscopy (Burns, 1978; Kim et al., 1986; and others).

Rod shaped bridges which occur between MT are of at least two sorts. Motion producing protein "arms" which consume ATP hydrolysis energy to generate force are analogous to myosin bridges of skeletal muscle. Moving arms attached to MT are made of proteins called kinesin and dynein. Dynein arms on MT which contract in organized sequences to produce collective movements were first described and characterized in cilia and flagella by Ian Gibbons (1968). The arms reside at periodic intervals along the outer MT within cilia and flagella. They possess "ATPase activity," that is they .contract conformationally due to hydrolysis of ATP. Dynein arms contract in waves and mediate sliding between the adjacent outer tubules to drive ciliary and flagellar motility. Dynein arms attached to MT in neuronal axons act collectively to pass material in "bucket brigade" axoplasmic transport. These and other MT "bending sidearms" will be discussed in Section 5.5.2.

Other MAPs are bridges between parallel MT, filaments, organelles and the "microtrabecular lattice" and appear to integrate microtubules with the rest of the cytoplasm (Figure 5.6). MAPs from mammalian brain neurons have been characterized into three main groups. These are high molecular weight (greater than 100 kilodalton) MAP 1 and MAP 2, and a number of closely related 56-62 kilodalton proteins designated as "tau." Studies in rats show an increase in number and an altered distribution of the various forms of tubulin, high molecular weight MAPs, and tau which correlate with rat brain development and learning. Both MAP 2 and tau are heat stable, stimulate MT polymerization (lower Cc) and share binding domains on intact MT; they are heterogeneous protein families whose functions are unknown. MAP 2 is concentrated in cell bodies and dendrites of neurons, found in a ratio of MAP 2 to tubulin of one to twelve, occurs in only small amounts in axons and is absent in glia. Conversely, brain tau is largely confined to axons. Both MAP 2 and tau are phosphorylated by cyclic AMP dependent protein kinases, proteins which amplify effects of calcium ions. Intracellular levels of calcium ions, in turn, are regulated by diverse extracellular signals including neurotransmitters, hormones and electrical factors to regulate cell shape, motility, secretory processes and other functions. Thus these MAPs can mediate diverse inputs into the cytoskeleton. Each MAP attached to an MT may function like a "synapse" in a parallel, laterally interconnected network.

Figure 5.14: MAP attachment patterns.

Figure 5.15: MAP attachment patterns.

Figure 5.16: MAP attachment patterns.

Tau heterogeneity varies during brain development and these changes result in the predominance of different tau polypeptides in mature cerebral cortex and cerebellum. Binder, Frankfurter, and Rebhun (1986) propose that MAP 2 binding sites result from genetically determined forms of tubulin which are predominant in neural cell bodies and dendrites, while the tau binding domains are specified by axonal tubulins. They suspect that different tau species appearing during development may herald the cytoskeletal differentiation of unique axonal subpopulations whose MT are committed to slightly different tasks. Variability and responsibilities of MAPs may be extensive. They are functional appendages, structural and communicative links ("arbiters"), and essential integrators of cytoskeletal and cellular function.

Figure 5.17: MAP attachment patterns as part of Koruga (1986) microtubule information code (Chapter 8). With permission from Djuro Koruga.

5.3           Intermediate Filaments

The major filamentous components of the cytoskeleton are MT, actin filaments, intermediate filaments (IF), and a class of delicate interconnecting fibrils called the microtrabecular lattice (MTL) which will be described later in this chapter. Here the "unknown" members of the cytoskeleton, intermediate filaments are reviewed (Lazarides, 1980). Intermediate filaments represent the most nebulous and chemically variable subgroup among the cytoskeleton. Five classes of IF have been distinguished which are built from polypeptides containing mostly alpha helix rod domains (Chapter 6). Under the electron microscope, IF appear as relatively featureless, 8-12 nanometer wide unbranched filaments. Treatment with certain fixative agents can cause IF to unravel into several 2-3 nanometer protofilaments, or 4-5 nanometer protofibrils whose number may vary from filament to filament, and even along the same filament. IF may form from parallel coiling of the alpha helix domains and under some circumstances, can form a polygonal meshwork with a 52 nanometer repeat of 8-10 nanometer wide filaments. These can also aggregate into crystal-like arrays with 24 nanometer spaced transverse bands. As such, IF may be involved in structures otherwise described as microtrabecular lattice, or cytomatrix.

Lazarides (1980) has shown that different types of IF associate with specific types of cells. For example, subunit structure defines five major classes of IF: 1) keratin ("tonofilaments"), which are found in epithelial cells, 2) desmin filaments predominantly found in smooth, skeletal and cardiac muscle cells, 3) vimentin filaments, found in mesenchymal cells, 4) neurofilaments, found in neurons (Figure 5.18), and 5) glial filaments, found in glial cells. Often two or more of these classes co-exist in the same cell.

Neurofilaments appear to function as a three dimensional structural lattice providing tensile strength to axons. Extruded axoplasm is a highly structured gel rich in neurofilaments; exposure of the gel to calcium ion results in degradation of neurofilaments and conversion of the gel to a more watery sol state, a phenomenon generally attributed to dissolution of actin filaments.

All IF including neurofilaments appear to be phosphorylated although the function that this could serve is not understood. IF are a distinct class separate from MT and actin filaments, and Lazarides argues their biochemical and morphological properties indicate they are involved in mechanically integrating the various components of the cytoplasmic space. IF remain poorly understood; their dense presence in neurons is mysterious, perhaps they participate in some way in the cognitive functions of the nervous system. In Chapter 8, Barnett's theory of neurofilaments as "string transform" memory banks coupled to MT will be described.

Figure 5.18: Cross section of small nerve axon. A. Microtubules with radial MAPs. B. Neurofilaments, outnumbering microtubules about 10:1. C. Vesicle being transported by microtubule axoplasmic transport. D. Crosslinked actin/neurofilament gel (microtrabecular lattice). E. Myelin layers. F. Filamin bracketing actin. By Paul Jablonka.

5.4           The Cytoplasmic Ground Substance

MT and IF are not the finest texture of cytoplasmic organization. Smaller, more delicate structures branch and interconnect in "gel" state networks which comprise the substance of living material.

Three distinct cytoskeletal component systems have been well characterized: MT, IF and actin filaments. Actin is the most versatile component. In conjunction with other proteins it can polymerize in string-like filaments, form dynamic branching nanoscale meshworks or geodesic polyhedrons. Even more evanescent than labile MT, assembly of actin and associated proteins create transient configurations of cytoplasm for specific purposes. The roots of intelligence may well be grounded in dynamic cytoplasmic expressions such as contractile rings which divide the cytoplasm in cell division, probing lamellipodia, and dendritic spines and synapses in neurons.

The nature and structure of the "ground floor" of organization, the cytoplasmic ground substance, has been explored from a number of orientations resulting in overlapping descriptions. These include the microtrabecular lattice, cytomatrix, and cytoplasmic solid state.

5.4.1      The Microtrabecular Lattice (MTL)

New techniques in electron microscopy developed by Keith Porter and colleagues (1981) at the University of Colorado at Boulder have led to observation of an irregular three dimensional lattice of slender strands throughout the cytoplasm, interconnecting nearly everything in the cell. The interlinked filaments appear to suspend the various cell systems, organelles, and larger cytoskeletal elements such as microtubules and filaments with a matrix material continuous with the individual lattice filaments. The lattice is suggestive of the trabecular structure of spongy bone and it was named the microtrabecular lattice (MTL, Figure 5.19).

Porter and colleagues took advantage of the properties of the high voltage electron microscope at Boulder. This massive device is capable of accelerating electrons across a potential drop of a million volts, ten times that of standard electron microscopes. The extra voltage gives the electrons sufficient energy to penetrate thick specimens or even intact cells up to several thousand nanometers thick. Previously, cells had to be sliced into sections thinner than 200 nanometers, and artifacts due to cell destruction were more prevalent. The high voltage electron microscope provides more information about depth, giving a three dimensional view of cell organization. Another innovation, the critical point drying method (Ellisman, 1981) avoids the distorting effect of surface tension which causes cells to collapse when they are dried in air. High voltage electron microscopy and critical point drying have now demonstrated the MTL in all eukaryotic cells which have been examined.

The MTL is a three dimensional lattice of slender strands called microtrabeculi. They range in diameter from 4 to 10 nanometers with lengths of 10 to 100 nanometers. The MTL is a finely organized meshwork that divides the ground substance into two phases, a protein rich polymerized phase comprising the MTL, and a water rich fluid phase that fills the intratrabecular spaces. At high magnification, microtrabeculi of the ground substance are seen to crosslink many elements in the cytoplasm. For example, they connect microtubules with the smooth endoplasmic reticulum. The MTL not only crosslinks, but appears to be dynamically active in moving elements through the cytoplasm. The dynamic MTL moves material in a saltatory manner at velocities equivalent to axoplasmic transport. A co-pioneer with Porter in the observation of cytoplasmic ground structure, Mark Ellisman (1981) of the University of California at San Diego has observed that MT and filaments are analogous to rigid skeletal elements such as bone, and that the microtrabecular lattice may be appropriately equated with work producing components such as skeletal musculature. Thus Ellisman suggests a more appropriate description of the MTL might be "cytomusculature."

In neurons, wispy MTL strands radiate from MT and neurofilaments at right angles into the cytoplasm, often interconnecting MT with other MT and neurofilaments with neurofilaments. At the synapse, the MTL appears intimately involved in both neurotransmitter release and post synaptic receptor mechanisms. Presynaptic terminals demonstrate notable MTL crosslinking among synaptic vesicles and among vesicles and synaptic membranes. In post synaptic dendrite MTL, linkages are visible among filamentous subsynaptic densities and the other elements of dendritic cytoplasm. Where axonal cytoplasm enters synaptic regions, transitions occur in the form of the MTL. In axons, cross linkages are clearly exhibited between MT, filaments, and other components. However, in presynaptic terminals and dendrites, the MTL network is more similar in structure to isolated actin-myosin gels observed in non neuronal cells. Raymond Lasek (1981) of Case-Western Reserve University has shown that the cytoplasm of nerve axon growth cones, which differs from axonal cytoplasm, is rapidly converted from the axonal variety to the growth cone variety in axons severed from their cell bodies. Thus "local" factors appear able to transform axoplasm to synaptic terminal cytoplasm, or axoplasm to growth cone cytoplasm. The MTL is the fine structure and texture of cytoplasm.

MTL activities appear to be dependent on calcium ion concentration. Ellisman's work shows that high calcium exposure causes the MTL to shorten and deform, leaving free ends or "nubbins." In normal axoplasm, different forms of MTL corresponding to both high and low calcium ion concentration appear to be found concurrently. These may correspond with "sol" and "gel" states determined by other techniques. Ellisman suggests that calcium may be the coupling agent for the dynamic aspects of the MTL in terms of location, extent and form of crossbridging of cellular constituents. Calcium regulated by cytoskeletal structures, or membranes, and the MTL itself may be locally dynamic and patterned. Standing waves, dissipative patterns, or holograms "hardwired" in MTL could be representations of calcium coupled states in cytoplasm.

Figure 5.19: Microtrabecular lattice (MTL) in PtK2 cell cytoplasm. Microtubules can be seen (one rising left to right on bottom) in the background of microtrabecular lattice (MTL). Two 40 nanometer gold particles are trapped in a vesicle within the MTL. With permission from DeBrabander (1985).

Ellisman raises the question of the importance of the cytoskeleton and MTL in the nervous system. One consideration is modulation of membrane related events such as receptor activity and neurotransmitter extrusion. Ellisman proposes that the MTL is triggered by calcium ions to release neurotransmitter vesicles. In addition, the MTL participates in other cytoskeletal functions also involving MT and filaments. These are axoplasmic transport, turnover and maintenance of membrane proteins and receptors, and the availability of neurotransmitters and enzymes at synapses. Ellisman has predicted some of the functions and/or behavior of the cytoskeleton and MTL within the nervous system. These are: 1) the MTL is a substrate to maintain cell shape changes including synaptic formation, and to regulate excitable properties of neurons. One example would be changes in distribution and volume of dendritic spines and synapses with implications for synaptic connections and perhaps learning and memory. Thus the MTL and other cytoskeletal elements can modify synaptic function by participating in trophic maintenance and turnover of membrane proteins to modulate membrane excitability and neuronal signaling. 2) The MTL, according to Ellisman, can also buffer small molecules and ions, maintaining these in specialized locations and patterns for metabolic and electrical functions. Thus the MTL may compartmentalize the cell, forming regions whose environments may vary. Buffering and control of calcium ion flux may be particularly important, and directly relate to cognitive functions. 3) The MTL can differentiate specific zones of cytoplasm and membrane, for example controlling the types of receptors or channels at synaptic zones by specific cytoskeletal linkages. The MTL and cytoskeleton also control the distribution of organelles, for example keeping the endoplasmic reticulum from entering axons, restricting the Golgi apparatus to perinuclear zones and keeping the synaptic bouton the right composition of axoplasm. 4) The MTL can mediate embryological development or morphogenesis through linkages with specific hormone receptors or tissue factors resulting in variations in developmental patterns. 5) The MTL can transduce chemical or mechanical work for intracellular transport and processes such as axoplasmic transport and translocation of synaptic vesicles to release sites on the presynaptic membrane. To Ellisman, the MTL regulates the rest of the cytoskeleton and the cell at large. In his view, it is the dynamic ground substance capable of intelligent behavior. One could argue, however, that MT regulate the MTL. What is most significant is the question of how they communicate.

The most labile and transient of the cytoskeletal levels of organization, the MTL is the current microfrontier of living material organization. The MTL is a network within a network of cytoskeletal proteins which, in the case of the nervous system, is a network within a network of neurons. In Chapter 8, a model of information processing will be discussed in which the MTL represents standing wave patterns of calcium coupled sol-gel states resulting from dynamic excitations of the cytoskeleton. Coherent excitations in the cytoskeleton could result in "holographic" standing waves which may be the bottom level of an information hierarchy: "infoplasm." An analog picture in which the MTL is both paint and canvas could be the texture of consciousness.

5.4.2      The Cytomatrix

Others have viewed the fine structure of cytoplasm and described it in different terms. Among these are Peter Satir (1984) of Albert Einstein University whose work has focused on the protein makeup and functional organization of the MTL, or "cytomatrix." Satir sees a functional integration of the cytomatrix, noting that protein-protein interactions have geometrical as well as biochemical and biophysical consequences. Certain interactions occur only at the ends of cytoskeletal polymers, others require or produce parallel arrangements of such elements by interacting only with their lateral surfaces, and still others may require orthogonal arrangements. In concert with centrioles, MTOC, and MT, cytoplasmic structures appear and function where and when needed.

The primary building block of the cytomatrix/MTL appears to be actin, a ubiquitous protein whose versatility describes the very nature of cytoplasm. Interaction with a variety of other proteins ("actin-binding proteins") unleash actin's full capabilities. When crosslinked by proteins such as fimbrin, actin can form rigid bundles which provide structural support. When associated with myosin (a "mechanoenzyme"), useful muscle-like contraction occurs. Also important are proteins such as talin, spectrin, vinculin, ankyrin and fodrin which connect the cytomatrix with membrane proteins, and calmodulin which mediates effects of calcium ion fluxes on the cytomatrix.

Proteins such as alpha actinin, troponin, and filamin link actin in networks which cause a gelatinous consistency to cytoplasm-a "gel" (Figure 5.20). In the presence of calcium ions and actin fragmenting proteins such as villin and gelsolin, the gel network liquefies to a solution-"sol." Other actin regulatory proteins include actin capping proteins, which stabilize polymerized actin and promote a "gel" condition, and profilin which binds actin subunits, prevents polymerization and maintains "sol" conditions. The layer of cytoplasm immediately below cell membranes is in a continuous gel state ("cortical gel"), but elsewhere dynamic transitions can occur. In the presence of myosin, actin gels can undergo contraction and streaming which are important in cytoplasmic movement such as amoeboid locomotion. The myosin appears to pull actin filaments against each other. A rise in calcium ion causes a sharp drop in viscosity of actin networks, through actin fragmenting proteins such as gelsolin. The same rise in calcium activates myosin to pull actin filaments against one another, resulting in gel contraction and vigorous streaming in adjacent "sol" regions. George Oster (1984) of the University of California at Berkeley has described a mechanism for amoeboid movement in which waves of calcium ion trigger transient sol/gel ,regions. The net flow of material is towards the calcium flux, which serves to pull the cell onward.

Figure 5.20: Components of the cytomatrix. Assembly of actin and related proteins form cytoplasmic "gel" states. A) monomeric actin, B) assembled actin filaments which are helical pairs of monomeric strands; center-cross section of actin filament, C) the presence of filamin (dark strands) causes actin cross-linking and dense "gel state," D) calcium and/or actin fragmenting proteins causes liquification to a "sol state." By Paul Jablonka.

Cytomatrix structures give rise to amoeboid and other types of cytoplasmic movement as well as polyhedral structures. Calcium mediated sol-gel transition and gel contraction can be important in many cell functions, including perhaps the transient representation of short term information and memory within neurons.

5.4.3      Cytoplasmic Solid State

Molecules such as proteins and RNA are heterogeneously distributed throughout cytoplasm and passively flow at rates below that expected for normal diffusion. Actively transported molecules such as those carried by axoplasmic transport travel at velocities far in excess of diffusion. Initially, the slow diffusion of biomolecules which are not actively transported was explained on the basis of cytomatrix barriers and channels. However, Gershon, Porter and Trus (1985) studied molecular diffusion through cytoplasm and came to a different conclusion. They found that the cytoskeleton/cytomatrix/MTL comprised from 16 to 21 percent of cytoplasmic volume, and that the cytoskeleton/cytomatrix/MTL surface area was from 69,000 to 91,000 square microns per cell (69 to 91 billion square nanometers). Their data has been interpreted to suggest that the cell "solid state" (cytoskeleton/cytomatrix/MTL) is not a barrier to diffusion since the aqueous phase occupies 4/5 of cell volume. They conclude that proteins and other molecules are dynamically bound to the solid state, accounting for the slow diffusion (Figure 5.21).

Biologist James Clegg (1981) has discussed how the binding of "soluble" enzymes to the solid state could account for the efficiency of various enzymatic processes. A sequence of enzymes in a complex biochemical pathway is much more efficient if physically arranged so that a cascade of reaction products occurs. If the product of one enzyme is the precursor for its neighbor enzyme, transfer time is minimized and the reactions are usefully facilitated. This also allows the possibility of dynamic regulation of these enzymes by the solid state structures. Clegg has also accrued evidence regarding the role of water within cytoplasm, particularly surrounding solid state structures. Using neutron diffraction and other techniques, Clegg has found that water adjacent to the cytomatrix is "ordered," that is aligned with polar bonds on the protein surfaces. Thus a layer of ordered water extends at least 3 nanometers from the billions of square nanometers of solid state surface area within each cell. This ordered water may be coupled by dipole oscillation to dynamics of the solid state, inhibit the thermal dissipation of protein oscillation energy from within the solid state, and shield calcium and other ions from random solid state interactions. Clegg also suggests that additional layers of slightly less ordered water ("vicinal water") extending further from the solid state would limit true "aqueous" water within cells to narrow cellular "sewage channels."

Figure 5.21: "Soluble" intracellular enzymes (circles) governed by the microtrabecular lattice (MTL: solid dark) and bordering region of ordered and vicinal water (within thin line). The dark circle is part of a cross sectional view. Clegg (1981) has proposed that dynamic activity within the MTL can regulate the enzymatic activity. By Paul Jablonka.

Is the MTL/cytomatrix the final microfrontier of biological organization? Current imaging techniques would not reveal smaller levels of organization, just as it took advances in electron microscopy to discover first the MT cytoskeleton and then later the MTL. Perhaps smaller, more intricate networks ranging down to the level of single peptide filaments might be present. With layers of ordered water and charged ions, such a solid state "infoplasm" could occupy nearly the entire volume of biological material.

5.5           Cytoskeletal Motility

Albrecht-Buehler (1985):

Cell movement ... appears to be determined by some kind of chemical computer, the nature of which is beyond our present understanding.

Cell motility was discovered by van Leeuwenhoek who observed flagella propelled sperm cells swimming in the 17th century. A variety of cytoplasmic movements include "amoeboid" creeping over a surface, internal streaming, axoplasmic transport, muscle contraction, ciliary and flagellar bending, and cell shape changes. These maneuverings are all engineered by a relatively small group of proteins whose net collective effects can be quite spectacular. For example, lymphocytes and macrophages are white blood cells which combat infection by migrating from the blood stream into an open wound to engulf invading bacteria or foreign material. Cells of developing embryos perform precisely choreographed movements that give rise to different tissues. Internal movement such as the streaming of cytoplasm, secretion of cell product vesicles (i.e. neurotransmitters), engulfment of matter, and the separation of paired chromosomes in cell division are routine functions whose complexity, organization, and precision generally boggle biologists.

Also, well controlled muscle contraction which depends on the conformational bending of myosin head molecules can result in strenuously running a 4 minute mile, or delicately painting a Mona Lisa. Four categories of cytoplasmic movement and force generation will be considered with attention to their regulatory mechanisms. These are: cytoplasmic probing, bending sidearms, ciliary and collective movements, and geodesic tensegrity structures.

5.5.1      Cytoplasmic Probing

In the early 20th century, W. H. Lewis, an embryologist at Carnegie Institute of Washington, took time lapse motion pictures of cells grown in culture (Lazarides and Revel, 1979). Lewis treated the fibroblasts (active cells which repair wounds) with protein cleaving enzymes to break up adhesive contacts and networks of extracellular material holding the cells together. He separated the resulting clumps and grew individual cells in a nutrient medium. After a few hours, Lewis' fibroblasts had acquired a polygonal shape. Along the shortest side of the polygon were lamellipodia, delicate sheet-like extensions of the cytoplasm. He exclaimed: "the ruffled lamellipodia slowly bend back and forth like the ruffles of a dress in a slight breeze" (Lazarides and Revel, 1979). Later, Ingram and Abercrombie at University College London studied the membrane coated ruffled lamellipodium, fibroblasts' main locomotion organ. The lamellipodium forms strings and contacts with the substrate on which the cell rests, stretching portions of the rest of the cell among several temporary adhesions. When the cell is spread out to its fullest extent it maintains one or two primary ruffles, each of which tend to lead the cell in a particular direction. The cell then migrates with changes in direction which typically come at intervals of several hours. If a ruffle does not stick and the cell move in another direction, the ruffle folds back on the upper surface of the cell, collapses onto it and disappears into the cell (Figure 5.22, 5.23 and 5.24).

Figure 5.22: Amoeboid movement in a time-lapse series by U. P. Roos at the University of Zurich. 1a) Probing lamellipodia (arrow-heads) in direction #a of cell movement. Retraction fiber (arrow) shows trailing end of cell. b) Cytoplasm has flowed to fill out lamellipodia. c) Finger-like filopodia (arrow) probe direction. d) Cytoplasm advances, moving cell. With permission from U. P. Roos (1984).

Using the electron microscope, Lazarides and Revel (1979) described the lamellipodium as a somewhat irregular sheet of cytoplasm about one tenth of a micrometer (100 nanometers) thick. It is decorated along the edge by small protrusions resembling stubby fingers which attach themselves to the surface of the dish, and can quickly extend several times their initial length into rod-like filopodia. These are highly dynamic structures that can change from a fluid state to a rigid one in less than a second by virtue of the sudden assembly of actin filaments. Growing from the cell surface, they provide spatial guides between which the cell membrane and cytoplasm flows to form the ruffle. The filopodia either attach themselves to the substrate and become rigid, or melt back into the cell. The cell adheres to the surface below it at only a few distinct sites, rather like a hand contacting a table top only with finger tips. Permanent contacts of the ruffle with substrate result in adhesion plaques; as the cell moves the plaques are continuously formed and broken. In addition to their role in cell adhesion and motility, the ruffles and particularly the filopodia serve a sensory function. In the developing embryo when the filopodium of one migrating nerve cell makes contact with the surface of another, the cells stop moving toward each other. Similarly, when the filopodium of a cell spreading in tissue culture touches the surface of a cell that is already flattened, the membrane of the first cell will flow in a direction opposite to the point of contact. This type of sensitive regulation of growth processes is defective in malignant cancer cell.

Figure 5.23: Cytoplasmic organization of a cultured cell moving to the right. Microtubules (MT) radiate from the centrosome (C), located near the nucleus (N), Golgi elements (G), and lysosomes (L). Mitochondria (M) and endoplasmic reticulum (ER) are aligned along MT. Phagocytic and pinocytic vacuoles (P) and secretory vesicles (V) move along MT between the centrosome and periphery. Intermediate filaments (IF) roughly parallel MT. Stress fibers (SF) are the cytoplasmic anchors for extracellular matrix fibers (MF) which contact the substrate. At the leading edge of the moving cell, lamellipodia and filopodia, comprised of actin bundles and meshwork, probe the future. At the trailing end, retraction fibers (RF) contain actin bundles and intermediate filaments. With permission from Marc DeBrabander (1982) and Janssen Pharmaceutics Research Laboratories.

Northwestern University biologist Guenter Albrecht-Buehler (1977, 1980) has studied isolated cytoplasm and beholds intelligent behavior. Examples include the movements of single cell organisms alternating between tumbling and smooth motions (Adler, 1969), swimming of motile bacteria in chemotactic environments (Macnab and Koshland, 1972), backward swimming of paramecium after collision with an object, and the amoeboid directed locomotion of cultured 3T3 mouse cells. Albrecht-Buehler classifies three major types of amoeboid motion: 1) force generation (contraction of an actin-myosin complex, assembly-disassembly of filament polymers, dynein-MT sliding, filament rotation), 2) transmission of motile forces to the framework work of a cell (cytoskeleton in general, anchorage of fibrous elements to the cell surface in particular) and 3) generation of traction (adhesion plaques, extracellular matrix, basement membranes).

Figure 5.24: Cell treated with microtubule assembly inhibiting drug. The cell has lost its polarity. Lamellipodia form around the periphery of the "blob"ular cell. The centrioles (C) are separated from the nucleus. Golgi elements (G), lysosomes (L), mitochondria (M) and endoplasmic reticulum (ER) are disorderly scattered throughout the cytoplasm. With permission from Marc DeBrabander (1982) and Janssen Pharmaceutica Research Laboratories.

Albrecht-Buehler further divides the control of cell migration into three subtopics: 1) generation of a front-rear axis (polarity involves MTOC and MT), 2) cell body coordination-how the forty billion protein molecules comprising each cell act cooperatively to move as a unit, and 3) logic of migration-the rules of changing direction. Migrating cells make decisions and globally assess their environment. Albrecht-Buehler asserts that the control of amoeboid cell migration provides evidence of cytoplasmic intelligence because it requires coordination of many cell domains and navigation which involves assessment of the environment.

But the cell is not the indivisible unit of intelligent behavior. Albrecht-Buehler contends there are hierarchical strata of information processing within cells. He has shown that small fragments of cytoplasm comprising about two percent of cell volume can be torn out of living fibroblasts (Albrecht-Buehler, 1980). These "microplasts" are devoid of any genetic input, yet can move filopodia, ruffle lamellipodia and produce blebs. Some fragments are so small they appear as isolated active filopodia, ruffles, or blebs and suggest that cells contain cytoplasmic domains capable of autonomous amoeboid movement. Albrecht-Buehler asserts that these cytoplasmic activities within cells are controlled by a superior stratum of organization to permit coherent locomotion. This level of control is not located in the genes since cells with their nuclei removed still exhibit migration behavior. There is intelligence in the cytoplasm. The cell has a driver's license.

5.5.2      Bending Sidearms

Several important cytoplasmic movements occur due to bending of contractile proteins attached along rigid cytoskeletal filaments. In muscle, arrays of parallel, interdigitating "thin" filaments (actin) interact with "thick" filaments (myosin). Tiny sidearms or crossbridges ("myosin heads") attached to the myosin thick filaments extend across a gap of about 13 nanometers and "cyclically row like banks of tiny oars" to move the filaments relative to each other. The energy from hydrolysis of ATP drives the conformational changes which causes the myosin molecules to curl. The precise utilization of ATP hydrolysis energy to cause contractility and other conformational changes remains poorly understood, but will be discussed in Chapter 6.

Each myosin filament carries about 500 heads, each of which cycles about 5 times per second. There is some evidence of cooperativity in that once a myosin head has detached, it is carried along by the action of other myosin heads along the thick filament. The myosin head rowing is initiated and coordinated by waves of calcium ion released from a reservoir (the sarcoplasmic reticulum) triggered by membrane electrical activity. Rises in calcium ion causes an actin-bound regulatory protein called troponin to shift its position and allow actin-myosin ratcheting.

Many MT related activities generate force, locomotion and movement of vesicles and other material; axoplasmic transport is one well studied example (Lasek, 1981; Ochs, 1982). Parallel MT within axons are polarized with their fast growing "plus-ends" distal from the cell body facing the synapse. Force generating sidearms occur about every 16-18 nanometers along MT lengths. These contractile crossbridges generate directional movement of material along MT by undergoing a sequence of conformational changes involving attachment of crossbridges to vesicles, ATP dependent force generation by the crossbridges, and detachment of crossbridges from vesicles. Detachment occurs only at "plus" ends near synapses. The process is similar to rowing of myosin heads to slide actin and myosin filaments past each other and shorten muscle fibers. MT based dynein activities, however, are far more variable, flexible, and interactive than the repetitive nanoscale events in muscle. For example, in MT dependent axoplasmic transport different material is simultaneously transported in the opposite direction, from the synapse to the cell center. This "retrograde" axoplasmic transport is thought to provide feedback to the protein synthesis machinery as to what enzymes or material are required, and/or to allow "recycling" of some molecules (Figure 5.25).

Robert Allen (1985) was among the first to suspect that MT and MAPS served as intracellular conveyor belts. He and his colleagues studied isolated MT and MT fragments which, with .available biochemical energy in the form of ATP, "glide" along glass cover slips at velocities of 150 to 450 nanometers per second. The velocity is independent of MT fragment length, occurs essentially in a straight path, and is in the direction of retrograde axoplasmic transport. The straight paths of gliding MT segments suggest that the force generating enzymes are deployed in linear, rather than helical paths along the MT, and that the stroke that causes gliding is parallel to the MT with a spacing interval of about 17 nanometers. Reducing the available ATP concentration slows the gliding speed significantly, but does not affect the number or behavior of gliding MT. Gliding MT almost never interact when they cross paths, and when the forward progress of a gliding MT is blocked by an obstacle, it "fishtails" slowly from side to side through a series of serpentine maneuvers. Allen and colleagues concluded that the forces observed in their slithering free MT as well as in axoplasmic transport and ciliary bending are due to "force generating enzymes" directly attached to MT. Dynein, which functions to cause binding in cilia and flagella, is one MT force generating enzyme and kinesin is another motor for organelle transport along microtubules. Latex beads coated with kinesin translocate along microtubules similar to organelles, although at a slower velocity. Purifled kinesin can increase the frequency of axoplasmic organelle movement along purified MT.

Allen and colleagues proposed the "backstroke hypothesis" which states that the force generating enzyme (dynein or kinesin) makes an elliptical stroke which imparts some force in both directions. Allen's dynein backstroke model is capable of carrying vesicles in opposite directions simultaneously through sufficiently separated pathways so that they seldom collide. Further, the motion generated can be continuous, not interrupted by cycles of attachment and detachment. The mechanical cycle of each sidearm includes a radial stroke that moves vesicles in the anterograde direction toward the microtubule plus end. This part of the cycle also causes isolated MT to glide "retrograde." The return stroke is tangential to the MT surface and transports the larger organelles in a retrograde pathway, and propels gliding MT toward their_. "anterograde" plus end.

The mechanisms by which the force generating protein arms may use ATP energy to contract will be discussed in Chapter 6. Even less well understood is the signaling and communication which orchestrates contractile activities of rows of arms spatially arrayed on MT lattices. Collective communication among MT lattice subunits (solitons, coherent excitations, lattice vibrations) could explain this orchestration.

The backstroke model is currently favored more than another model: microstreams. Shimizu and Haken (1983) had proposed a dynamic cooperativity of cytoskeletal elements which generated hydrodynamic microstreams conveying cellular materials. They specifically focused on actin-myosin interactions to generate these microstreams. New techniques such as Nanovideo Microscopy developed by Marc DeBrabander and colleagues (1986) at Janssen Pharmaceutica Research Laboratories in Belgium show direct tracking of immunolabeled particles along MT, questioning the significance of microstreams. Particles are seen to travel in opposite directions along the same MT, passing each other like two railroad trains on adjacent tracks. Microstreaming does not appear dominant in axoplasmic transport, but could be important in other phenomena. The primary site of coordinated transport and its complex orchestration appears to rest solely in the province of MT.

5.5.3      Ciliary and Collective Movement

Figure 5.25: Axoplasmic transport occurs by coordinated activities of sidearm, contractile proteins ("dynein"), which cooperatively pass material in a "bucket brigade." The orchestration mechanism is unknown, but shown here as the consequence of signaling by "soliton" waves of tubulin conformational states. By Fred Anderson.

The structure of cilia and flagella is a core of parallel microtubules in a cylindrical "9+2" arrangement of doublet or triplet microtubules. The arrangement is similar to centrioles which are also cylinders of 9 MT triplets, but without the additional central pair. Motor cilia and flagella have actin wound around their central pair of MT. The side arm proteins which connect the parallel microtubules are called links, spokes, or sidearms and are comprised of dynein, the contractile protein which utilizes ATP energy to produce force. Cilia and flagella are anchored inside the cell to basal bodies which are also composed of parallel microtubules and form a short cylinder with the same outer diameter and nine fold symmetry as cilia, flagella and centrioles. It has been clearly shown that ciliary mechanisms can function without influence from the cell to which they are attached. Flagella or cilia severed from cells by laser beams continue to propagate normal bending movements, if the surrounding medium contains ATP and either magnesium or calcium ions. Cilia function to move fluid over the surface of a cell or to propel single cells through a fluid. Single cell organisms use cilia for the collection of food particles and for locomotion. In the human lung and respiratory tract, epithelial lining contains about a billion cilia per square centimeter which act to sweep layers of mucous, trapped particles, and dead cells towards the mouth where they may be coughed up or swallowed. The cilia bend in coordinated unidirectional waves in which each cilium moves as a tiny whip. There is a forward stroke in which the cilium is extended and exerts maximal force on the surrounding liquid medium, followed by a recovery phase when it returns to its initial position. The movement is a rolling motion which minimizes viscous drag and requires about 0.1 to 0.2 seconds. Cycles of adjacent cilia are not quite in synchrony, and the small delay produces wave-like patterns of the entire ciliary complex. Simple flagella of sperm and single cell organisms are much like large cilia in their internal structure but are usually longer and propagate in quasi-sinusoidal waves rather than faster whip-like movements. The mechanism of flagellar beating in eukaryotes (but not of bacteria) is very similar to that of cilia.

Muscle contraction, axoplasmic transport, and ciliary and flagella motion all occur by the contractile bending of sidearm proteins attached along cytoskeletal filaments. In the case of muscle contraction, the stable cytoskeletal filaments are myosin with appendages called myosin heads crawling along parallel actin filaments. In the case of axoplasmic transport and ciliary and flagellar bending, microtubules are the stable filaments from which dynein contractile sidearms crawl or row along other MT (in the case of cilia and flagella), or pass along material or vesicles (in the case of axoplasmic transport). The energy for all of these mechanisms is supplied by the hydrolysis of ATP, but its precise utilization, transfer of conformational states and the temporal orchestration required to control these sidearm appendages are unknown. Figure 5.25 shows one possible cooperative control mechanism in which a wave of tubulin conformational states (soliton) travels along the MT cylindrical surface lattice to trigger the dynein activities.

5.5.4      Geodesic Tensegrity Gels

Actin-myosin contraction also occurs in non-muscle cells. Soon after actin and myosin were identified in muscle, Loewy found that extracts of slime mold cytoplasm responded to ATP with a decrease in viscosity. He hypothesized that ATP provided chemical energy which was converted into mechanical work required for slime mold locomotion. After H. E. Huxley and colleagues at University College London showed that muscle contraction was achieved by ATP dependent sliding of actin and myosin filaments past one another, Hatano and Osawa at Nagoya purified actin from slime mold. Subsequent investigations turned up myriads of cytomatrix proteins including myosin which were related to contractility and its regulation (Lazarides and Revel, 1979).

Using fluorescent antibodies to "light up" specific proteins, Lazarides and Revel (1979) have observed actin filaments organized into strikingly regular networks looking remarkably like geodesic domes. They describe icosahedral geodesic networks that encompass the area above and around cell nuclei and other cell regions after the cells are treated with fluorescent antibodies illuminating actin, alpha actinin and tropomyosin. Alpha actinin is localized primarily at the vertices of the geodesic network and tropomyosin is localized along the actin fibers connecting the vertices. Longer fibers attach to network vertices and extend into filopodia, lamellipodia, and membranes. The geodesic network and vertices act as organization centers involved in maintaining cell structure (Figure 5.26).

Figure 5.26: Geodesic actin cytomatrix gel surrounding cell nucleus, from Lazarides and Revel (1979). Vertices of the icosahedral nuclear dome attach filaments which extend to the cell membrane. By Paul Jablonka.

Some actin networks are transient dynamic structures which serve fleeting, but vital functions during the cell cycle. During the final stages of cell division, after duplicated chromosomes have been pulled apart by the mitotic spindle, a ring of constriction encircles the equator of the mother cell perpendicular to the spindle axis. Work by this "cleavage furrow" constricts the cytoplasm until it is divided into two daughter cells. The constricting tension has been estimated from observations in sand dollar and sea urchin eggs to be about 3 x 10⁵ dynes per square centimeter, a value comparable to the force generated in muscle (Lazarides and Revel, 1979). The cleavage furrow contractile ring is a temporary structure that exists for only about 10 minutes; it rapidly assembles and disassembles. Although the width and thickness of the contractile ring remain constant during constriction, the volume decreases. The ring must disassemble even as it contracts, meaning that any sliding interaction of the actin and myosin filaments must be followed by disaggregation of some of the filaments. This disassembly must take place uniformly through the ring during its entire brief lifetime. Therefore, a simple sliding filament model is insufficient to explain the cell cleavage function of the contractile ring.

Steve Heidemann and colleagues (Joshi, Chu, Buxbaum and Heidemann, 1985) at Michigan State University have examined compressive and tensile properties of cytoplasm. They find that semi-rigid microtubules are under compressive forces generated by interwoven contractile actin filaments. A balance between parallel compressive and tensile forces leads to a selfsupporting property characterized by Buckminster Fuller (1975) as "tensegrity." Tensegrity can provide cell support even if the rigid parallel element are not in direct contact. Tensegrity in the cytoskeleton might explain the self-supporting structural properties of cytoplasm in which the rigid parallel elements are not in direct contact.

Robert Jarosch (1986) has proposed that contractile actin winds and unwinds microtubules by a "torque drive," causing rotational oscillations and perhaps tuning and detuning of the microtubule system. Dynamic compression/tension may also be important in the regulation of membrane receptors whose mobility are limited by anchoring MT. Conversely, contractile actin filaments can redistribute the receptors unless they are restrained.

Dynamic tensegrity (Chapter 8) may be an important mechanism in many biological functions. In the cytoplasm, complex structures assemble, perform, and vanish into soluble subunits.

5.6           The Cytoskeleton and Development

The cytoskeleton performs critical functions in reproduction and development. These include meiosis (division of duplicated chromosomes in sperm and egg cells), sperm motility, mitosis, cell proliferation, cell migration and changes in cell shape which accompany differentiation, the expression of cell form and function. All cells in a given organism have the same genetic capabilities, but take on the roles of specific tissues by the mysterious processes of trophism and differentiation. Cells within developing organisms or embryos can be moved and will grow and assume the characteristics of the new tissue in which they are placed.

The cytoskeleton is crucial to all steps in reproduction, growth, trophism and differentiation. If chromosomes are maldistributed in meiosis or mitosis, nonviable or abnormal offspring can occur. Such maldistribution may be related to cytoskeletal dysfunction, an early theory for the cause of cancer (Boveri, 1929). Variability in tubulin isozymes and MAPs could explain tissue and cell specificity based on differences in the molecular composition and activities of the cytoskeleton.

The origins of form, growth patterns, and differentiation comprise the biological science of embryology, first addressed by the Greek Aristotle. Two possible explanations for the development of form and patterns from what appeared as nothingness were considered by Aristotle. The first notion was that embryos derive from formless, tiny masses through a process of continuous unfolding he termed "epigenesis." The second notion was that individual patterns exist in miniature within the parent, and development consists in steady growth of its dimensions, the process of "preformation." Aristole (Book VI of Historia Animilium) described his experiments in which he interrupted the incubation of a hen's egg in various stages. After three days,

the heart appears like a speck of blood in the white of egg, ... [it] beats and moves as though endowed with life and from it two vein ducts with blood trend in a convoluted course.

Only seven days later are the "chick and all its parts distinctly visible." Aristotle favored epigenesis, however the concept of preformation dominated science for centuries until 1759 when Friedrich Wolff, a German physician living in St. Petersburg published "Theoria Generationis." With better optics than Aristotle, Wolff noted that the chick started as "globules" or cells that seemed to have no functional (relation to one another. With time, distinct groups of cells emerged to form the structures that become the hen's principle organs. Further evidence against the preformation theory came in 1900, when Hans Adolf Driesch demonstrated that a sea urchin embryo, when cut in half, develops into two complete embryos (Burnside, 1974).

Aristotle's epigenesis theory of differentiation states that homogeneous spheres begin to divide, yielding more spheres which themselves divide. The "cells" begin to exhibit differences in structure and function and a "collective coherence." Each cell knows where to go, when to go there, which cells to congregate with, and how to work cooperatively. Excluding the possibility that a transcendental intelligence personally oversees the development of each organism, the conclusion is that the original cell contains enough information to orchestrate the entire process. Aristotle would probably be even more amazed to discover that during early embryological development each cell is totipotential: it can take on the role of any tissue in the body. We now know that the chromosomal DNA in each cell contains all genetic information. We are beginning to understand how specific genes are turned on and off. Cells appear to take on the role of their environment due to morphological "trophic" influence conveyed by cytoskeletal axoplasmic transport within their "innervating" nerve cells.

Much of the mystery of biological form and differentation thus focuses on the growth, pathfinding and trophic capabilities of embryological neurons. The form, architecture, and connections which nerve processes make not only establish tissue and body shape, but synaptic connections and brain "hardware."

How do neuronal processes know where and when to send their processes and establish connections? The mystery has been likened to the "Indian rope trick" fable in which a "fakir" tosses a rope into the air where it mysteriously stays rigid. He then climbs up the rope, pulls the lower portion up, and disappears! The embryological Indian rope trick depends on neuronal shape and orientation: the number of young axons and dendrites ("neurites") which arise from the cell body, their direction, degree of branching, how far they grow, and where and how they form synapses. All these factors are manifested by the cytoskeleton. The centriole/MTOC establishes cell polarity and orientation and presumably "launches" the extending processes. The tips of lengthening neurites are specialized areas of cytoplasm called "growth cones" which not only grow outward, but participate in decisions about direction, branching, and termination of extension. Growth cones can sense changes in local environment and, acting in concert with other cytoskeletal elements within the neurite, shift direction, branch, or form synapses. Growth cone activities are remarkably similar to movement in amoeba and other simple organisms. They continually probe their surroundings by sending out and then retracting delicate ruffles known as lamellipodia, and finger-like projections called filopodia or microspikes. These dynamic micro-appendages are comprised of meshworks and parallel arrays of actin filaments. MT and neurofilaments from the neurites splay into the growth cones, but generally stop short of the actin-rich areas (Bunge, 1986).

The roles of MT and actin in axonal extension have been studied by Yamada, Spooner and Wessells (1970). Addition of cytochalasin B, which is known to alter actin filaments, blocks neurite outgrowth if added before extension has begun. Treatment of extending axons with cytochalasin B results in cessation of the "ruffling" activity of the growth cone with little immediate effect on the neurite shaft. In contrast, antimicrotubule agents such as colchicine leave growth cone activity unaffected but block neurite extension. Continued exposure results in retraction of the cell process with subsequent formation of one or more growth cones from the cell body. These observations together with ultrastructural evaluations of treated cells, show that MT are necessary for the growth of neurites, while actin filaments are essential to growth cone protrusion, a phenomenon similar to amoeboid motion. A complex interplay of dynamic activities of actin and MT are required for neurite sprouting and growth cone extension. A composite view of growth cone activities is that, under direction of the MT cytoskeleton, actin assembly generates protruding lamellipodia and filopodia. Upon contact with an appropriate external substrate, filopodia adhere and actin bundles form from the filopodium tip into the growth cone. Myosin colocalizes with actin and the actin-myosin interaction produces a "muscle-like" tension which provides an anchorage for the growth cone to the filopodium adherence site. Lamellipodia, sheet-like ruffles, dart out among the finger-like filopodia, particularly near points where decisions about branching are required.

Growth cone activities related to embryological differentiation are at the very ground floor of intelligence. Evidence suggests that expression of tubulin, MAPS, and other cytoskeletal elements are also important for determination of cell form and function. Barra and colleagues (1974) have shown changes in alpha and beta tubulin during brain development. The relative amount of alpha and beta tubulin in rat brain peaks at about the 14th day after birth and then declines to adult levels as a result of reduction in the rate of synthesis. The pattern of alpha and beta tubulin genetic variants (isozymes) undergoes marked changes during brain development with an increase in the variety of tubulin. For example, 3-4 different types of beta tubulin are observed in embryonic mouse brain compared to 13 types in adult mice. Modification of alpha tubulin isotypes begins in the embryonic brain whereas the beta tubulin modification begins to occur after birth and coincides with a period of extensive outgrowth of processes and synaptogenesis in the developing brain.

MAPs are also implicated in development and expression (Barra et al., 1974). In the case of rat brain, two tau polypeptides are present in the first few days of birth. By 35 days the adult pattern has emerged in which four major tau polypeptides are apparent. The tau proteins of maturity are more adept at promoting MT assembly than are the tau proteins of immaturity. MAP 1 can be resolved into three groups of MAP 1A, 1B, and 1C. In chicken brain development, MAP IA is initially present at low levels and increases during late embryonic development and post hatching. Similarly, MT from 10 day old rat brains are depleted of MAP 1A in comparison to adult brains. MAP 2 is restricted to dendrites and cell bodies in adult brain and is particularly concentrated in dendritic tips, Purkinje cell dendrites and all neuronal cell bodies, but absent from axons. Localization of MAP 2 to the dendritic cytoskeleton begins at the earliest times of appearance of dendritic outgrowths. In the adult brain, MAP 2A and MAP 2B show up at different times and brain regions and are localized in neurofilament rich axons.

All these changes are due to localized cytoskeletal mechanisms in addition to genetic expression. The symphony of alterations in tubulin and MAP isozymes has the score written in DNA, but its performance requires collective cooperative interactions among the conductor and orchestra. For example, "tyrosination" of beta tubulin occurs due to signaling and conditions present in the cytoplasm. Modifications are involved in the control of the outgrowth of axonal and dendritic processes, transport of constituents to the tips of the growing processes, cell migration and division, and establishment and maintenance of synapses and the adult form of the neuron. The temporal relationships between changes in neuronal MT proteins and differentiation is essential to the final product: the brain/mind.

5.7           The Cytoskeleton and Medicine

Defects related to microtubules are specifically linked to several human diseases. One example is "immotile cilia syndrome" (Afzelius, 1979) which is caused by altered dynein and results in an inability to expel secretions from the lungs, leading to recurrent bacterial infections. Another is developmental disability in infants which is caused by abnormal MT function induced by defective MAPs (Purpura, 1982). The cytoskeleton participates in the effects of various diseases (malignancy, Alzheimer's disease, viral infections), drugs, toxins and the body's response to disease.

The pathway to understanding MT led through the disease gout, a painful swelling of joints caused by the body's response to accumulation of urate crystals. Lymphocytes and macrophages, the body's immune cells, leave the bloodstream and migrate by amoeboid locomotion towards the urate crystals which often lodge in a joint of the big toe. Gout may be precipitated by purine containing foods which are metabolized to urate. The urate crystals are not particularly harmful except for the painful inflammatory immune response they trigger. By luck, a drug called colchicine was found to be helpful in relieving the pain and tenderness. Later it was discovered that colchicine worked by depolymerizing microtubules and preventing the locomotion and engulfment behavior of the lymphocytes and macrophages.

In addition to cell migration, the cytoskeleton is clearly involved in the establishment of normal cell architecture, function, and control of cell division and growth. In malignancy, control of cell reproduction is lost and growth proceeds without regard to the needs of the organism. Cancerous cells exhibit a tendency to break away from their anchorage and set up growth elsewhere in the body. Malignant cytoskeleton is disorganized with formation of oscillating aggregates of contractile material that aids dislodgement of the cell from its anchorage, instability in chromosome number and loss of growth regulation. All these effects could be primarily cytoskeletal in origin, and Puck (1977) has proposed that malignancy is a disease of the cytoskeleton. Clearly, the cytoskeleton is involved in the expression and processes of malignant cells. In cancer, cell division goes out of control: multipolar or asymmetric mitotic spindles are commonly observed. Boveri observed in 1929 that such aberrant distribution of genetic material could result in any combination or permutation of genes, most of which would be nonviable. However, some permutations may be sufficiently viable and have the abhorrent traits of malignancy. Many other factors leading to genetic alterations can account for the same results, so the precise etiology cannot be pinpointed. Indeed, cancer is probably caused by a number of etiologies including viruses which infiltrate and usurp the genetic apparatus and cytoskeleton.

Much of our current knowledge of the cytoskeleton has been learned from experimental perturbations by toxins, poisons, or drugs. Claude Bernard said in 1875:

The poison becomes an instrument which dissociates and analyzes the special properties of different living cells; by establishing their mechanisms and causing cell death or changes in cell function we can learn indirectly much about the relation between molecular structure in the physiological process of life.

Peripheral nerve MT and axoplasmic transport are vulnerable to toxin and drug effects. Vinca alkaloids (vincristine, vinblastine) are commonly employed in battling cancer because they disrupt MT mitotic spindles as they polymerize. Because the cancerous cells are dividing so much more rapidly than normal cells, the mitotic spindle poisons are effective against malignancy. However, they can cause side effects including peripheral nerve damage by injuring MT dependent axoplasmic transport. This results in peripheral nerve damage in many patients who receive the anti-microtubule drugs. Sometimes severe neurological dysfunction limits the use of the vinca drugs. Fortunately, neither vinca alkaloids nor colchicine cross the blood brain barrier so central nervous system problems are limited.

No agents are known to selectively disrupt intermediate filaments (although 2,5 hexane dione may act in this way). In intact cells, vanadate combined with drugs that disassemble microtubules cause vimentin type intermediate filaments to collapse around the nucleus. A toxin known as beta, beta prime iminodiproprionitrile (IDPN) disrupts microtubule/neurofilament organization in axons, which results in colocalization of certain MAP II subgroups with intermediate filaments. Wisniewski and colleagues (1966) showed that injection of aluminum salts into the brain or cerebrospinal fluid of experimental animals induced marked accumulation of 10 nanometer filaments in cell bodies, dendrites and initial axon segments of large neurons. When silver stained, these accumulations superficially resemble the neurofibrillary tangles that are encountered in a number of human disease states, notably Alzheimer's disease, Parkinsonism dementia and senility. The filamentous accumulations are not precisely identical to those of the human diseases but raise interesting questions about the role of cytoskeletal proteins in these afflictions. Alzheimer's disease and related forms of senile dementia are characterized by these neurofibrillary tangles which result in various symptoms including a shortage of acetylcholine at synapses of "cholinergic" neurons. This shortage may be due to a breakdown in the cytoskeletal transport mechanisms which deliver acetylcholine precursors to the synapses. The cognitive dysfunction which characterizes the dementias is somehow related to disruption of the cytoskeleton. Other toxins whose mechanisms have been ascribed to cytoskeletal effects include hexanedione and hexacarbons, carbon disulfide, acrylamide, methylmercury, and mineral fiber asbestos.

When cells are injured by lack of oxygen, acidosis, toxins, or other causes, there is an irreversible point beyond which cell death is inevitable. Recent evidence points to a pathological elevation of cytoplasmic calcium ion as the irreversible point (Farber, 1981). The excess calcium may originate outside the cell, diffusing across damaged membranes, or may be released from cytoplasmic reservoirs including the cytoskeleton. Regardless of its source, the elevated calcium causes depolymerization of microtubules and other cytoskeletal components. The net result is a loss of cytoplasmic organization, enzyme function, and structural integrity leading to cell death. Dimethysulfoxide (DMSO) is a solvent which stabilizes polymerized microtubules, and which has been shown to preserve integrity of cells and tissues against damaging effects of radiation, low temperature, and other insults. Other compounds (taxol, poly-lysine, etc.) also fortify or preserve MT and the cytoskeleton and could be important in future therapies.

The effects of a number of other drugs may be mediated via the cytoskeleton; among these are anesthetics which cause a reversible cessation of consciousness. In the 19th century, Claude Bernard noted that an anesthetic (chloroform) inhibited "protoplasmic streaming" in slime molds, a function of the cytoskeleton. Allison and Nunn (1968) showed that sufficient concentrations of the general anesthetic halothane reversibly depolymerized MT. Sleep producing barbiturates, local anesthetics, and major tranquilizers such as thorazine also bind to brain MT. Chapter 7 will describe how anesthesia is related to inhibition of cooperative activities related to information processing in the cytoskeleton and connected membrane proteins. The future emergence of nanotechnology (Chapter 10) may permit medical intervention to be more specifically tuned to functions and dysfunctions of the cytoskeleton.

5.8           Intelligence in the Cytoskeleton

Cytoskeletal gel networks have complex repertoires. Motile events within non-repetitive muscle cells are dynamic, often transient and variable and not strictly analogous to muscle contraction. Cells contain from two to five different types of actin and these may combine up to ten different ways depending on the presence of binding proteins and other factors. Certain actins polymerize in the presence of calcium or magnesium ions whereas other actins polymerize only in their absence. Non-muscle cells possess control mechanisms that dynamically switch between monomeric actin, actin in a filamentous bundle form, and actin in a geodesic gel meshwork form. Some cytoplasmic motility is the result of actin myosin crawling, whereas examples depend on explosive polymerization of actin, rapid disaggregation of actin filaments, or interconversion of filament bundles into a meshwork (Satir, 1984). Microtubules, filaments, and centrioles are also involved in these same aspects of cell movement and are particularly important to orientation and directional guidance.

How can these diverse modes be coordinated? Guenter Albrecht-Buehler (1985) cites two basic requirements for cytoplasmic intelligence. These are compartmentalization, which separates components engaged in various functions to prevent chaos, and the information content. According to Shannon (1948) information is not concerned with the meaning of the message but only with its formal structure. As Marshal MacLuhan said: "the medium is the message"; the cytoplasm is both!

Information can have spatial and temporal content: spatially a message can be in the form of a letter or magnetic tape, and/or can have a temporal structure such as a radio signal or movie. It is the very coupling of spatial and temporal components in a dynamic sense that provides the medium of information. Shannon suggested that intended signals be "unpredictable." For example, a meaningful text is a sequence of words and letters that the reader cannot anticipate. In contrast, text consisting of an uninterrupted string of a single letter doesn't carry much information. Similarly in temporal messages such as radio signals, the strictly periodic and therefore predictable carrier wave of a transmitter carries no information. Only after the periodic oscillations of the carrier wave have been modulated with unpredictable changes of frequency (FM), or amplitude (AM) can speech or music be heard. At the opposite end of the spectrum, random stochastic noise is equally devoid of information.

Albrecht-Buehler observes that cytoplasm is neither totally regular, like a periodic crystal, nor totally random like a boiling liquid, but is an organized piece of matter. Complex, intricate activities occur "in the midst of drowning thermal noise all around and within." The cytoplasm not only keeps its cool against the thermal background, it routinely couples spatial and temporal components to manifest information. One example of cytoplsamic information which is independent of DNA/genetic control is the pattern of ciliary orientation in paramecium (Figure 5.27). Extrinsically altered, "nongenetic" patterns are maintained through one hundred mitotic generations (Aufderheide, Frankel and Williams, 1977).

Albrecht-Buehler describes three possible and progressively enlightened approximations to account for the intelligent actions of cytoplasm. 1) "The secret of cytoplasmic complexity is sought in the very randomness of thermal chaos combined with the observed specificity of biochemical reactions." This view is doubtful since the cytoskeleton and other structures take on elaborate, nonrandom forms. 2) Cytoplasmic intelligence stems from "supramolecular topology, architecture and dynamics rather than freely swarming inhibitors and promoters with their competing binding constants." The complex spatial arrangements of protein subunits and other molecules in macromolecular assemblies strongly suggests cooperativity between biochemical events over large intracellular distances. This leads to consideration of cytoplasm as a "giant multienzyme complex" based on cooperative actions of actin, IF, and MT. This implies an automatic, robot-like machine function of the cytoplasm, presumably set in motion and directed by the genetic apparatus. This view is embraced by many biologists who deify DNA as the prime mover in all biological activities and neglect the "real time" cytoplasmic activities of organisms. 3) Albrecht-Buehler suggests an inherent intelligence within cytoplasm. Intelligence implies the ability to collect and process data and make decisions on the basis of these data. Also important are intrinsic criteria that distinguish between desirable and nondesirable outcomes. Intelligence implies the ability to assess global situations, not merely reacting to local stimuli whenever and wherever they occur, and it implies communication of data with other intelligent objects and appropriate adjustment of actions. Albrecht-Buehler suspects that computers were discovered, rather than invented, and that cytoplasm is a "chemistry based gel or even liquid data processing system."

Cytoplasmic intelligence may depend on collective dynamics of cytoskeletal subunits. Parallel arrays of MT and neurofilaments provide a framework around which microtrabecular lattice structures could form with varying durations of existence as correlates of learning, information, memory, and consciousness. Mechanical contractility of actin-myosin and other proteins within the cytomusculature/cytomatrix could impart mechanical vibrations and cooperative resonances, solitons, or interference wave patterns. Geodesic tensegrity nets of MT, actin and their ordered water may be pulsating in the nanoscale at this very moment within all living cells. Polymerization patterns of actin and other proteins may also be regulated by calcium induced sol-gel state phase differences or harmonic coupling with other microtrabecular and cytoskeletal structures. Coherent nanosecond excitations and propagating solitons are additional mechanisms which have been proposed to occur within the cytoskeleton. In the brain, reinforcement from higher levels of parallel processing (neuron level, neural net, brain) could fortify specific substructures such as wave patterns of calcium coupled sol-gel states in neural cytoplasm.

The "genetic code" was decrypted by Marshal Nirenberg and colleagues (1961, 1964) who were able to equate DNA base pair patterns with specific amino .acid sequences in synthesized proteins. The "real time" information codes in the cytoskeleton may be understood when cytoskeletal nanoscale events just beyond our current capabilities become approachable with the advent of nanotechnology in the next decades.

Figure 5.27: Paramecium mitosis and cytoskeletal "heredity." Top: a single paramecium elongates and divides into two daughter cells. The surface of paramecium are covered with hair-like cilia, each of which is a collective assembly of microtubule doublets or triplets. Cilia bend cooperatively to propel the paramecium, and to propel liquid environment past the organisms. All the cilia are oriented in the same direction, to the left. Second row: Experiments done by Aufderheide, Frankel and Williams (1977) are illustrated. A segment of cell membrane and underlying cytoskeleton which anchors cilia is transplanted in reverse orientation to a sister paramecium. The abnormal cilia orientation persists in the next generation, and for 100 generations to follow! By Paul Jablonka.

6         Protein Conformational Dynamics

Proteins are the structural and organizational elements of "the living state." Their essential functions are intrinsically linked to their structure and dynamic switching among different conformational states. For example, ion channel proteins embedded in a membrane may be either in an "open" conformation, through which specific ions diffuse along a gradient, or they may be "closed." This type of conformational switch does not require biochemical energy such as ATP hydrolysis, but utilizes energy stored in transmembrane voltage gradients and occurs in response to an appropriate trigger. Proteins such as enzymes, receptors, cytoskeletal filaments, muscle myosin and hemoglobin undergo important conformational changes in response to a variety of stimuli. Dynamic patterns of conformational states among cytoskeletal subunits may represent information, exert control over routine biological functions, and provide the "grain of the engram."

6.1           Protein Structure

Figure 6.1: Hydrogen bond between hydrogen and oxygen in amide one resonance bond. With permission from Bolterauer, Henkel and Opper (1986).

Proteins have several hierarchical levels of structural organization which determine their dynamic and functional capabilities. Protein primary structure is determined by a sequence of 22 possible amino acids held together by peptide bonds. An amino acid consists of carbon atoms bound to nitrogen atoms (peptide bond) and their attendant side groups. The 22 amino acids found in proteins differ in their side groups although each contains a carbon-oxygen double bond known as "amide 1." Strings of amino acids called "peptides" perform many physiological functions including acting as neurotransmitters and circulating hormones. The amino acids impart specific properties according to the sequence of their incorporation into the peptide string. Amino acids differ in their size depending on their side groups; their molecular weights can range from 75 daltons (a dalton is the mass of a hydrogen atom, a twelfth of a carbon atom) for glycine, to about 200 daltons for tryptophan which contains a double aromatic ring. Functional proteins also range in size: 55 kilodaltons (one kilodalton = one thousand daltons) for tubulin monomers to 630 kilodaltons for thyroglobulin, to several million kilodaltons for protein assemblies which comprise large viruses and infectious agents of certain diseases like psittacosis and lymphogranuloma venereum (Harper,1969). Thus proteins are comprised of several hundred to millions of amino acids. The precise sequence of amino acids is determined by DNA and they are assembled on ribosomes. A polypeptide "backbone" of 200 amino acids would have 22²⁰⁰ different possible primary structures. The mass required to include one example of each of this number of protein structures would far exceed that of the universe!

Amino acids which comprise polypeptide chains and primary protein structure can form linkages with other amino acids within the polypeptide by hydrogen bonds and disulfide bonds. These linkages cause bending of polypeptide chains into coiled or folded structures which determine protein secondary structure. The most stable and commonly observed secondary structure is a right handed coil called an "alpha helix" in which hydrogen bonds (Figure 6.1) form between an oxygen and a hydrogen separated by 3 or 4 amino acids on the polypeptide chain. Alpha helices can interact among themselves in a protein, stacking in parallel or antiparallel arrangements or forming left handed "coiled-coils." Alpha helices (Figure 6.2) are common occurrences in a wide variety of proteins; tubulin is about 40 percent alpha helix in most circumstances (Dustin, 1978). The alpha helix has been proposed as an important site of energy and information transfer in proteins. Davydov has proposed that the energy of ATP hydrolysis utilized in actin-myosin muscle contraction and many other biological events is conveyed along alpha helix pathways via propagating "solitons" which occur due to enharmonic coupling in the carbon-oxygen double bonds (Section 6.7).

Hydrogen bonds forming among amino acid-side groups on polypeptide chains in parallel result in planar secondary protein configurations called beta-pleated sheets. First proposed by Linus Pauling in 1951, beta pleated sheets may themselves align in parallel, antiparallel or mixed formations.

Alpha helices, beta pleated sheets and other secondary structures interact to define protein tertiary structure, upon which most protein functions depend. Hydrophobic forces, charge distribution, disulfide bridges and secondary structure result in folding into globular regions which define the shape of single protein units. Alpha helix and beta pleated sheets pack into arrangements which help determine the tertiary structural domains which often have specific functions like binding a molecule, or associating with other domains to create larger structures. Assembly of groups of tertiary structure determines quaternary structure, like tubulin subunits assembling into microtubules. Generally, hydrophobic interactions like Van der Waals forces are important in quaternary protein assemblies.

Levels of protein assemblies include, at the simplest level, monomeric enzymes. Weighing from 20 to 90 kilodaltons, these enzymes have a single active site at which specific molecules undergo chemical reactions facilitated ("catalyzed") by the enzyme. Some enzymes may require metal ions, organic molecules or specific cofactors to function. Reduced enzymatic activity can result from occupancy of the active site by molecules resistant to enzymatic action, a phenomenon called "competitive inhibition." Active site binding corresponds with specific conformational states of enzymes.

A more complex system is the oligomeric enzyme, an example of which is the acetylcholine receptor: a four subunit oligomer which is activated by binding of acetylcholine. Being composed of several subunits leads to collective properties which arise from the organization and interactions of the components. Many or perhaps most enzymes are oligomeric with molecular weights ranging from 35 to several thousand kilodaltons. Oligomeric subunits often have two binding sites; one is the active site for its enzyme action and the other is a regulatory site which controls the active site by a change in subunit shape or conformation. Regulatory, or effector molecules can change not only the catalytic site activity on that subunit (allosterism) but also on adjacent subunits (cooperativity). Oligomer subunits may be alike (homopolymers) or different (heteropolymers) and coenzymes may be necessary. A single coenzyme molecule can "turn on" the binding capabilities of multiple subunits within an oligomeric enzyme (Roth and Pihlaja, 1977).

Examples of oligomeric enzymes include glutamate dehydrogenase, composed of six 55 kilodalton subunits forming a complex with approximate dimensions of 100 by 100 by 80 nanometers. An example of a more complex oligomer is aspartate transcarbamylase. It contains six regulatory units of about 34 kilodaltons, each composed of 3 dimers. An additional six 100 kilodalton catalytic subunits are each composed of two trimers. Six zinc atoms are necessary in each of the regulatory subunits. Another cooperative oligomer is hemoglobin, composed of two "alpha" and two "beta" subunits; many other oligomeric enzyme patterns have also been observed. Oligomeric enzyme subunits have cooperative interactions which means that the function of one subunit facilitates the conformational actions of other subunits. In hemoglobin, conditions sufficient for binding of one oxygen molecule to one subunit causes the remaining subunits to bind oxygen more easily.

More complex protein assemblies include cytoskeletal polymers and virus coats. The cooperative interactions of their subunits can lead to information processing and thus to intelligent behavior.

6.2           Protein Conformation

At the tertiary structural level, most protein molecules are able to shift reversibly between several different but related stable conformations and are known as allosteric proteins. Such proteins may be able to form several alternative sets of hydrogen bonds, disulfide bridges and Van der Waals forces of about equal energy among their constituent amino acid side chains. Each alternative set of intraprotein bonds and charge distribution requires a change in the spatial relationships between components of the polypeptide chains and thus motion occurs among the states. Only certain distinct conformations are energetically favorable and any intermediate conformations are unstable and unlikely. Charge redistribution (i.e. dipole oscillation) can also couple to conformational switching. Thus step-like, nonlinear jumping occurs between specific conformations so that only a discrete number of alternative conformations exist for a given protein, and random switching from conformation to conformation is limited.

Figure 6.2: Alpha helix with three "spines" formed by hydrogen bonds (dashed lines) between hydrogen and oxygen atoms. With permission from Bolterauer, Henkel and Opper (1986).

Each distinct conformation of an allosteric protein has a different surface and a different ability to interact with other binding molecules or "ligands." Only one of several conformations may have a high affinity for a particular ligand and the presence or absence of that ligand can determine the conformation that the protein adopts. Two distinct ligands may bind specifically to different surfaces of the same protein, so that the concentration of one ligand may change the affinity of the protein for the other. Such allosteric mechanisms facilitate fine tuning and regulation of many biological processes in which proteins transduce, or integrate various modes of signaling and information. Allosteric proteins are especially effective signaling devices when they exist as aggregates of identical subunits. The conformation of one subunit can influence that of neighboring subunits, producing a collective effect similar to amplification or switching in a computer. The collective behavior of hemoglobin in its binding of oxygen is one example; state transformations in microtubules may be another.

The conformational state of a specific protein is regulated by a variety of factors and may have profound importance for functional activity. Hemoglobin is an iron containing protein in red blood cells which carries oxygen from the lungs to the various body tissues like brain, heart, or muscle. Hemoglobin which binds an oxygen molecule is in a different conformation ("oxyhemoglobin") than hemoglobin without oxygen ("deoxyhemoglobin"). The conformational state of hemoglobin found inside red blood cells determines the protein's capacity to bind or release oxygen. Hemoglobin conformation is determined by factors which include the availability of oxygen, temperature, pH, presence of certain other molecules (i.e. 2,3 DPG), and the amount of nearby oxyhemoglobin. The latter describes a "collective" effect; when a critical amount of hemoglobin binds oxygen, the oxyhemoglobin conformation is easier to attain for the remaining protein molecules. This results in a "sigmoid" shape to the curve which describes oxygen/hemoglobin binding. The nature of this collective phenomenon is an indication of the behavior of protein assemblies.

Different frequencies of conformational changes coexist cooperatively in the same protein (Table 6.1). Some totally reversible conformations like oxyhemoglobin persist for tens of seconds; others can be very short-lived (i.e. femtoseconds: 10^-15 seconds), or very long (minutes to hours). The oxyhemoglobin conformation persists until the oxygen molecule is delivered to the tissue whose mitochondria use it to produce ATP and GTP. To reach their binding sites within hemoglobin's central core, oxygen molecules diffuse through transient packing defects in the protein's structure. Nanosecond scale, conformational "breathing" permits the oxygen molecules to slip in and out, dependent on surrounding pH, temperature, hormones and other factors. There thus appear to be at least two levels of conformational states in hemoglobin. The "functional" conformation (binding vs no binding of oxygen) are relatively long lasting (long enough to carry oxygen from the lungs to tissue for delivery). In addition there are more rapid conformational vibrations such as the nanosecond "breathing" which facilitates diffusion of oxygen molecules through hemoglobin to reach their binding sites.

Table 6‑1: Protein Conformational Dynamics-Motions of Globular Proteins at Physiological Temperature (Modified from Karplus and McCammon, 1984).

The vast majority of processes of biological interest are in the time scale greater than one nanosecond, which also lies in the collective mode realm for protein dynamics (Karplus and McCammon, 1984). Protein conformational changes in the nanosecond time frame are able to be coupled to a stimulus and result in a functional conformational change. Three fundamental features appear to control these functional states: hydrophobic interactions, charge redistribution and hydrogen bonding. Appropriate stimuli including neurotransmitters, voltage alterations, hormones, ions like calcium, and enzyme substrates can induce conformational changes within specific proteins. Conformational transduction is the sensitive link in the mechanism of anesthetic gas molecules (Chapter 7) and indicates that cognitive functions relating to consciousness depend in some way on protein conformational regulation. In many cases, proteins "integrate" a number of factors to result in a new conformation (Figure 6.3). Portions of proteins may be dynamically active with functional importance. For example, electron transfer processes such as that which occur in the mitochondrial production of ATP may rely on vibrational coupling and fluctuations which alter the distances between electron donors and acceptors. Relative motions of distinct structural domains within proteins are important in their activities related to muscle contraction, enzyme activities, antibody functions, and assembly and activities of supra-molecular structures such as viruses and cytoskeletal proteins. Dynamic conformational changes of proteins are the dynamics of living organisms.

Figure 6.3: Protein switching between two different conformational states induced by binding of ligand, calcium ion, or voltage change. One mechanism proposed by Fröhlich is that dipole oscillations (e) within hydrophobic regions of proteins may be a trigger, or switch for the entire protein.

6.3           Proteins and Energy

All movements within cells depend on forces generated by proteins. A typical allosteric protein can adopt two alternative conformations-a low energy form and a high energy form that differ by about the energy available from forming a few hydrogen bonds on a protein surface. The low energy conformation will be favored by about 1,000 to 1, and the protein will tend to be in this "inactive" conformation unless influenced by other factors. It can be "pulled" into the active high energy conformation by binding to a ligand which binds only to the high energy state. Also, an input of chemical energy can be used to "push" the protein into the high energy conformation. A common mechanism involves the transfer and covalent linkage of a phosphate group from biochemical energy sources like ATP or GTP to one of several amino acid side chains (serine, threonine or tyrosine) in the protein. This "phosphorylation" can create a distribution of charged amino acid side chains which are more favorable to one conformation than another, leaving the nonphosphorylated conformation unavailable. Ten percent of all mammalian cell proteins undergo phosphorylation as a regulatory or programming mechanism (Alberts et al., 1983).

The laws of thermodynamics demand that free energy be depleted when work is performed. Therefore, protein molecules cannot show net movement (as do microtubules and other cytoskeletal proteins) without some added source of energy. Phosphorylation is one way by making one step irreversible. Another is to drive allosteric changes by the hydrolysis of an ATP molecule, converting it to ADP (or hydrolyzing GTP to GDP). The energy released in the hydrolysis reaction is imparted to the protein, pushing it to a higher energy state. The precise mechanism by which this energy is utilized and transferred by proteins is unknown.

Besides generating mechanical force, allosteric proteins can use the energy of ATP phosphorylation and hydrolysis to do other forms of work, such as pumping specific ions into or out of the cell. An allosteric protein known as "sodium potassium ATPase" which is found in the membrane of all animal cells including nerve cells, pumps three sodium ions out of the cell and two potassium ions in during each cycle of conformational change driven by ATP mediated phosphorylation. This ATP driven pump creates ion gradients across the cell membrane. The energy stored in ion gradients is harnessed by conformational changes in other membrane proteins: ion channels. When these are triggered to open in nerve membranes, a conformational change initiated by a voltage change, ligand binding, or allosteric effect from membrane or cytoskeletal protein permits ions to passively diffuse. The passage of charged ions depolarizes the membrane as part of a local gated potential or traveling action potential.

The ion flow may also drive other membrane bound protein pumps that transport glucose or amino acids into the cell. Energy available in proton/hydrogen ion gradients across the inner mitochondrial membranes is used to synthesize most of the ATP used in the animal world. Actin and myosin and other proteins create contractile force in muscle by cooperative conformational changes induced by ATP hydrolysis. Within bending cilia, contractile protein bridges which span between microtubules hydrolyze ATP to drive their mechanical activities. ATP and other energy producing molecules are the fuel of biological protein engines. Less well understood, however, is their control, guidance and orchestration.

6.4           Protein Cooperativity-Historical View

An interesting idea regarding the control of protein conformational state and utilization of energy was proposed by Szent-Gyorgyi (1948). He suggested that proteins behave as semiconductors and that electrons "hop" between specific intraprotein regions. Experimental data, however, showed that the intraprotein energy band gap was too great to support such a concept. Utilization of dynamic biological and protein conformational energy remained enigmatic through the 1950's and 1960's.

A focal point in the history of attempts to understand protein conformational dynamics was a 1973 meeting of the New York Academy of Science (which also hosted a landmark meeting concerning microtubules in the same year). The twin mysteries of how ATP energy was utilized to produce mechanical protein events, and how energy and information were transferred in proteins and organelles were addressed by a gallery of scientists. The prevalent theme was "a crisis in bioenergetics," in that cooperative processes of obvious importance in biological systems were unexplained. One idea which generated controversy was that electromagnetic resonance energy was transferred between periodically arrayed excitation sites. At that meeting, C. W. F. McClare (1974) proposed that ATP induced excited states ("excitons") were coupled by infrared dipoles in protein biostructures to provide a communicative medium. McClare concluded:

there is yet another level of organization in biological systems: a tuned resonance between energy levels in different molecules that enables bioenergetic machines to operate rapidly and yet efficiently.

This concept was supported by several allies including Wei (1974) who suggested dipole coupling among membrane proteins to explain nerve functions. McClare's general model of resonant dipole coupling was greeted by skepticism, however. Highly respected biochemist Gregorio Weber (1974) raised two objections. He noted that at least some of the emitted energy following molecular excitation is emitted in 10^-12 seconds, too short to be utilized by biomolecules. The second objection referred to the transfer of quantum vibrational energy through the surrounding medium. The process of dipole-dipole coupling involves a similarity of the energies emitted and received, and requires that the interacting oscillators be surrounded by a medium transparent to the wavelength of the transferred energy quantum. Water surrounds every biomolecule and provides a medium which appeared to be opaque to the infrared energy McClare described. Weber implied any emitted energy would be dissipated as heat to the water environment. McClare countered, unconvincingly, that systems could have evolved to be able to slow down the relaxation and energy emission, and that such systems may be somehow separated from the aqueous phase. A consensus was that energy needed to be stored for a nanosecond or longer to be useful.

The mode of energy and information transfer was described in other comparable ways by different scientists. Several considered propagating packages of conformational or acoustic energy: "phonons." A phonon can be defined as a propagated lattice vibration, an intranuclear vibration of a carbon-nitrogen bond that propagates in a protein lattice. The transmission of phonons occurs at the speed of sound, so they are not resonance transfer phenomena but a propagated vibration in a periodic lattice. In 1967 Straub postulated that protein lattices contain phonons which must be isolated from their aqueous environment. At the New York Academy meeting in 1973 he suggested that hydrophobic interactions like Van der Waals forces could contain phonons in the lattice and shield them from the aqueous bath. The concept of "conformon," a quantum of protein conformational energy coupled to discrete protein states was independently described by Green (1970) and Ji (1974) in America, and Volkenstein (1972) in the Soviet Union. Later, A. S. Davydov of the Soviet Union proposed that mechanical conformational movements in proteins were nonlinearly coupled to electronic bond distortion or charge movement, resulting in propagating waves called "solitons." These models all faced a common problem of shielding from the aqueous environment. One possible resolution of this problem is that the water surrounding proteins may be "ordered" and resonantly coupled rather than thermally dissipating protein excitation energy. Another is that hydrophobic interactions exclude water from areas where important bioenergetic interactions occur.

6.5           Living Water and Hydrophobic Interactions

Biomolecules have evolved and flourished in aqueous environments, and basic interactions among biomolecules and their pervasive hosts, water molecules, are extremely important. The properties of intracellular water are controversial. Many authors believe that more than 90 percent of intracellular water is in the "bulk" phase-water as it exists in the oceans (Cooke and Kuntz, 1974; Schwan and Foster, 1977; Fung and McGaughy, 1979). This traditional view is challenged by others who feel that none of the water in living cells is bulk (Troshin, 1966, Cope 1976, Negendank and Karreman, 1979). A middle position is assumed by those who feel that about half of "living" water is bulk and the other half "ordered" (Hinke, 1970; Clegg, 1976; Clegg, 1979; Horowitz and Paine, 1979). This group emphasizes the importance of "ordered" water to cellular structure and function.

Many techniques have been used to study this issue, but the results still require a great deal of interpretation. Nuclear magnetic resonance (NMR), neutron diffraction, heat capacity measurement, and diffusion studies are all inconclusive. Water appears to exist in both ordered and aqueous forms within cells. The critical issue is the relation between intracellular surfaces and water. Surfaces of all kinds are known to perturb adjacent water, but within cells it is unknown precisely how far from the surfaces ordering may extend. We know the surface area of the microtrabecular lattice and other cytoskeleton components is extensive (billions of square nanometers per cell) and that about one fifth of cell interiors consist of these components. Biologist James Clegg (1981) has extensively reviewed these issues. He concludes that intracellular water exists in three phases. 1) "Bound water" is involved in primary hydration, being within one or two layers from a biomolecular surface. 2) "Vicinal water" is ordered, but not directly bound to structures except other water molecules. This altered water is thought to extend 8 to 9 layers of water molecules from surfaces, a distance of about 3 nanometers. Garlid (1976, 1979) has shown that vicinal water has distinct solvent properties which differ from bulk water. Thus "borders" exist between water phases which partition solute molecules. 3) "Bulk water" extends beyond 3 nanometers from cytoskeletal surfaces (Figure 6.4).

Drost-Hansen (1973) described cooperative processes and phase transitions among vicinal water molecules. Clegg points out the potential implications of vicinal water on the function of enzymes which had previously been considered "soluble." Rather than floating freely in an aqueous soup, a host of intracellular enzymes appear instead to be bound to the MTL surface within the vicinal water phase. Significant advantages appear evident to such an arrangement: a sequence of enzymes which perform a sequence of reactions on a substrate would be much more efficient if bound on a surface in the appropriate order. Requirements for diffusion of the substrate, the most time consuming step in enzymatic processes, would be minimal. Clegg presents extensive examples of associations of cytoplasmic enzymes which appear to be attached to and regulated by, the MTL. These vicinal water multi-enzyme complexes may indeed be part of a cytoskeletal information processing system. Clegg conjectures that dynamic conformational activities within the cytoskeleton/MTL can selectively excite enzymes to their active states.

The polymerization of cytoskeletal polymers and other biomolecules appears to flow upstream against the tide of order proceeding to disorder which is decreed by the second law of thermodynamics. This apparent second law felony is explained by the activities of the water molecules involved (Gutfreund, 1972). Even in bulk aqueous solution, water molecules are somewhat ordered, in that each water molecule can form up to 4 hydrogen bonds with other water molecules. Motion of the water molecules (unless frozen) and reversible breaking and reforming of these hydrogen bonds maintain the far miliar liquid nature of bulk water. Outer surfaces of biomolecules form more stable hydrogen bonding with water, "ordering" the water surrounding them. This results in a decrease in entropy (increased order) and increase in free energy: factors which would strongly inhibit the solubility of biomolecules if not for the effects of hydrophobic interactions. Hydrophobic groups (for example amino acids whose side groups are non-polar, that is they have no charge-like polar groups to form hydrogen bonds in water) tend to combine, or coalesce for two main reasons: Van der Waals forces and exclusion of water. Combination of hydrophobic groups "liberate" ordered water into free water, resulting in increased entropy and decreased free energy, factors which tend to drive reactions. The magnitude of the favorable free energy change for the combination of hydrophobic groups depends on their size and how well they fit together "sterically." A snug fit between groups will exclude more water from hydrophobic regions than will loose fits. Consequently, specific biological reactions can rely on hydrophobic interactions. Forma, tion of tertiary and quaternary protein structure (including the assembly of microtubules and other cytoskeletal polymers) are largely regulated by hydrophobic interactions, and by the effect of hydrophobic regions on the energies of other bonding. A well studied example of the assembly of protein subunits into a complex structure being accompanied by an increase in entropy (decrease in order) is the crystallization of the tobacco mosaic virus. When the virus assembles from its subunits, an increase in entropy occurs due to exclusion of water from the virus surface. Similar events promote the assembly of microtubules and other cytoskeletal elements.

Figure 6.4: Microtrabecular lattice (MTL-solid dark) with layers of ordered and vicinal water. "Soluble" enzymes (circles) are held within ordered or "vicinal" water and may be functionally regulated by the MTL. Modified from Clegg (1984) by Paul Jablonka.

The attractive forces which bind hydrophobic groups are distinctly different from other types of chemical bonds such as covalent bonds and ionic bonds. These forces are called Van der Waals forces after the Dutch chemist who described them in 1873. At that time, it had been experimentally observed that gas molecules failed to follow behavior predicted by the "ideal gas laws" regarding pressure, temperature and volume relationships. Van der Waals attributed this deviation to the volume occupied by the gas molecules and by attractive forces among the gas molecules. These same attractive forces are vital to the assembly of organic crystals, including protein assemblies. They consist of dipole-dipole attraction, "induction effect," and London dispersion forces. These hydrophobic Van der Waals forces are subtly vital to the assembly and function of important biomolecules.

Dipole-dipole attractions occur among molecules with permanent dipole moments. Only specific orientations are favored: alignments in which attractive, low energy arrangements occur as opposed to repulsive, high energy orientations. A net attraction between two polar molecules can result if their dipoles are properly configured. The "induction" effect occurs when a permanent dipole in one molecule can polarize electrons in a nearby molecule. The second molecule's electrons are distorted so that their interaction with the dipole of the first molecule is attractive. The magnitude of the induced dipole attraction force was shown by Debye in 1920 to depend on the molecules' dipole moments and their polarizability. Defined as the dipole moment induced by a standard field, polarizability also depends on the molecules' orientation relative to that field. Subunits of protein assemblies like the tobacco mosaic virus have been shown to have high degrees of polarizability. London dispersion forces explain why all molecules, even those without intrinsic dipoles, attract each other. The effect was recognized by F. London in 1930 and depends on quantum mechanical motion of electrons. Electrons in atoms without permanent dipole moments (and "shared" electrons in molecules) have, on the average, a zero dipole, however "instantaneous dipoles" can be recognized. Instantaneous dipoles can induce dipoles in neighboring polarizable atoms or molecules. The strength of London forces is proportional to the square of the polarizability and inversely to the sixth power of the separation. Thus London forces can be significant only when two or more atoms or molecules are very close together (Barrow, 1966). Lindsay (1987) has observed that water and ions ordered on surfaces of biological macromolecules may have "correlated fluctuations" analogous to London forces among electrons. Although individually tenuous, these and other forces are the collective "glue" of dynamic living systems.

6.6           Electret, Piezo, and Pyroelectric Effects

Assemblies whose microscopic subunits and macroscopic whole both possess permanent electric dipoles are known as electrets. They exhibit properties known as piezoelectricity and pyroelectricity which may be useful in biological activities. In these crystals, dipolar elementary subunits are arranged in such a way that all the positive dipole ends point in one direction and all the negative dipole ends are oriented in the opposite direction. In microtubules, the positive ends of tubulin dimer subunits point away from microtubule organizing centers (MTOC) toward the cell periphery, and the negative ends point toward MTOC. Electrets can store charge and polarization and have now been identified in a variety of nonbiological materials such as ionic crystals, molecular solids, polymers, glasses, ice, liquid crystals and ceramics. Biological tissues demonstrating electret properties include bone, blood vessel wall materials, keratin, cellulose, collagen, gelatin, artificial polypeptides, keratin, DNA, cellulose and microtubules (Athenstaedt, 1974). An electret effect accounts for specific properties such as anti-blood clotting in biomaterials and the non-stickiness of teflon. Sources of polarization or charge storage in macromolecules are dipoles, ionic space charges, or ordered surface water.

Electret materials are piezoelectric (Gubkin and Sovokin 1960). Piezoelectric materials change their shape or conformation in response to electrical stimuli, and change their electrical state in response to mechanical stimuli. Koppenol (1980) has shown that the dipole moment orientation of an enzyme changes in concert with its functional activity. Electrets are also "pyroelectric," in that any change in temperature alters the electrical and conformational characteristics of the molecule. The permanent electric dipole moment of pyroelectric bodies results from the parallel alignment of elementary fixed dipole moments. Any change in temperature modifies the length of the pyroelectric body and alters its elementary dipole moments (pyroelectric effect). In the same way, any mechanical change in length or any deformation of a pyroelectric body produces a modification of its dipole moment (piezoelectric effect). Thus every pyroelectric body is at the same time piezoelectric. The two requisites for such behavior are an electric property which causes a permanent dipole moment in the molecules and a morphological property that favors a parallel alignment. Microtubules and other cytoskeletal structures appear to be appropriately designed electret, pyroelectric, piezolectric devices.

The electret state within bone has been well studied and is able to store large amounts of polarization of the order of 10^-8 coulombs per square centimeter (Mascarenhas, 1974, 1975). The limit of charge separation (equivalent to maximal information density) has been calculated (Gutman, 1986) to be about 10¹⁷ electronic charges per cubic centimeter, while there may be about 10²¹ total molecules per cubic centimeter. One cubic centimeter of parallel microtubules densely arrayed 100 nanometers apart contains about 10¹⁷ tubulin subunits and therefore may contain 10¹⁷ dipoles equivalent to the maximal density of charge! Electret and related properties can impart interesting and potentially useful properties to biomolecules including cytoskeletal polymers. Among these are the potential capacity to support the propagation of conformational waves such as solitons.

6.7           Solitons/Davydov

Important biological events involve spatial transfer of energy along protein molecules. One well known example is the contractile curling of myosin heads in muscle contraction, fueled by the hydrolysis of ATP molecules. These quanta of biological energy are equivalent to 0.43 electron volts, only 20 times greater than background energy and insufficient to excite molecular electronic states. Consequently, under usual conditions biological systems do not emit photons. This implied to Davydov (1977) during the 1970's that ATP hydrolysis energy is transferred by vibrational excitations of certain atomic groups within proteins. Davydov focused on the alpha helix regions of proteins and identified the "amide 1" (carbon-oxygen double bond) stretch vibration of the peptide group as the most likely "basket" in which energy may be carried. His selection was based on the "quasi-periodic," or crystal-like arrangement of amide 1 bonds, their low vibrational energy (0.21 electron volts-half the energy of ATP hydrolysis) and their marked dipole moment (0.3 Debye). According to linear analysis, energy transported by this means should spread out from the effects of dispersion and rapidly become disorganized and lost as a source of biological action. However Davydov analyzed nonlinear aspects of the amide 1 stretch and concluded that amide 1 vibrations are retroactively coupled to longitudinal soundwaves of the alpha helix, and that the coupled excitation propagates as a localized and dynamically self sufficient entity called a solitary wave, or "soliton." Davydov reasoned that amide 1 vibrations generate longitudinal sound waves which in turn provide a potential well that prevent vibrational dispersion, thus the soliton "holds itself together."

Solitary waves were first described by nineteenth century naval engineer John Scott-Russell in 1844. While conducting a series of force-speed experiments for boats on a Scottish canal, an accident happened. A rope broke and a canal boat suddenly stopped, but the bow wave which the barge caused kept on going. Russell galloped alongside the canal and followed the wave for several miles. He described the exceptional stability and automatic self-organization of this type of wave. Mathematical expressions of solitary waves can be given as particular solutions of some nonlinear equations describing propagation of excitations in continuous media which have both dispersive and nonlinear properties. These solitary wave solutions have "particle-like" characteristics such as conservation of form and velocity. Such traits led Zabusky and Kruskal (1965) to describe them as "solitons." As an answer to the problem of spatial transfer of energy (and information) in biological systems, Davydov applied the soliton concept to biological systems in general, and amide 1 vibrations within alpha helices in particular. For solitons to exist for useful periods of time and distance, certain conditions must be met. The nonlinear coupling between amide 1 bond vibrations and sound waves must be sufficiently strong and the amide 1 vibrations be energetic enough for the retroactive interaction to take hold. Below this coupling threshold, a soliton cannot form and the dynamic behavior will be essentially linear; above the threshold the soliton is a possible mechanism for virtually lossless energy transduction.

Computer simulation and calculation of solitons have led to assumptions about the parameters necessary for nolinearity and soliton propagation. A critical parameter is the "anharmonicity," or nonlinearity of the coupling of intrapeptide excitations with displacement from equilibrium positions (Figure 6.5). Anharmonicity is the nonlinear quality which determines the self capturing of the two components of the, soliton. The degree to which the electronic disturbance nonlinearly couples to the mechanical conformational change of the protein structure is the crux of the soliton question. If the two are coupled as a step-like functioning switch (nonlinear) rather than a direct linear correlation, they can provide a "grain" to represent discrete entities capable of representing and transferring information. An index for soliton viability, the anharmonicity parameter, is known as χ. For χ greater than 0.3 x 10^-11 newtons, solitons in computer simulation do propagate through the spines of an alpha helix at a velocity of about 1.3 x 10³ meters per second. The distance of 170 nanometers corresponding to the length of a myosin head in striated muscle would then be traversed by a soliton in about 0.13 nanoseconds. Computer simulations by Eilbeck and Scott (1979) demonstrate, for above threshold values for the coupling parameter χ, soliton-like excitations propagating along alpha helix spines in the form of a local impulse with a size of a few peptide groups. Experimental data supporting such biological solitons is slowly emerging. Although results from biomolecular light scattering (Webb, 1980) seemed to provide confirmation (Lomdahl et al., 1982), follow up work (Layne, et al., 1985) indicated another source of the experimental observations. Optical experiments on crystalline acetanilide (a hydrogen bonded, polypeptide crystal) however, do provide an unambiguous demonstration of a localized state similar to that discussed by Davydov (Careri, et al., 1984; Eilbeck, et al., 1984; Scott, et al., 1985; Scott, 1985). Definitive resolution of the existence of solitons in biological materials may await the imminent advent of nanotechnology (Chapter 10).

Davydov has considered other types of solitons such as those in solid three-dimensional crystals which have phase transitions. He contends that sufficient anharmonicity in these lattice structures will produce soliton type excitations representing themselves as local displacements of the equilibrium positions moving along the molecular chains. These are called acoustic solitons. Other, "topological" solitons are described as symmetry "kinks" which travel through an ordered medium.

Toda (1979) invoked the concept of solitons to describe local displacements from equilibrium positions of molecules in one dimensional lattices. He studied molecular chains and assumed that displacement of individual molecules within the chain interacted with neighboring molecules. Mathematical evaluation of Toda lattices by Davydov show localized excitations described by a bell shaped function characterizing a reduction in the distance between molecules in the excitation region of the lattice. These are called "supersound acoustic solitons," or "lattice solitons" and have been modeled in proteins by Bolterauer, Henkel and Opper (1986).

Davydov's work further suggests that excess electrons can be captured by supersound acoustic solitons and conveyed along with them, giving rise to "electrosolitons." Electron transfer between donor-acceptor pairs of proteins are found in photosynthesis, cell respiration (ATP generation) and the activity of certain enzymes. These arrangements of structures are often called electron transport chains. Davydov observes that electron transfer has traditionally been assumed to be accomplished by quantum mechanical tunneling as first proposed by Britton Chance and colleagues (Devault, Parkas, Chance, 1967). In these systems, electrons are generally transferred between centers spaced about 3-7 nanometers apart. Davydov argues that this electron transfer can be better explained by an electrosoliton.

Figure 6.5: Computer simulation showing energy (vertical axis) localization along distance (southwest to northeast axis) as function of anharmonicity (southeast to northwest axis). Over a specific range, energy becomes localized and travels as solitary wave, or soliton. With permission from Bolterauer, Henkel and Opper (1986).

Concrete evidence exists for solitons as giant waves in or underneath the ocean, as optical solitons in laser fiber optics and in other systems. Current technologies are incapable of proving or disproving biological solitons. If Davydov is correct about myosin heads, then solitons are responsible for the molecular level filamentous contractions that drive every move we make. Propagating solitons in the cytoskeleton could be the dynamic medium of biological information processing. If so, solitons would be to consciousness what electricity is to computers.

6.8           Coherent Excitations /Fröhlich

Protein conformational states can register dynamic biological information and control the real time functions of cytoplasm. The mechanisms of conformational regulation are not clearly understood, primarily because technology has not (quite) yet reached the nanoscale. Proteins are clearly vibrant, dynamic structures in physiological conditions. A variety of recent techniques (nuclear magnetic resonance, X-ray diffraction, fluorescence depolarization, infrared spectroscopy, Raman and Brillouin laser scattering) have shown that proteins and their component parts undergo conformational motions over a range of time scales from femtoseconds (10^-15 sec) to many minutes.

The most significant conformational vibrations are suggested by Harvard's Karplus and McCammon (1984) to be in the middle of this range: nanoseconds. Such fluctuations are appropriate for conformational motion of globular proteins (4 to 10 nanometer diameter), consistent with enzymatic reaction rates and "coupled modes" like solitons. As an example, Karplus and McCammon describe a rotation of a tyrosine ring deep inside a globular protein called bovine pancreatic trypsin inhibitor. The side chain of the amino acid tyrosine includes a six carbon "aromatic" hexagonal ring with an electron resonance cloud. The rotation of the ring has been studied experimentally by nuclear magnetic resonance and Karplus and McCammon have done a computer simulation based on that data which shows the protein changing conformational state as the tyrosine ring rotates 90 degrees. The switch occurs in the nanosecond time scale and is collectively coupled to movement in the polypeptide backbone chain.

Collective nanosecond conformational states have been elegantly woven in a theory of coherent protein excitations by Professor Herbert Fröhlich who presently divides his time between Liverpool University and the Max Planck Institute in Stuttgart. Recognized as a major contributor to the modern theory of superconductivity, Fröhlich turned to the study of biology in the late 1960's and came to several profound conclusions. One is that changes in protein conformation in the nanosecond time scale are triggered by a charge redistribution such as a dipole oscillation within hydrophobic regions of proteins (Fröhlich, 1975). Another Fröhlich (1970) concept is that a set of proteins connected in a common voltage gradient field such as within a membrane or polymer electret such as the cytoskeleton would oscillate coherently at nanosecond periodicity if energy such as biochemical ATP were supplied. Fröhlich's model of coherency can explain long range cooperative effects by which proteins and nucleic acids in biological systems can communicate. A major component of Fröhlich's theory suggests that random supply of energy to a system of nonlinearly coupled dipoles can lead to coherent excitation of a single vibrational mode, provided the energy exceeds a critical threshold. Frequencies of the order of 10⁹ to 10¹¹ Hz are suggested by Fröhlich, who maintains that the single mode appears because all others are in thermal equilibrium. Far reaching biological consequences may be expected from such coherent excitations and long range cooperativity.

Conformation of proteins and their dipole moments in aqueous, physiological environment are dominated by interaction of their charge groups with surrounding water and ions. Some biomolecules may possess excited states with very high dipole moments. These levels, according to Fröhlich, tend to become stabilized (become "metastable states") through internal and external deformations and through displacement of "counter" ions like calcium. Metastable states, which correlate with functional conformations, are thus collective effects involving the molecule and its surroundings. A molecule might be lifted into a metastable state through the action of electric fields, binding of ligands or neurotransmitters, or effects of neighbor proteins. Thus rapid, nanosecond oscillations may become "locked" in specific modes which correspond to useful conformations of a protein. For example an ion channel, receptor, enzyme, or tubulin subunit may stay in one conformational state for relatively long periods, on the order of milliseconds. Fröhlich characterizes these conformations as "metastable" states.

Fröhlich observes that the high electric field of the order of 10⁷ volts per meter maintained in many biological membranes (100 millivolts / 10 nanometers = 10⁷ volts/meter) requires an extraordinary dielectric property of the membrane components including lipids and proteins. Similar requirements would exist for cytoskeletal proteins in an electret. Ordinary material would suffer dielectric breakdown in such fields unless specially prepared. Fröhlich contends the biological evolution of this dielectric strength on a molecular scale must have strong significance. Biological organisms are relatively impervious to effects of electromagnetic radiation, yet can be exquisitely sensitive to it in some circumstances. Some biological functions border on the limit imposed by quantum mechanics. Our eyes are sensitive to single photons and certain fish are sensitive to extremely weak electric fields. Such performances, according to Fröhlich, require the use of collective properties of assemblies of biomolecules and certain types of collective behavior such as coherent vibrations should be expected.

If coherent oscillations representing dipole vibrations within molecular systems do coherently oscillate in the range of 10⁹ to 10¹¹ Hz, it should be possible to excite these modes by electromagnetic radiation. In order to couple and excite the biological vibrations, radiation should be matched to the biological frequency and the wavelength should be large compared to the dimension of the oscillating object. A significant amount of evidence supports this notion. Irradiation of a great variety of biological objects with coherent millimeter waves in the frequency region of 0.5 x 10¹¹ Hz can exert great influences on biological activities provided the power supply lies above a critical threshold (Grundler and Keilman, 1983). According to Fri hlich, the biological effects are not temperature effects. They show very sharp frequency resonances which indicates that localized absorption in very small spatial regions contributes to the biological actions.

The sharp resonance of this sensitive window has a frequency width of about 2 x 10⁸ Hz. The layer of ordered water and ions subjacent to membranes and cytoskeletal structures (the "Debye layer") absorbs in the region of 10⁸ Hz. This suggests that the Debye layer is closely involved with the dynamic functional activities of the biostructures which they surround. Green and Triffet (1985) have modeled propagating waves and the potential for information transfer in the dynamics of the Debye layer immediately beneath membranes and cytoskeletal proteins. They have hypothesized a holographic information medium due to the coherent vibrations in space and time of these biomolecules. The medium they consider is the ordered water and layers of calcium counter ions surrounding the high dipole moments in membranes and biomolecules. Thus they have developed a theory of ionic bioplasma in connection with nonlinear properties which relates to the existence of highly polar metastable states. The small scale and ordering would minimize friction in these activities. Fröhlich observes: "clearly the absence of other frictional processes would present most interesting problems." He suggests the possibility of propagating waves due to the lack of frictional processes ("superconductivity") in the biomolecule itself as well as the layer of ordered water or Debye layer (Kuntz and Kauzmann, 1974). Until recently, superconductivity has been considered to occur only in certain ordered materials at temperatures near absolute zero. Recent discoveries, however, have shown that superconductivity can occur in materials at higher temperatures due apparently to coherent ordering and coupling among localized and collective lattice vibrations (Maddox, 1987; Robinson, 1987).

Expanding on Fröhlich's work, Wu and Austin (1978) conclude that oscillating dipoles within a narrow band of resonance frequencies with large enough coupling constants may be expected to cause strong long range (about 1 micron) attractive forces among dipoles. In a dense microtubule array, 1 cubic micron (one billion cubic nanometers) would encompass about 160,000 tubulin subunits-an array sufficiently large for collective effects.

Evidence for such "long-range" effects have been observed in the behavior of red blood cells. Discoids of eight micron diameter (8 thousand nanometers), red blood cells tend to array themselves in stacks called "Rouleaux formation." Rowlands (1983) has studied Rouleaux formation and found that attractive forces begin when the red cells are about four microns (4 thousand nanometers) apart, a distance several orders of magnitude greater than the range of attractive chemical forces. Rowlands views this behavior as consistent with Fröhlich's coherent excitations and long range cooperativity. Rowlands also projects the significance of Fröhlich's theory to communication in the nervous system.

Rowlands (1983) notes that

A communication band extending from 10¹⁰ to 10¹¹ Hz ... could pack over a million FM radio stations ... or 150,000 television broadcasts ... the action potential may be just a crude fast transmitter of urgent messages ... . Fröhlich vibrations might be transmitted along the membrane of the nerve fibers, but they would be interrupted by action potentials. It is more likely that microtubules in the axon are used.

6.9           Massless Bosons, Cytoskeletal Self-Focusing

The complexity of biological systems has attracted the interest of nonbiologists who possess mathematical tools useful for "many body problems." In addition to Fröhlich, these include a group of scientists from the University of Milan (Del Giudice, Doglia, Milani and Vitiello, 1986). Viewing living matter as a sea of electric dipoles, they have taken advantage of mathematical and computer tools used to keep track of the countless particles involved in nuclear reactions. Using a mathematical approach called quantum field theory, the Milan group considers electret states and the consequent ordering of water around biomolecules as sets of dipoles whose states, order and symmetry have collective properties. In a typical nonbiological system, component dipoles are random and disordered, resulting in an overall symmetry.

Such a system would look the same when viewed from any angle ("rotationally invariant"). In living systems, order is induced by reduction of tridimensional symmetry to a rotational alignment along filamentous electrets such as cytoskeletal structures. According to the Milan group quantum field theory and the "Goldstone theorem" require that the symmetry breaking ("Bose condensation") results in long range interactions among system components (dipoles) conveyed by massless particle/waves ("Goldstone bosons"). The Milan group argues that the energy required to generate massless bosons is invested in the electret states of biomolecules and correlated fluctuations of their surrounding water and ions.

Celaschi and Mascarenhas (1977) showed that electret activation energy of biomolecules (0.2-0.4 electron volts) is equivalent to the hydrolysis of one ATP or GTP molecule and what Davydov predicted for initiation of solitons. Consequently solitons, massless bosons, and Frolich's coherent polarization waves may be synonymous.

Pursuing their quantum field approach, Del Giudice and his colleagues came to an astounding concept of self-focusing of electromagnetic energy within cytoskeletal filaments. Electromagnetic energy exceeding a threshold and penetrating into cytoplasm would be confined inside filaments whose diameters depended on the original symmetry breaking ("Bose condensation") of ordered dipoles. Any electric disturbance produced by thermally fluctuating dipoles or by any other source would be confined inside filamentous regions. Ordering is preserved outside the filaments and is disrupted only inside where energy becomes concentrated (the "Meissner effect"). The diameter of the self focusing energy filaments depends on the polarization density, or ordering of biological water. Del Guidice's group calculated a self focusing diameter of about 15 nanometers, precisely the inner diameter of microtubules! Del Guidice and colleagues feel the cytoskeleton is the material consequence of dynamic self focusing of polarization waves in the cytoplasm. The observed diameters of self focused optical beams in simple nonbiological liquids are of the order of microns; correlation among components is created by propagation of waves rather than as a specific property of the material itself. The Milan group concludes that focusing occurs in cytoplasm of eukaryotic cells due to the spatial coherence and ordering imparted by cytoskeletal electret behavior.

The self-focusing predicted by the Milan group would have interesting capabilities. Energy is refracted into beams which become surrounded by cylindrical waveguides: the Indian rope trick. Coherency imparted to the refracted energy by either a Fröhlich-type mechanism or periodic structure of a cytoskeletal waveguide biomolecule could lead to holographic mechanisms. A rudimentary theory of waveguide/holographic behavior in microtubules has been described (Hameroff, 1974). Photorefractive crystals can be used to generate dynamic, real time holography (Gower, 1985) and MT could be projecting dynamic cytoplasmic holograms.

Models of cooperative protein dynamics described by Davydov solitons or Fröhlich coherent oscillations may be different perspectives of the same phenomena. Tuszynski and co-workers (1984) have compared the two approaches and their respective emphasis. They observed that Fröhlich's model concentrates on time-independent effects, or stable states, to explain the establishment of order, whereas Davydov's model highlights time dependent propagation of order via solitons.

The overlapping cooperative models of protein conformational dynamics (coherence, resonance, solitons, electrets, self focusing) are of interest when applied to specific structural elements with relevant properties. For example, Davydov's model of soliton propagation was originally applied to contractile coupling between actin and myosin. The cytoskeleton appears uniquely suited to take advantage of cooperative dynamics related to information processing. Chapter 8 reviews evidence and models of cytoskeletal information processing based on cooperative dynamics. The next chapter describes anesthesia-the result of inhibition of collective, cooperative protein conformational dynamics in the brain.

7         Anesthesia: Another Side of Consciousness

The net collective effect of protein dynamics and other factors in the human brain is consciousness. Since the time of ancient Sumerians, Egyptians, Assyrians and Greeks, opiate derivatives of the poppy (Papaver somniferum) and other drugs have been used to obtund or ablate consciousness in the face of pain, or for surgical procedures. In the 19th century, anesthetic gases including nitrous oxide ("laughing gas") and diethyl ether were developed. These inhalation anesthetics, when administered properly in a narrow range of concentration, were able to cause a reversible cessation of consciousness. As early anesthetists tragically discovered, excess anesthetic caused inadequate breathing, cardiovascular failure and death. However, when properly used, anesthesia became a boon to mankind. In addition to alleviating the suffering of countless surgical patients, anesthetics also became an important tool in the investigation of consciousness. A trail of experimentation and observation has led to the conclusion that anesthetics inhibit collective dynamic conformational changes in brain proteins.

7.1           Levels of Anesthesia/Consciousness

With the widespread advent of diethyl ether, anesthesia became somewhat standardized and it became useful to delineate "stages" in the continuum from the awake state through anesthesia to respiratory paralysis, cardiovascular collapse, and death. In 1847 John Snow published his pioneer monograph, On the Inhalation of the Vapour of Ether in which he described five empirical levels through which an anesthetized patient progressed from consciousness to respiratory paralysis. Surgery could be performed in "stage three" characterized by analgesia (pain relief) and amnesia (lack of memory storage), or in "stage four" characterized by muscular relaxation and regular, automatic breathing. Snow's scheme was expanded in 1920 by Arthur Guedel who published codified stages and signs of anesthesia, later detailed in his 1937 monograph, On Inhalation Anesthesia-A Fundamental Guide. Guedel predicated his stages on obvious physical signs involving muscle tone, respiratory patterns, and eye signs. He enumerated four levels: stage of analgesia, stage of delirium, surgical stage (subdivided into four planes), and stage of respiratory paralysis. Others have added a fifth stage, anesthetic overdose: lack of tissue oxygen, convulsions, and imminent death.

Figure 7.1: Levels of anesthetic depth from different systems displayed on a common axis. Guedel's (1937) stages and planes serve as a standard. EEG levels (Courtin, Bickford and Faulconer, 1950), jaw and forearm relaxation (Galla, Rocco and Vandam, 1958), Woodbridge's (1957) classification of nothria, and stage one for ether (Artusio, 1955) are also represented. With permission from Grantham and Hameroff (1985).

In Guedel's stage one (Figure 7.1), the patient progresses from alertness and sensibility to pain to total amnesia, analgesia, and sedation. Breathing is slow and regular, with use of both diaphragmatic and rib muscles; the eyelid reflex (twitching of the eyelids in response to gentle brushing) is intact. Stage two has been variously termed the stage of delirium, excitement, unconsciousness, or the "dream state." The patient may pass through this stage sedately or may manifest wild, uninhibited activity. Breathing is irregular and unpredictable, pupils of the eye may be dilated, ocular muscles are active, and eyelid reflex active. In this stage the patient is at risk for unwanted reflex activity such as vomiting, spasm of the airways and cardiac dysrhythmias. The stage two excitement phase caused by anesthetics is somewhat puzzling. Early explanations described inhibition of neocortical "inhibitory" brain circuits, leading to unchecked primitive activity. However, anesthetic effects on single neurons may also show an excitatory phase, thus anesthetic induced "excitation" may be a molecular level effect at low concentrations. Guedel's stage three has four different "planes" of progressively deeper anesthesia during which surgery may be performed. Muscle relaxation is slight in plane one, but progresses to complete abdominal muscle relaxation in planes three and four. Breathing is very irregular and periodic in plane one, as in normal sleep. With plane two, a pause develops between inhalation and exhalation, and inhalation becomes shorter relative to exhalation. Paralysis of rib muscles begins in plane three, and diaphragmatic breathing is prominent. In plane four, paradoxical movement of the rib cage occurs with inhalation, breathing is irregular, and if anesthetic depth is not lightened breathing will stop altogether. Eye muscles are active initially in plane one, however the eyes become immobile when plane two is reached. The eyelid reflex disappears in plane three. The pupils tend to become dilated with progression to plane four. The fourth stage of anesthesia is respiratory arrest and ensuing cardiovascular collapse. Muscles are flaccid, and pupils are widely dilated. Death will occur if anesthetic depth is not decreased from stage four (Grantham and Hameroff, 1985).

Because of the risks of anesthetic overdose and the variable requirements of individual patients, anesthesiologists have sought methods to judge anesthetic depth in addition to the clinical signs enumerated by Snow and Guedel. Since the early 20th century, electroencephalography (EEG) has been recorded from anesthetized patients. EEG waves recorded at the scalp are thought to emanate from summated dendritic synaptic potentials ("dipole fields") generated by pyramidal cells of the cerebral cortex. Scalp potentials generally range from 10 to 200 microvolts, and in frequencies from several Hz to about 50 Hz. The frequency spectrum of the EEG is usually divided into 4 major classifications, delta: less than 4 Hz, theta: 4-8 Hz, alpha: 8-13 Hz, and beta: greater than 13 Hz. Generally, power at higher frequency decreases as anesthetic depth increases. Accordingly, efforts to derive an appropriate index of anesthetic depth have included attempts to quantify an EEG frequency below which most EEG power occurs. This involves a Fourier transform of raw EEG data into a frequency/power spectrum. One example is "spectral edge," the frequency below which 95 percent of EEG power occurs (Rampil, 1981). As anesthetic depth increases, spectral edge roughly decreases. Other attempts to quantitatively assess EEG and anesthetic depth have included the plotting of EEG voltage in "phase space," yielding phase portraits and chaotic attractors. Phase space plotting involves choosing an arbitrary "phase lag," and plotting EEG amplitude at any point in time against the amplitude at that particular time plus the phase lag. This results in geometric phase portraits which may be analyzed for complexity, or "dimensionality" on a continuum of order and chaos. As patients become more anesthetized, the dimensionality of their EEG becomes more ordered, and less chaotic (Figures 7.2 and 7.3, Watt and Hameroff, 1987). Consciousness may thus be described as a manifestation of deterministic chaos somewhere in the brain/mind.

In addition to observing activities of the brain, anesthesiologists must closely monitor other physiological functions of their patients during and immediately after surgery. Cardiovascular, pulmonary, neuromuscular, kidney, blood clotting, and other factors are carefully followed. A variety of technological advances in monitoring permit safer care of sicker patients for more complex procedures. Future monitoring may utilize even more advanced technologies. One possible example is the "biosensor" field effect transistors which are tiny membrane covered chips which can detect a variety of ions, molecules, drugs or hormones. Small enough to fit on the tip of a small catheter harmlessly inserted into a blood vessel or tissue, these biosensors connected to a computer can yield "on-line" monitoring of blood chemistry and many other functions. With the advent of nanotechnology, even tinier, more profoundly sensitive biosensors will materialize. The capability for monitoring nanoscale conformational dynamics of neural proteins (telemetrically or via very small implants) will lead, not only to more sensitive observation of brain function during anesthesia and surgery, but perhaps eventually to the manipulation of consciousness.

Figure 7.2: EEG from awake patient prior to anesthesia. Top: 4 seconds of "conventional" EEG which shows low voltage, high frequency waves. Bottom: same 4 seconds plotted as phase space trajectory with digitization of 300 Hz and phase lag of 5/300 sec. Phase portrait of awake state is densely centered. From Watt and Hameroff (1987).

7.2           Memory

Occasionally patients have reported awareness or recall during anesthesia, an abhorrent event to both patient and anesthetist. Research into this area has illuminated the mechanisms of memory consolidation and led to specific amnesia producing anesthetic drugs. Accordingly, awareness and recall during anesthesia is currently exceedingly rare. In an excellent review of anesthesia and memory processes, Cherkin and Harroun (1971) described a two-stage theory of memory which postulated that information is first perceived and stored in an unstable dynamic form (short-term memory), which may then be consolidated into a stable physical memory trace (long-term memory).

Figure 7.3: EEG from patient after induction of general anesthesia with thiopental and isoflurane. Top: 4 seconds of conventional EEG show higher voltage, lower frequency waves. Bottom: same 4 seconds plotted as phase trajectory as in Figure 7.2. Phase portrait of anesthetized patient is unraveled with "looser," geometric contour resembling an "attractor" Dimensional analysis reveals a less "chaotic," more "ordered" phase portrait in the anesthetized state compared to the awake state. >From Watt and Hameroff (1987).

Perception and short-term memory storage may constitute awareness, but do not necessarily cause a physiological response or become long-term memory to produce postoperative recall.

Figure 7.4: Sensory information may be perceived and register short term memory and awareness. A second step which takes a finite period of time results in long term memory storage and recall. Modified from Cherkin and Harroun (1971).

Consolidation of short term memory to long term storage appears to take a finite period of time, on the order of 45 seconds (Figure 7.4). The establishment of long-term memory traces depends to a great extent on the initial impact of the information input. Many of us who are old enough can remember exactly where we were when President Kennedy was shot. Similarly, threatening or derrogatory remarks are more likely to be heard and consolidated to long-term memory and recall in partially anesthetized patients (Bitnar, 1983). Amnesia (lack of recall) results from prevention of memory consolidation at any point from input to long-term memory storage. Anesthetic drugs can act at various stages of this process: narcotic painkillers prevent noxious input, amnestic tranquilizers block consolidation to long term memory, the general anesthetics inhibit these and all other cognitive functions. The consolidation process appears to occur in the brain's hippocampal region but memory storage and awareness are more diffusely distributed.

7.3           Mechanisms of Anesthesia

A variety of quite different molecular structures have anesthetic activity. Other molecules, quite similar in structure to anesthetics, may have opposite effects and cause convulsions. Despite these paradoxes, imaginative attempts have been presented to unify a single mechanism for anesthesia and represent clues to the mechanism of consciousness.

Figure 7.5: Anesthesia results from prevention of protein switching between two or more different conformational states induced by binding of ligand, calcium ion, or voltage change. Anesthetic gas molecules bind by Van der Waals forces within hydrophobic pockets within which electron dipole oscillations may be a trigger, or switching mechanism for protein conformation.

Several neural functions and structures are inhibited by anesthetics, although some are inhibited at concentrations higher than that required for anesthesia. Propagation of action potentials resulting in nerve conduction as well as microtubule dependent axoplasmic transport are blocked by anesthetics such as halothane, which also causes depolymerization of MT. However, the measurable neural function which is sensitive to anesthetics at concentrations barely sufficient to erase consciousness is synaptic transmission. Neurotransmitter release and/or neurotransmitter receptor activation thus appear to be the specific actions whose inhibition results in anesthesia. Inhibition of collective communicative activities within the cytoskeleton and attached membrane proteins related to synaptic transmission are presently undetectable with current technologies and their inhibition may still account for anesthesia. Anatomical localization of sites of anesthetic action has focused on regions with many synapses. Accordingly, the reticular activating system which is involved with regulation of wakefulness and attention and is "polysynaptic" was originally viewed as a major site of anesthetic effect. However, many other brain regions are inhibited by anesthetics at concentrations relevant to anesthesia. Further, different anesthetics which have the same end result have dominant actions at differing areas of the brain, and have differing characteristics and side effects.

The mechanism of anesthesia, the "other side" of consciousness, is a beguiling enigma. Efforts to understand the actions of anesthetics began in mid-nineteenth century; Claude Bernard observed that the anesthetic chloroform inhibited "protoplasmic streaming" in slime mold. Around the turn of the twentieth century Meyer (1899) and Overton (1901) discovered that the anesthetic potency of a group of compounds directly correlated with their solubility in a lipid environment, specifically olive oil. Because membranes are largely lipid, the natural conclusion was that anesthetics exerted their effects through actions on lipids in membranes. Much later it was determined that critical, dynamic effects in membranes occurred via proteins, and that hydrophobic regions within proteins were "lipid-like" and hence able to bind anesthetic gas molecules by weak Van der Waals forces. Most contemporary theories agree that anesthesia results from alteration of dynamic, conformational functions of important brain neural proteins: membrane ion channels, synaptic receptors, cytoskeleton, neurotransmitter releasing mechanisms, and/or enzymes (Koplin and Eger 1979; Kaufman, 1977; Eyring, Woodbury and D'Arrigo, 1973). Opinions and theories disagree as to whether anesthetics alter these dynamic protein functions directly via intra-protein hydrophobic pockets, or indirectly via membrane lipids surrounding proteins, or at lipid protein interfaces (Franks and Lieb, 1982). There is further disagreement as to whether a single, unitary mechanism can explain actions of a wide range of anesthetic compounds acting on a presumably wide range of neural proteins, as well as explain reversal of anesthesia by increased pressure, and the paradoxical relationship between anesthetics and convulsants (Halsey, 1976).

Correlations of anesthetic potency with solubility in "lipid-like" solvents (olive oil, octanol, lecithin) are consistent with anesthetic effects occurring either in lipids or in "lipid-like" hydrophobic pockets within proteins (Eger, Lundgren, Miller and Stevens, 1969). Wulf and Featherstone (1957) were among the first to demonstrate binding of anesthetics within hydrophobic regions of whale myoglobin and other proteins. They suggested that anesthetic binding caused a protein conformational change with a resultant increase in exposure of charged groups at protein surfaces which sufficiently altered protein function to cause anesthesia. Altered charge groups can change protein binding of surface water and can explain the findings which led Pauling (1965) and Miller (1969) to propose that anesthetic induced water-protein complexes ("clathrates") were the cause of anesthesia. Other research including Brammall, Beard and Hulands (1974) demonstrated that anesthetics can cause conformational changes in functional proteins, but at concentrations significantly higher than that required to cause reversible clinical anesthesia.

A logical inference is that anesthetics, rather than causing protein conformational changes, prevented those that were necessary for normal function. Looking for model systems of functional dynamic activity in proteins, a number of researchers have focused on a seemingly obscure group of systems which, on the surface, are distantly removed from any semblance of cognitive function: anesthetic inhibition of luminescence from photoproteins in firefly and a variety of bacteria (Harvey, 1915; Halsey and Smith, 1970; White and Dundas, 1970). Firefly luciferase is a protein dimer of about 100 kilodaltons (not unlike tubulin). When combined with a 280 dalton hydrophobic molecule called luciferin, and in the presence of ATP and oxygen, an excited electron state is induced which results in photon emission. The light emission from the luciferase/luciferin complex is exquisitely sensitive to anesthetic gases in proportion to their anesthetic potency. Studying the light emission from firefly luciferase, Ueda (Ueda, 1965; Ueda and Karmaya, 1973) proposed that anesthetic binding at a hydrophobic site in the luciferase protein induced an inactive conformational state which prevented the ATP activated excited state which would normally result in luminescence. In more recent anesthetic studies on firefly luminescence, Franks and Lieb (1984) corroborated earlier work that anesthetic potency correlated with luciferase inhibition potency, much like the earlier correlations with solubility in "lipid-like" hydrophobic environments. They also reported that anesthetic binding did not cause a luciferase conformational change, and that the anesthetic effects occurred by competitive binding with luciferin for a hydrophobic site in luciferase. Franks and Lieb suggested that anesthesia occurs due to displacement of endogenous ligands from brain neural protein hydrophobic pockets which bear some resemblance to the luciferin site within firefly luciferase. Displacement of endogenous ligands would not explain, however, anesthetic inhibition of functional protein conformational changes which occur in response to stimuli other than hydrophobic ligands (i.e. voltage change, calcium, other ions).

Ultimately, anesthesia results from impairment of protein conformational dynamics. As described in Chapter 6, mechanisms which regulate protein states are not totally understood, but collective, cooperative mechanisms appear important. The conformational regulation of anesthetic sensitive neural proteins is schematically summarized in Figure 7.5. Under normal, non-anesthetic conditions, there is a class of neural proteins which undergoes functional conformational change in response to appropriate stimuli. These proteins may be membrane bound ion channels which become permeable to sodium, potassium, chloride, or calcium ions; membrane bound receptors for acetylcholine, gamma-amino butyric acid (GABA), glycine, aspartate, glutamate or other neurotransmitters which are coupled to ionic channels; cytoskeletal proteins which perform a wide range of dynamic intracellular functions; or various enzymes. Stimuli which induce functional conformational change in these proteins are thought to include binding of ligands (i.e. neurotransmitters), changes in voltage, or ionic flux (i.e. calcium ions). These functional conformational changes may either facilitate neuronal excitatory activity or have inhibitory effects. The common anesthetic sensitive link for both excitatory and inhibitory effects appears to be a protein conformational change.

The coupling of conformational states to appropriate stimuli is not clearly understood; possible explanations were discussed in Chapter 6 and appear to involve collective, nonlinear coupling of electronic mobility to mechanical movements. Conformationally coupled electron mobility within neural protein hydrophobic pockets may thus be essential to highest cognitive function and sensitive to the effects of anesthetic molecules. Evidence from gas chromatography (McNair and Bonelli, 1969) and corona discharge experiments (Hameroff and Watt, 1983) suggest that anesthetic gases can directly alter electron energy, capturing, retarding, or enhancing their mobility by Van der Waals-London forces (instantaneous dipole coupling). Thus regulation of protein conformational state, as proposed by Fröhlich's theory of coherence, electret behavior or Davydov's soliton model, would be directly inhibited by anesthetic occupancy of protein hydrophobic pockets because the environmental medium for electron mobility would be significantly altered. Hydrophobic pockets vary in size and shape among different proteins which would account for the variability among different anesthetic gas molecules. The weak, reversible Van der Waals interactions by which anesthetics bind within hydrophobic regions explain the reversibility of anesthesia. The flip side of anesthesia, consciousness, may be inferred to occur due to dynamic protein conformational effects among a distributed class of brain neural proteins. Because consciousness is the brain function most susceptible to anesthetics (which can spare breathing control and many reflexes at appropriate concentrations) it appears related to collective effects of protein conformartional dynamics. Inhibition of a subset of the collective dynamics would therefore inhibit consciousness.

Conversely, excitation of a specific subset involved in collective protein dynamics could lead to alteration or enhancement of consciousness. Psychoactive drugs such as amphetamines and LSD can exert profound effects on the brain/mind at extremely low concentrations. They are thought to bind and to exert their effects at membrane protein receptors such as the serotonin receptor in the case of LSD. Kang and Green (1970) correlated the potency of psychoactive compounds with their electronic bond structure and molecular orbitals. They found that the drug molecules' electronic orbital energy available to the receptors (and connected membrane and cytoskeleton) was an index of drug potency. Thus collective effects related to electron mobility within key proteins in a cooperative assembly may be important to consciousness.

8         Models of Cytoskeletal Computing

Cooperative, collective effects of dynamic protein conformational states are a likely substrate for biological intelligence ranging from cytoplasmic probing to human consciousness. The activities, functions, and structures of microtubules and other cytoskeletal components appear suited to information processing and have led at least a dozen author groups to publish theoretical models of rudimentary cognition within MT and the cytoskeleton. The concepts range from passive MT signal transduction, to descriptive patterns among MT subunit states, to dynamic cooperative "automaton" effects among coherent oscillations of centrioles and the cytoskeleton, to cytoplasmic/cytoskeletal "sol-gel field" effects utilizing holographic imagery. If correct, these proposals could explain many aspects of information representation and dynamic organization in biological systems. Hopeful metaphors, these models are non-exclusive and may be overlapping and complementary.

Cytoskeletal information processing would require some mechanism of cooperativity, long range order, coherence, and/or energy transfer among cytoskeletal components and their subunits. Such possible mechanisms were discussed in the previous chapter. Specific evidence which supports transfer of energy and information within microtubules will be discussed here followed by thirteen models of cytoskeletal information processing.

8.1           Energy and Information in Microtubules

Direct support for the propagation of signals in MT has been generated by Vassilev, Kanazirska, and Tien (1985) who reconstituted bilayer membranes from brain lipids and studied their electrical excitability. They suspended membranes as parallel unconnected plane surfaces separated several millimeters apart in a buffer solution which contained depolymerized tubulin, GTP, and other physiological components. Each membrane was monitored electrically and baseline recording of the two membranes showed no electrical coupling; when one membrane was electrically stimulated it depolarized, but the other membrane remained silent. When the tubulin was caused to polymerize into MT (by lowering calcium ion concentration) MT bridges formed between the two membranes and electrical coupling between the two membranes was observed. Electrical stimulation of one membrane then resulted in depolarization in both membranes. The addition of the MT destabilizing drug colchicine prevented coupling, demonstrating that intact microtubules were necessary. The authors concluded that intermembrane signaling occurred by electrically induced polarization and conformational changes of MT components which linked the two membranes. They suggested that similar communication functions occurred routinely within the cytoskeleton.

Another series of experiments which supports the notion of MT mediated signaling is fluorescence resonance transfer among MT and membrane components. Becker, Oliver and Berlin (1975) developed a technique to study energy transfer among fluorescent groups separately attached to different MT subunits or to membranes. Resonance energy transfer occurs when a fluorescent portion of a molecule ("chromophore") which is electronically excited by the absorption of light energy transmits that energy to another "acceptor" chromophore some distance away. This transmission requires the overlap of the emission spectrum of the "donor" chromophore with the absorption spectrum of the acceptor, without involving the actual reabsorption of light by the acceptor. The process is therefore referred to as "nonradiative" resonance energy transfer and occurs if the distance between the chromophores is relatively close, not to exceed about 10 nanometers. In the study by Becker, Oliver, and Berlin, fluorescein isothiocyanate (FITC) was used to fluorescently label one population of unpolymerized MT subunits or membranes, and another fluorescent label, rhodamine isothiocyanate (RITC) was used to label a second population of tubulin. They chose these chromophores because they bind covalently to tubulin or membranes, and because the emission spectrum of FITC "donors" extensively overlaps the absorption spectrum of RITC acceptors. Recordings of fluorescence spectra reveal the "resonance transfer" when it occurs. When MT were depolymerized, a mixture of donor labeled and acceptor labeled tubulin did not show resonance transfer. With polymerization or aggregation of MT subunits, fluorescent excitation of fluorescein labeled tubulin resulted in fluorescent emission by rhodamine labeled tubulin, as the chromophores were brought sufficiently close together in a common lattice to permit resonance energy transfer. The energy transfer occurred not only among tubulin subunits in MT, but among MT subunits and membrane components.

Evidence for another mode with communicative implications in microtubules is suggested by the parallel alignment of MT in applied electric and magnetic fields (Vassilev, Dronzine, Vassileva, and Georgiev, 1982). They cite the postulated existence of low intensity electric fields (Jaffe and Nuccitelli, 1977; Adey, 1975) in the range of 20 to 500 millivolts per centimeter within cells (one millionth of the field strength across polarized membranes). Vassilev and colleagues isolated rat brain tubulin and created polymerizing conditions in the presence of pulsed electric fields of about 25 millivolts per centimeter. Electron micrographs showed that the MT polymerized in perfect parallel alignment with the applied field. Similar results were obtained when low intensity (0.02 Tesla) magnetic fields were applied. If assemblies of MT can also generate electric and/or magnetic fields of similar intensity via an electret effect, then a cooperative communication comparable to the "Indian rope trick" may be utilized in cellular growth, differentiation, and synaptic plasticity. MT could then generate their own pathways for cytoplasmic movement.

Other data (Matsumoto and Sakai, 1979; Alvarez and Ramirez, 1979) suggest that the intraneuronal cytoskeleton is necessary for nerve membrane excitability and synaptic transmission. Nerve membrane proteins including ion channels and receptors which are anchored to the cytoskeleton may be the "tips of an iceberg" of a cytoskeletal communicative medium which could utilize a number of possible modes to achieve collective cooperativity and intelligent cellular behavior. The following models suggest some possible strategies.

8.2           Cytoskeletal Information Processing

The following models have been roughly grouped by authors and significant concepts.

8.2.1      MT Sensory Transduction/Atema

Several authors have discussed the transduction of sensory information by the mechanical distortion of cilia: membrane covered centriole-like structures which protude from cells. Lowenstein, Osborne, and Warshall (1964) suggested that the "kinocilium" of the hair cells in the inner ear served as motile cilia in reverse. They reasoned that motile cilia produced movement using chemical energy provided by ATP hydrolysis, so mechanoreceptor cilia should transduce mechanical deformation caused by environmental stimuli to provide the cell with "patterns of chemical energy representing information." Lettvin and Gestalind (1965) also proposed that receptor cilia operated "by virtue of transmitting mechanical signals to the base of the cilium."

Biologist Jelle Atema (1973) of the Wood's Hole Oceanographic Institute, whose work had focused on acoustical perception at the cellular level, also linked microtubules and sensory transduction. Noting common cilia-like structure in sensory receptor organelles among a wide variety of organisms, Atema proposed that sensory cilia conveyed environmental information to the rest of the cell. He suggested that transduction occurred by propagated conformational changes in the microtubule subunits which constituted these cilia. He argued that microtubules were active functional units in reception and transduction of sensory information.

Atema reviewed conformational changes in MT tubulin subunit dimers observed by a variety of authors and suggested that these occurred under physiological conditions. For example, oscillations of sperm flagella are apparently not controlled by their cell membrane but rather are direct properties of their microtubules in the presence of ATP (Lindemann and Rikmensboel, 1972). Atema concluded that a sequence of subunit conformational changes is likely to occur in microtubules. In motor cilia and flagella, the distortion would originate at the base of the structure and be propagated distally, resulting in wavelike or whiplike motions which propel the organism. In sensory cilia, the distortion apparently originates near the distal end of the ciliary MT and propagates in either direction or at least proximally towards the cell body. There the signal may propagate via either an excitable membrane or through the anchoring basal body and cytoskeleton. Because mechanical deformation and/or local chemical distortion are sufficient stimuli to start a propagating signal in sensory cilia, Atema's microtubule theory assumed that distortion of tubulin conformation by any number of sources was sufficient to propagate a conformational wave. Thus light energy, chemical bond energy, and mechanical forces could be transduced by sensory cilia.

Atema's view of MT information processing was an "all or none" propagation by allosteric conformational changes along tubulin protofilaments. His was the first theory to look beyond the global behavior of cilia to consider conformational effects in tubulin components. Subsequent theories became more elaborate to consider localized analog functions, switching, and collective neighbor interactions among MT subunits.

8.2.2      MT Mechano-lonic Transducers/Moran and Varela

Biological sensory systems ranging from human inner ears to honey bee hairplate receptors use sensory cilia to transduce mechanical deformation into cognitive information like position in space ("proprioception"). Harvard biologists Moran and Varela (1971) studied the proprioceptive transduction of mechanical deformation in the legs of cockroaches. There, tactile spines contain mechanoreceptor structures called "campaniform sensilla" which consist of a single bipolar neuron from whose dendritic tip extends a modified cilium containing 350 to 1000 microtubules. Moran and Varela determined that this parallel bundle of MT was directly involved in the transduction of mechanical deformation to the neuron.

All neurons are mechanical receptors, being stretch sensitive to cause ionic currents. In the cockroach campaniform sensilla, the cilium is the only bridge and transduction does not occur when MT are incapacitated with tubulin binding drugs like colchicine and vinblastine (Moran and Varela, 1971). MT are thus directly involved in proprioceptive determination of the system's spatial relation to its environment. Moran and Varela saw two possible roles for MT in their mechanoreceptor function: as passive translation rods, or as generators of ionic/electrical currents. They suggested that MT behaved as "mechanochemical engines driven backward." Compression or bending of MT would cause release of bound ions from MT subunits, resulting in an ion flux. Moran and Varela saw these ionic currents capable of generating membrane depolarization, being perhaps the first to suggest that MT can regulate membranes. Each tubulin dimer reversibly binds 16 calcium ions so ionic fluxes of significant current could result from an active assembly of parallel MT. This process may be analogous to the coordinated release of calcium ion waves by sarcoplasmic reticulum in muscle cells which triggers actin-myosin contractile interactions. Calcium waves are also thought to regulate the bending and waving of cilia and flagella, and can regulate the cytoskeletal "ground substance" by coupling to sol-gel states. Moran and Varela's contribution was to observe that MT could release ions such as calcium in a controlled and modifiable manner useful for intracellular communication.

8.2.3      Cytomolecular Computing/Conrad and Liberman

Wayne State University computer scientist Michael Conrad and Soviet information scientist E. A. Liberman have collaborated to consider aspects of biomolecular computing including the cytoskeleton. They consider that the "computing power of the brain is primarily based on intracellular processes." They propose that the cyclic nucleotide system (energy rich molecules such as cyclic AMP) sculpts three dimensional dynamic patterns in the cytoplasm of neurons and other cells which are the analog texture of real time information processing. Conrad and Liberman view reaction diffusion patterns of cyclic AMP, regulated and perceived by both cell membranes and the cytoskeleton, as a link between macroscopic neural activities and molecular scale computing. Conrad, known for his conceptualization of protein enzymes as possible computer components (Chapter 1), has also advanced the notion of molecular automata within cells (Conrad, 1973). He and Liberman suggest that the intracellular cytoskeleton perceives mechanical stretch or distortion subsequent to membrane events and accordingly regulates cyclic AMP and other biomessengers.

Conrad and Liberman (1982) suggest:

in neurons, mechanical stimulation appears on movement of the intraneuron tubule skeleton and micromuscle ... details of the skeleton and micromuscles (are) suitable for constructing the molecular analog ... of the real physical field in which this system moves.

Conrad and Liberman view a molecular analog within the cytoskeleton as a representation of the external world. Extrapolated to complex systems like the brain, such a cytoskeletal analog could suffice as a medium of cognition.

Conrad (1985) notes that

highly parallel signal processing and vibratory behavior on the part of microtubules and other cytoskeletal elements could play a significant role.

8.2.4      MT Signal Processing/DeBrabander

Cell biologist Marc DeBrabander has extensively studied activities of the cytoskeleton in mitosis and cellular organization in general. He and his colleagues at Belgium's Janssen Pharmaceutica Research Laboratories (DeBrat bander, DeMey, VandeVeire, Aerts and Geuens, 1975, 1986) have examined the distribution and movement of cell membrane proteins and gathered evidence that they are linked to the cortical actin filament system. In turn, the ordered activity of the filaments appears to be controlled by the microtubule system, analogous to its function in cell movement and cell shape. DeBrabander (1977) observes that in these phenomena MT act beyond their capacity as skeletal support elements and perform as signal transducers from the cell center to the periphery, and vice versa. Interactive signaling in the cytoskeleton fits with observations that local events at one site in the cell often lead to a global cellular rearrangement. DeBrabander raises several MT features which could favor signaling: MT intrinsic polarity, the existence of "centrifugal and centripetal microtubules," the conformational propagation mechanism proposed by Atema (1973), directional organelle movement and topography, and transport of ions such as calcium along microtubules.

DeBrabander and his associates (1986) have also innovated new techniques in the examination of cytoskeletal structure and dynamics. These include "Nanovid Microscopy," a nanometer scale video microscopy in which transport of labeled particles along individual MT may be visualized, and a method of labeling individual tubulin subunits within MT which are either tyrosinated or glutamated (Geuens, Gundersen, Nuydens, Cornelissen, Bulinski, DeBrabander, 1987). As described in Chapter 5, polymerized MT may be detyrosinated in the cytoplasm subsequent to DNA directed genetic input. Tyrosine, the terminal amino acid in tubulin, may be removed to expose glutamate as the new terminal amino acid. Thus all tubulins within polymerized MT are either tyrosinated, or glutamated. A variety of cytoplasmic factors determine whether or not a particular tubulin is tyrosinated. The precise significance or function of tyrosination/glutamation is unknown but could serve as a convenient programming or memory function. DeBrabander and colleagues developed a double labeling technique in which immunogold particles bind to tubulin subunits. Large immunogold particles (10 nanometers) identify glutamated tubulin and smaller particles (5 nanometers) bind to tyrosinated tubulin. Patterns of tyrosinated/glutamated tubulin within MT show heterogenous distribution, and suggest the potential for a coding mechanism. MAP attachment, tubulin conformation, calcium binding and other factors could be coupled to MT function via such patterns. Although predisposition to detyrosination may be genetically linked to tubulin isozymes, the actual patterns of tubulin detyrosination are determined by "real time" cytoplasmic factors. DeBrabander and colleagues have provided the first direct evidence of modifiable patterns of tubulin variability in intact microtubules. Consequently, MT appear capable of signal processing in addition to signal transduction.

Figure 8.1: Microtubules from PtK2 cells double labeled with antibody and immunogold under high magnification. Large circles (10 nanometer gold particles, arrows) tag glutamated tubulin subunits, small circles (5 nanometer gold particles) label tyrosinated tubulin subunits. Left: interphase microtubule, Right: spindle microtubule. With permission from Geuens, Gundersen, Nuydens, Cornellisen, Bulinski, and DeBrabander (1986), courtesy of Marc DeBrabander and Janssen Pharmaceutica Research Laboratories.

8.2.5      Cytoskeletal String Processors/Barnett

Figure 8.2: Barnett's string processor model of microtubule-neurofilament memory storage. 1) a processing channel (microtubule-MT) is laterally connected to a parallel storage unit (neurofilament-NF), 2) information traverses the processing channel from right to left; on the storage channel, synonym pairs move from left to right, 3) as the word "chips" moves by its own self in storage, it is deleted, 4) "chips" is replaced by its synonym "french fries" and 5) the replacement is complete. By Paul Jablonka.

Brooklyn College computer and information scientist Michael P. Barnett has pursued the design of computer components suited to molecular scale devices. In addition to the development of tiny binary switches to be assembled into circuits on digital architecture, Barnett (1987) has also sought an associative memory which can analyze imprecise analog data as well as conventional digital information. Fabricated systems with these features could be versatile, powerful and a boon to the goals of artificial intelligence. Like many AI oriented researchers, Barnett turned to biology for clues. However, unlike most Al researchers, Barnett scoured the subcellular biological realm in search of molecular scale information processing concepts. His fancy was captured by cytoskeletal microtubules and neurofilaments! He has proposed information representation as patterns in the subunits of cytoskeletal polymer subunits. He proposes that specific subunit states may be characterized by "electrons transferred from delocalizable molecular orbitals," but his basic premise would also be supported by other causes of conformational state variability among MT and neurofilament polymer subunits. Barnett suggests that filamentous cytoskeletal structures operate like information strings analogous to word processors.

In Barnett's conceptualization (Figure 8.2), information strings move from right to left along processing channels, which run parallel to one dimensional memory channels in which character strings can be stored. Barnett's string transformers can perform global replacements on sequences of characters like common word-processors. His model assumes the existence of processing channels (MT) along which strings of information can move, and memory channels (neurofilaments) which consist of a succession of locations, each of which can hold a single character. Parallel array and lateral interconnectedness of MT and neurofilaments could qualify these cytoskeletal elements as string processors, assuming that information may be represented in the polymers. Barnett's model is thus compatible and complementary with other models of conformational patterns within MT and the cytoskeleton.

8.2.6      Microtubule "Gradions"/Roth, Pihlaja, Shigenaka

Roth, Pihlaja, and Shigenaka (1970), and Roth and Pihlaja (1977) have considered information processing in two types of biomolecular assemblies: membrane rosettes and microtubules. Citing cooperative allosteric effects among adjacent proteins or protein subunits, they propose that conformational gradients in protein arrays represent information by patterns of conformational states among near neighbors in protein lattices.

Rosettes are ordered rings which consist of from seven to twelve membrane embedded proteins important in membrane fusion in lower organisms.

One example of rosette function is in "suctorian feeding", in which certain protozoa affix themselves to a host cell membrane and feast upon its cytoplasm by sucking its contents through a rosette common to both membranes. Bardele (1976) suggested that rosettes utilize concepts of cooperativity and allosterism because the triggering of conformational dynamics within the complex does not require contact with all particles. Stimuli at only one or a few subunits followed by allosteric changes in the rest can cause activation of the total complex. Satir (1973) showed that rosettes in membranes of tetrahymena have rosette pairs in register with each other: internal and external rings of eight proteins each. Induction of a conformational change by binding of an effector molecule at the external rosette causes allosteric cooperative changes in the conformation and functional states of all sixteen subunits. In mutants with aberrant rosettes, a minimum of 5 subunits is required before rosettes functionally respond.

Roth, Pihlaja, and Shigenaka considered the next level of complexity in protein assemblies to be exemplified by microtubules. They viewed MT as highly oriented patterns of tubulin dimers and anticipated at least three different conformational states of each tubulin dimer, based on studies of tubulin binding to vinblastine and GTP (Luduena, Shooter, and Wilson, 1976). Biologists Roth, Pihlaja, and Shigenaka (1970) studied the patterns of linkage among MT within the axopod of a simple organism called Echinosphaerium, a complex spiral assembly of hundreds of MT. The precisely patterned, interwoven spiral arrays of MT which comprise the axopod are explained by inter MT bridges of two different lengths. The authors considered that these two types of linkages can not only account for the complex structure of the axopod, but also indicate two different conformational states of the tubulin dimers to which the bridges attach. Along the lengths of the axopod, microtubule linkages are found at intervals that reflect binding at approximately every fourth dimer. These precise linkage sites represented a code of some sort to Roth, Pihlaja, and Shigenaka who proposed in 1970 that recurrent patterns, or "gradients" existed in microtubules to localize these precise binding patterns. They defined these patterns as "gradions": repeating conformational sequences or fields in which multiple varying patterns can exist in the same molecular architecture simultaneously.

Figure 8.3: "Gradions" within microtubule lattice. Dark shaded dimers are MAP attachment patterns; numbered dimers are determined by proximity to MAP attachment sites. MAP binding and other "gradionators" are thought to induce coded patterns representing information. From Roth, Pihlaja and Shigenaka (1970) by Paul Jablonka.

Conformational states of individual tubulin subunits were thought to be controlled by factors Roth, Pihlaja, and Shigenaka termed "gradionators." These could include ligand induced conformation, tubulin isozyme, detyrosination, or binding of MAPs including inter-microtubule bridges. They defined discrete gradion fields within microtubule surfaces as the neighborhood areas around intermicrotubule MAP bridges. They further assumed five possible conformational states for each dimer determined by inter-MT linkage sites and their cooperative allosteric effects, as well as dimer binding by a number of different native substrates or foreign molecules. The pattern of tubulin dimer conformational states in a region of MT lattice "governed" by attachment of inter-MT linkages (or other MAPs) is defined as a "gradion," or conformational field which includes about seventeen dimers. Consequently, 5¹⁷ different gradion patterns could exist if the conformation of each subunit were independent of all others. Cooperativity and allosteric effects preclude independence, so the number of possible MT "gradions" is somewhat less, but still substantial. Roth and Pihlaja (1977) see formation of many "gradions" within axopod MT as a mechanism for position determination and coding for connections. Applied to neuronal cytoskeleton, such deterministic patterns would be generally useful for mental processes in the brain and be a viable candidate for "grain of the engram."

The gradion theory may be applied to any large field of allosteric particles. Allosterism and cooperativity in membranes, cell junctions, and protein particles in many cells could explain aspects of cooperative communication, however microtubules appear especially well suited for information functions. The MT gradion model of Roth, Pihlaja, and Shigenaka is an early specific theory of information processing in protein assemblies in general, and microtubules in particular.

8.2.7      Gyroscopic Centrioles/Bornens

French molecular biologist Michel Bornens (1979) has investigated cellular mechanisms of organization and argues for a dynamic stability based on organizational properties of centrioles and the cytoskeleton. Specifically, Bornens proposes that centrioles are animated by rapid oscillatory rotation about their longitudinal axis which results in a dynamic stability and inertia analogous to a spinning top or gyroscope. Peripheral movements throughout the cell are interconnected by the cytoskeleton, with the gyroscopic centrioles an inert point of reference which provides cellular gravity. Centrioles and microtubule organizing centers (MTOC) are the origin for the cell's spatial coordinates and cytoplasmic movement appears to occur relative to the MTOC. Bornens likens movement in each cylinder to a stepwise electric motor in which the central "cartwheel" (or "pinwheel") is equivalent to an ATP-dynein "stator," and the centriolar wall the moving "rotor." Continuous torque rotation of centrioles could serve to propel them through cytoplasm, like an "Archimedes screw" (Record, 1986). Bornens also considers the possibility of "back and forth" oscillation with utility for scanning the cell environment.

Reviewing Bornens' gyroscopic centriole model, Albrecht-Buehler (1981) questioned the high rate of rotation required for significant inertia. Albrecht-Buehler calculated that centrioles would have to rotate at frequencies of 2.3 million revolutions per minute before their kinetic energy matches one kT (the thermal energy of one molecular degree of freedom). Consequently, "much greater rotational frequencies would be required before the centriole could withstand the impact of thermally moving molecules around them and maintain stable axial orientation" (Albrecht-Buelhler, 1981). Bornens responded by suggesting a submicroscopic mechanism "allowing more independence of the centriole with respect to surrounding material." Factors which could support his contention include ordered water coupled to centriolar oscillation, some unknown property of the "pericentriolar material," ionic charge layer, or even superconductivity as suggested by Fröhlich and Del Giudice's group (Chapter 6). Rotatory oscillations in the range of 10⁷ per second would be consistent with the findings of several researchers (Chapter 9) who found collective energy absorption in the range of 10⁷ per second by protein assemblies such as virus coats.

Bornens views centrioles as the center of a dynamic cytoskeleton which communicates and integrates cellular information. In Bornens' view of cytoskeletal organization, microtubules are conductors of spatial centrifugal information, rigid organizers of cell space in which physical or electrical signals propagate as conformational modifications of MT subunits. Intermediate filaments and the microtrabecular lattice also participate in Bornens' vision of a dynamic network of pulsating polymers. He also suggests that ATP generated centriolar rotation triggers propagating impulses along microtubules by transitory contact/stimulus of the nine rotating MT doublet/triplets in the centriole wall with their surrounding satellite bodies. This would lead to rhythmic signals through the cytoskeleton with a frequency nine times greater than that of the centriole's rotation. Rhythmic, propagating signals are compatible with coherency, solitons, and other models. Their occurrence throughout the cytoskeleton would be mechanisms close to the nature of life itself.

8.2.8      Centriole-MT Signaling/Albrecht-Buehler

Northwestern University biologist Guenter Albrecht-Buehler has considered the general question of intelligence in cytoplasm (Chapter 5) as well as two specific models of cytoskeletal information processing: centriole signal detection, and propagation of MT impulses. The walls of centrioles are composed of nine MT doublets or triplets arrayed in a cylinder. Each MT triplet can consist of two incomplete C-shaped MT and a complete O-shaped one fused longitudinally. The triplets are arranged at an angle of 30-45 degrees from the main cylinder, pitched to form a blade which advances to a final increment of one ninth of the perimeter just below the position of the next blade. A line drawn through any blade connects with the inner edge of the preceding blade (Figure 8.4).

Collective behavior of cytoplasm would seem to require some communicative format, and Albrecht-Buehler suggests that centrioles are perfectly designed to detect both intensity and direction of linear signals. One possible example of a highly specific, yet ubiquitous signal propagated in a straight line in the cellular environment is infrared radiation of the molecules inside and around cells (Albrecht-Buehler, 1981). As discussed in Chapter 6, transmission of biomolecular infrared energy would require shielding from bulk water, and perhaps nonlinear coupling to structural conformational states. Both of these requirements may be met by the ordered water and ions surrounding the cytoskeleton and the electron-dense pericentriolar material.

Cellular navigators, centrioles are involved in directional orientation of moving cells, establishment of cell architecture in cell growth and differentation, and all dynamic rearrangements of cytoplasm. In an attempt to understand how centrioles could navigate and orient, Albrecht-Buehler asks how an optimally designed, technological spatial signal detector would appear. Instruments such as radar scanners can determine direction of signals by scanning different directions sequentially. However, such scanners miss signals which arrive from one direction while another is being scanned. A properly designed nonscanning instrument can listen simultaneously to all directions with no moving parts. Albrecht-Buehler points out some geometric features of an optimally designed, nonscanning "angular" detector. With signals arriving from arbitrary directions, a detector designed as a circle with a number of regularly spaced marks around its circumference would be accessible and capable of identifying direction. Nine fold symmetry provides small size with sufficient angular resolution. To locate a signal source, a detector must prevent an emitted signal from arriving at more than one receptor. A simple circular arrangement would fail to meet this requirement because about half the receptors are accessible to a signal emitted from any source. A simple way to improve the design is to attach blinds or "blades" to one side of each receptor to absorb or deflect a signal wherever it interacts with them. A radial arrangement of straight blinds would be inadequate because at least two receptors would remain accessible to signals from the same source. However, if the blinds are bent circumferentially, an optimal angle is achieved when the blinds prevent access to all but one receptor without producing "blank spots," areas from which signal sources cannot reach any of the receptors. Blinds that restrict access to single receptors are even better if their shape is concaved. Used by manufacturers of "Venetian blinds," this curvature averts the possibility that a signal located directly in line with a straight blind could reach two adjacent receptors. Consequently, a signal is received not by the receptor closest to the signal source, but by one located at a fixed angle off the incident direction. Centriolar "blades" extend above and below the centriolar cylinder and are pitched, or twisted. Albrecht-Buehler sees this torque as further angular resolution, but the "propeller-like" arrangement could also serve to screw centrioles through the cytoplasm, assuming a centriolar rotation as suggested by Bornens (Record, 1986). Two detectors are best placed at right angles to each other so that one of them can locate the "longitude" while the other locates the "latitude" of the signal source. Centriole-like basal bodies fixed perpendicularly into a two dimensional cell surface are usually not accompanied by a second basal body at right angles. Thus the right angle paired cylinder formation permits global exposure and three dimensional reckoning unnecessary in basal bodies. Albrecht-Buehler's view of centrioles as perfectly designed signal detectors is complementary with Bornens' concept of a gyroscopic oscillator and signaling center. Combination of the two models results in a dynamic cell center capable of piloting cytoplasmic activities.

Figure 8.4: Centriole in cross section is comprised of nine triplet MT angled like "Venetian blinds" from centriole axis. Centriole pairs consist of perpendicular cylinders. Albrecht-Buehler (1985) contends these features are ideally suited for signal detection. Bornens suggests rotatory oscillations of centrioles leading to gyroscopic function. By Paul Jablonka.

Albrecht-Buehler (1985) has also considered a mechanism for signal propagation along microtubules. He considers that each MT protofilament is a chain of alpha-beta tubulin dimers: AB, AB, AB, ..., AB. Within the wall of a microtubule each monomer is in contact with other monomers of the same protofilament and with those of adjacent protofilaments. Each monomer is consequently subject to attractive Van der Waals forces from surrounding tubulin monomers which hold together the protofilaments and MT cylinders. Albrecht-Buehler proposed that each of these interactions must weaken the A-B dimer bond; consequently the wall of a microtubule exists in a state of resonance as to the relative strengths of the intermonomeric bonds. For example, (A-B) (A-B) ... (A-B) (A-B) could resonate with (A) (B-A) (B-A) ... (B-A) (B). Such a resonating chain could propagate information at close to the speed of light.

Albrecht-Buehler is suggesting a coherent communicative resonance among protein conformational states within MT assemblies.

8.2.9      Dynamic Tensegrity/Heidemann and Jarosch

Buckminister Fuller proposed an interesting architecture constructed from components which may have a recursive or fractal structure (Fuller, 1975). Its macro level structure, a "tensegrity mast," is a rigid structure constructed from an assembly of tension and compression members. The compression members of solid struts are isolated from each other, held together by the tension members. In one of Fuller's variations, he notes that in the macro tensegrity mast, each individual solid strut may be replaced by a miniaturized version of the macro tensegrity mast. And then each one of the miniature solid struts may itself be replaced by a still smaller subminiature tensegrity mast, and so on down to the atomic level. Thus tensegrity structures may have a fractal substructure.

Joshi, Chu, Buxbaum and Heidemann (1985) have shown that cytoplasm has both compressive and tensile elements. Semi-rigid microtubules are under compression presumably due to tension generated by actin filaments and the microtrabecular lattice or "cytomusculature" (Chapter 5). In general, MT do not contact each other so that the self-supporting capability of cytoplasm may stem from tensegrity.

Robert Jarosch (1986) has published a series of papers describing actin-MT interactions which suggest that 1) contractile actin filaments are spirally wound around microtubules, 2) coordinated contraction of the actin filaments imparts a rotational torque to MT, somewhat like a spinning top, 3) actin filaments wound in opposite directions on the same MT can cause rotational oscillations of the MT. These two models fit together to provide a picture of a dynamic cytoplasmic tensegrity network in which the cytoskeleton may be twisting back and forth, even "rockin' and rollin'!" Perturbation of any part of such a tensegrity network could have dynamic consequences throughout its domain. Transient changes in tension, compression, or oscillatory rhythm caused by a variety of factors would be detectable and possibly amplified throughout the cytoskeleton.

8.2.10 Dynamic MT Probing/Kirschner and Mitchison

Figure 8.5: Robert Jarosch has proposed that actin filaments wind around microtubules and contract, causing MT to rotate and oscillate around their long axis. With permission from Robert Jarosch (1986).

Strong evidence supports the concept of dynamic instability in microtubule assembly (Chapter 5). Many MT exist in either growing or shrinking phases and MT stabilized at merely one end by centriole based microtubule organizing centers (MTOC) tend to predominate. Selective retention of MTOC based MT establishes cell polarity important in extension of axons and dendrites, elongation of cells in embryological development, and formation of lamellipodia and filopodia in locomotory cells. All are examples of the "Indian rope trick" in which cells somehow choose their direction of growth, and then grow in that direction. A cue presented at the cell periphery can lead to rearrangements of cell symmetry and polarity. Kirschner and Mitchison (1986) ask: "how can a peripheral clue lead to reorganization deep within a cell?" One possibility is that a signal is relayed to the microtubule organizing center leading to a change in its structure, orientation, and directed nucleation of microtubules. This would be consistent with a primary role for information integration and decision making within the MTOC. A simpler "non-hierarchical" idea is that a signal at the periphery affects distribution directly. Since the whole cytoskeletal array is very dynamic, it would only be necessary to stabilize or destabilize a particular subset of microtubules for the entire cytoskeleton to rapidly transform. Preferential stabilization of a microtubule (MTOC, GTP capping, binding to membrane related structures etc.) could be mediated by interactions at the cell periphery close to the site receiving environmental information. Kirschner and Mitchison (1986) have proposed that the dynamics of the microtubule array results in probing many regions at random and, by stabilizing certain conformations as they arise, the cell "can arrive at a structure that is not precisely defined by genetic information but fulfills a particular functional role as dictated by environmental factors." Dynamically unstable MT offer many possibilities for controlling the distribution of MT by selective stabilization. Kirschner and Mitchison suggest that the tendency for probing and transformation is a fundamental advantage which favored the evolution of dynamically active microtubules.

8.2.11 Sphere Packing Screw Symmetry/Koruga

There are 32 possible symmetry arrangements of packed spheres in a cylindrical crystal. Erickson (1973) used hexagonal packing of protein monomers to explain the form and patterns of viruses, flagella and microtubules. Djuro Koruga (1986) of the University of Belgrade's Molecular Machines Research Unit has analyzed the symmetry laws which describe cylindrical sphere packing and the structure of microtubules. Koruga has used both hexagonal packing and face centered cubic packing of spheres to explain microtubule organization. Koruga (1986):

The particular symmetry group which represents the packing of spheres in microtubules is 'Oh(6/4).' Hexagonal packing may be described by using fixed conditions if the centers of the spheres lie on the surface of the cylinder and if the sphere values in the long axis of the cylinder are the same as in the dimension of face centered cubic packing. The six fold symmetry and dimer configuration lead to screw symmetry on the cylinder: a domain may repeat by translocating it in a spiral fashion on the cylinder. From coding theory, the symmetry laws of tubulin subunits suggest that 13 protofilaments are optimal for the best known binary error correcting codes with 64 code words. Symmetry theory further suggests that a code must contain about 24 monomer subunits or 12 dimers.

Figure 8.6: Koruga's derivation of the symmetry of microtubules. With permission from Djuro Koruga (1986).

Koruga's symmetry arguments may be compared with the "gradion" concept of Roth, Pihlaja, and Shigenaka in which a field of about 17 monomers is thought to represent a basic information unit. Koruga also concludes that microtubule symmetry and structure are optimal for information processing.

Figure 8.7: Koruga's screw symmetry and optimal information unit in lattice wall of microtubule. With permission from Djuro Koruga (1986).

8.2.12 Cytoskeletal Self-Focusing/Del Giudice

As described in Chapter 6, a group of scientists from the University of Milan have applied the mathematical tools of "many body problems" to the activities of biomolecular dipoles. Del Giudice, Doglia, Milani and Vitiello (1985, 1982) have used quantum field theory to describe the electret state of biological systems (ordered water surrounding linear biomolecules) and determined that there exists a strong likelihood for the propagation of particle-like waves in biomolecules. Further, the ordering of water should lead to self-focusing of electromagnetic energy into filamentous beams excluded by the ordered symmetry. For ordered cytoplasm, they calculate the diameter for the confinement and propagation of particle-like waves (massless bosons, or solitons) in biomolecules to be about 15 nanometers, exactly the inner diameter of microtubules.

The proposal by the Milan group has a number of implications. Confinement within filamentous regions excluded from water would favor the propagation of electromagnetic energy in biological systems, and provide a mechanism for alignment and communication (the "Indian rope trick"). Further, cytoskeletal polymers may be capable of capturing and utilizing ambient or biologically generated electromagnetic energy. One possible example is infrared energy which is routinely generated by dipoles in biological molecules. This energy is generally believed to be dissipated into heat within the aqueous cytoplasm, however "self-focusing" could utilize this energy productively in a communicative medium. The Milan model also includes lateral force generation by focused energy within cytoskeletal filaments which would be useful in biomolecular maneuvering and communication.

8.2.13 MT Automata, Holography/Hameroff, Watt, Smith

The self-focusing of electromagnetic energy described by the Milan group is thought to occur by an electret induced increase in the refractive index of cytoplasm. A similar concept was proposed (Hameroff, 1974) in which microtubules were thought to act like "dielectric waveguides" for electromagnetic photons. Living tissue does transmit light more readily than nonliving material. Van Brunt, Shepherd, Wall, Ganong and Clegg (1964) measured penetration of sunlight into mammalian brain by routes other than the visual system. Stereotactically placed photoreceptors recorded intensities of 10^-3 lumens in sheep hypothalamus when surface intensity was 0.4 lumens, with a logarithmic diminution. The most light permeable areas were in the temporal regions of the skull, lateral to the orbits; the brain's temporal poles and hippocampus received maximum light intensity. When the animals were sacrificed, light penetration to the hypothalamus remained constant for about 30 minutes following which the brain opacity rose sharply. This suggests that some property of living brain tissue is relatively translucent to optical photons; polymerized MT acting as waveguides may be such a property.

Hameroff (1974) also proposed that the periodic array of MT subunits "leaked," or diffracted energy with 8 nanometer periodicity, resulting in a source of "coherent" energy (or calcium ions) from each MT. Cytoplasmic interference of the coherent sources from among multiple MT would lead to holographic imaging in cytoplasm. Coupling of calcium concentrations to cytoplasmic sol-gel states could "hardwire" holographic patterns into the microtrabecular lattice. In parallel arrays of MT within nerve fibers, graded potentials or traveling action potentials were thought to collectively activate "planes" of cytoplasm perpendicular to the long axis of the MT and nerve fibers. These traveling planes may be likened to image screens as in TV sets. In a TV picture tube, the screen is motionless and electron beams move to create a picture by their intersection with the screen. Perhaps imaging within neurons occurs on traveling screens generated by action potentials moving through parallel MT arrays. The content of such images would depend on programming mechanisms in the conformation of tubulin subunits which comprise the MT walls and which update with each successive action potential. Hameroff and Watt (1982) described a method of MT tubulin programming in which charge carriers (calcium ions, electrosolitons) or conformational waves such as phonons or solitons were steered through MT lattices by genetically or cytoplasmically programmed tubulins and specific MAP binding sites. MAP bridges to other MT, cytoskeleton, or other organelles were thought to act as "sinks" or "sources" which conveyed pulse trains of charge/conformation among MT throughout the cytoskeleton as a regulatory and communicative medium. Hameroff and Watt (1982, 1983) likened MT to microprocessors in which switching in a "Boolean matrix" was determined by programming factors intrinsic to the tubulin subunits (Figure 8.9).

Figure 8.8: Interference patterns in cytoplasm caused by coherent waves (e.g. Ca++, sol-gel state, MTL) generated by dynamic activities in microtubules may be a basis for holographic information imagery.

Figure 8.9: Boolean switching matrix in microtubule lattice from Hameroff and Watt (1982). Alpha and beta tubulin subunits in the MT lattice wall were considered to be transiently occupied by quanta of charge/energy/conformation. MAPs behaved as "left switches" or "sink" or "source" proteins. Sequences and patterns of conformational states traveled through the MT lattice and via "sink" and "source" inter-MT bridges throughout the cytoskeleton somewhat like a pinball machine. The model demonstrated a capability for tubulin-MAP "programming" of dynamic activities related to biological intelligence.

In subsequent papers, Hameroff, Smith and Watt (1984, 1986) utilized principles of cellular automata to explain information processing in MT. As described in Chapter 1, cellular automata are dynamical systems which can generate and process patterns and information, and are capable of computing. Cellular automata require a lattice structure of "like" neighbors with discrete states and neighbor rules, and a universal "clock" to which all neighbors are timed. Adopting Fröhlich's model of coherent nanosecond dipole oscillations coupled to conformational states as a clocking mechanism, the authors calculated MT lattice neighbor Van der Waals dipole interactions as rules for an MT-automaton computer simulation. Each tubulin dimer was considered to be in one of two possible states at each nanosecond "generation." The two states were related to Fröhlich's concept of dipole oscillation so that the dipole can be oriented either toward the alpha tubulin end ("a," Figure 8.2.13) or toward the beta tubulin end (represented by a dot in the Figure). The polarity and electret behavior of MT indicate that in the resting state, tubulin dimer dipoles should be oriented toward the beta monomer.

Dimer states at each "clock tick," or generation were determined by neighbor states at the previous generation.

where n = 7 as the number of neighbors, and f(y) the force from the "ith" neighbor in the y direction.

.

The dipole state of any particular dimer at each clock tick thus depends on the summation of the dimer's neighbor dipole states (including its own) at the previous clock tick. The neighbor influences are unequal because of the screw symmetry of the MT lattice. Distant dimers (more than one neighbor away) would be expected to have little influence because of the dropoff in force intensity (y/r³) with distance. However, collective influences from many "like" oriented dimers could lead to long range cooperativity. Using only near neighbor influences, computer simulation of an MT automaton yielded interesting patterns and behavior of dipole/conformational states. These included both stable and traveling interactive patterns capable of computing and regulation of cytoskeletal activities. For example, Figure 8.13 shows a "kink-like" pattern traveling through an MT lattice, leaving an altered "wake," or memory. Assuming nanosecond generations, these traveling patterns would travel at 8 nanometers per nanosecond (80 meters per second), a velocity consistent with propagating action potentials, or solitons. Variability in individual tubulin dimer isozymes, ligand bind-ing, or MAP attachments could "program" and "read out" information in routine cellular functions. As one example, propagation of MT conformational patterns could coordinate the activities of contractile MAP sidearms in axoplasmic transport. In a general sense, MT automata may be the information substrate for biological activities ranging from ciliary bending to human consciousness. Specific automata patterns distributed throughout wide volumes of cytoskeletal arrays within the brain could lead to cooperative resonance and collective effects resulting in a thought or idea similar to the manner in which coherence induced phase transitions in metals yield emergent collective properties such as superconductivity.

Figure 8.10: Top: MT-tubulin dimer subunits comprised of a and b monomers. Relative electron occupancy of either monomer may correlate with coherent dipole oscillations in the nanosecond time domain. Bottom: screw symmetry hexagonal packing leads to unequal neighbor rules based on lattice distances and Van der Waals dipole interactions. For dimers shown at right, x = 5 nm, y = 4 nm, r = 6.4 nm. The relative strength of each neighbor dimer dipole state is y/r³.

Figure 8.11: 30 "nanoseconds" of computer simulation of MT cellular automaton based on Fröhlich coherent oscillations and tubulin subunit neighbor rules based on dipole-dipole interactions. 13 protofilaments are arranged horizontally, 64 rows are shown vertically yielding a flat MT lattice. "Alpha" states of tubulin (Figure 8.10) are shown by "a," beta states by a dot. 4 contiguous beta states are highlighted by diamond patterns, 4 contiguous alpha states by darkened dots. The simulation sequence (generations 40, 45, 55 and 70) shows movement of patterns at velocity of 80 nanometers per nanosecond (80 meters per second). With permission from Hameroff, Smith, and Watt (1984).

Figure 8.12: Continuation of MT cellular automaton sequence of Figure 8.11. Alpha and beta patterns collide and generate bizarre shapes, leaving configuration altered from initial state. Such interactions could represent rudimentary "computing" and "memory." With permission from Hameroff, Smith and Watt (1984).

Figure 8.13: Cellular automaton model of information processing in microtubules. A "kink-like" pattern moves from left to right. From Hameroff, Smith and Watt (1984, 1986).

8.3           The Cytoskeletal Connection

Microtubules, centrioles, and the cytoskeleton evolved to take command of biology. By being at the right size scale and moving in the right time scale, they appear capable of utilizing many forms of available energy. MT polarity, periodic structure, and helical lattice array of conformationally programmable subunits qualify them as "ultimate computers" and nanoengines as well, generating purposeful force and movement. They may capture and self focus electromagnetic radiation, induce coherency, and propagate energy quanta with minimal loss. Propagation may manifest as coherent polarization waves as described by Fröhlich, massless bosons as described by Del Giudice or as solitons as described by Davydov. Charge density waves or proton transfer at MT surface layers of ordered water, and positively charged counter ions such as calcium are other possible modes available for cytoskeletal based information and energy transfer.

Figure 8.14: "Kink-like" pattern continues left to right through MT automaton. Such dynamic patterns can represent and compute information, serve as binding sites for transported molecules, designate MAP and inter MT bridge attachment sites, or generate coherent waves of calcium coupled sol-gel states.

MT electron surpluses occur due to an abundance of acidic amino acids. With the intrinsic polarity of each tubulin dimer subunit, MT reside in a strong polar field or "electret" which possesses piezoelectric properties. Each MT subunit can thus "integrate," being capable of absorbing and sensing input in the form of acoustical energy, optical photons, chemical ATP, altered potential changes, fluxes of calcium and other ions, and responding with conformational changes accordingly. Each subunit within a microtubule lattice can not only represent information, but can input and output information into an ongoing automaton. Coherent oscillations in MT tubulin subunit states among regional forests of microtubules can provide a communicative medium in which any subunits which are out of phase induce waves of phase uncoupling along MT structure. Alterations which disturb coherency could be amplified by collective oscillations among cytoskeletal macroassemblies like rotatory centrioles or tensegrity structures of MT wound by contractile actin filaments.

Several other possible modes of information management present themselves in the structure of MT. Could the gaps between tubulin subunits act like pores? The relative abundance of electronegative charges in the outer surface of cylindrical microtubules may create gradients across tubule walls if the MT interior is neutral or positive. Presently unmeasurable, even a small gradient across 4 nanometer MT walls would comprise a significant voltage field. Thus a propagating soliton, charge density wave, or transient osmotic swelling of nerve processes could open "cracks" among the tubulin subunits to allow for ion flux or radiation of electromagnetic energy focused and trapped within MT. Alternatively, an electronegative MT interior core might support Del Giudice's suggestion of cytoskeletal superconductivity. Self-focused energy exerting force perpendicular to the long axis of MT could account for mysterious effects such as the perpendicular birth of daughter centrioles and inter-MT MAP bridges. Energy or ion fluxes radiated from MT due to propagating waves would be coherent due to spatial periodicity of the gaps between the tubulin subunits, and could thus form the basis of coherent wave interference in a three dimensional hologram. Coupled to calcium/sol-gel states, holographic cytoplasm may be an "infoplasmic" canvas for dynamic biological information.

In the brain, highly parallel arrangements of cytoskeletal proteins within asymmetrical axons and dendrites suggest analogies to parallel computing. Propagation of action potentials or dendritic depolarization waves could induce sequences of alterations of MT subunit states due to influx of calcium, alteration of electrical fields, or direct mechanical effects as sodium and water transiently swell the nerve process. Signals may cooperatively reverberate throughout the cytoskeleton by waves of calcium release and uptake, mechanical/electrosolitons propagating through the cytoskeletal lattice, and/or lateral propagation through sidearms and other components of the cytoskeletal network. Transient fluxes of calcium resulting from conformational waves propagating down cytoskeletal lattices in parallel with action potentials or dendritic current waves can result in traveling frames of dynamic imagery. Sequences of image frames traveling through holographic cytoplasm and changing with each nerve firing may collectively be the "Mind's Eye, the "grain of the engram."

The current prevalent model of brain function is the "neural network," a collective dynamical system whose output is determined by the states of component neuronal synapses. In turn, the state of each synapse is determined by other neurons modulated by collective dynamic effects of the interneuronal cytoskeleton. In turn, the cytoskeleton is a collective dynamical system whose net activity is determined by the states of its component subunits. Their states, in turn, are determined by another level of organization including cytoplasmic factors, ordered water and ions, genetic isozymes of individual subunits, and lower level cytoskeletal elements such as the microtrabecular lattice. Each cytoskeletal subunit is a computer capable of integrating multiple inputs to a specific output state. Cognitive processes which have been ascribed to a neural net concept may thus be more accurately interpreted as a fractal hierarchy of dynamical systems which are highly parallel, highly interconnected, and of increasing capacity as they become more microscopic. Collective effects may occur at each level and at successively more macroscopic levels. The net effect may be consciousness.

Microtubules and the cytoskeleton created their place in evolutionary history by being problem solvers, organelle movers, cellular organizers, and intelligence circuits. Where do they go from here?

9         Viruses/Ambiguous Life Forms

9.1           What Is the Essence of Living Matter?

Oparin (1938) proposed that living matter be defined as having the following properties: metabolism, self-reproduction, and mutability. Eigen (1971) observed that these criteria could be met by decidedly non-biological entities such as von Neumann's "self-reproducing automata," or various robots. Even some simple biological systems are somewhat ambiguous regarding their "life-like" status. Primitive systems which blur the distinction between animate and inanimate include viruses, prions (protein crystals which may be causative agents in some human diseases) and proteinoids (self organizing protein assemblies) implicated in life's origins (Fox, 1972). Independent cytoskeletal elements also have characteristics of being "alive." The slithering microtubules which R. D. Allen and colleagues (1985) isolated from squid axoplasm ambulate, avoid collisions, and have a sense of direction. Are they alive? If future technologies lead to nanoscale replicating automata, the distinction between living and nonliving entities will be further clouded.

9.2           Virus (Mis)Behavior

Among the more basic varieties of life are viruses. Their simple structure and activities have prompted questions as to whether they are actually "alive," or are merely chemical robots which multiply and reside within truly living organisms. Viruses form spontaneously as their simple subunits assemble into complex three dimensional structures. The assembly process, driven by the increased stability or lowered energy of the completed virus, flows against the second law of thermodynamics which dictates that order proceeds to disorder. Data from the tobacco mosaic virus (Chapter 6) show that hydrophobic interactions which exclude water from unassembled subunits offset the increase in order (negative entropy) of the assembled virus.

Figure 9.1: Phage virus landing and injecting genetic material into host cell. The cylindrical collar undergoes a collective conformational contraction associated with the injection. By Paul Jablonka.

Generally, viruses enter cells and pirate their genetic machinery to cause virus multiplication; they then escape to begin another cycle. Some viruses quietly and harmlessly reside within host cells for extremely long periods of time whereas the sojourn of other viruses involves the total commandeering of cell machinery and can have extremely damaging consequences for the host. Virus induced human diseases range from trivial common colds and influenza to chicken pox, measles, hepatitis, polio, and deadly diseases such as smallpox, rabies, yellow fever, cancer, and acquired immunodeficiency syndrome ("AIDS"). Some viral diseases such as smallpox have been controlled by modern medicine but others such as AIDS remain significant threats to human health.

Once assembled inside a host cell, viruses can escape in several ways. One route involves causing the death and disintegration of the host cell, allowing the newly formed viruses to spill out and carry their infection elsewhere. Viruses which are covered by lipid membranes can also escape by "budding" or reverse endocytosis ("pinocytosis"), a mechanism similar to secretion or extrusion of neurotransmitter vesicles from synaptic boutons.

An important subclass of viruses are retroviruses which contain RNA without DNA. When arriving within a host cell, the retrovirus brings an enzyme known as reverse transcriptase which converts the virus single stranded RNA into a double stranded DNA copy. This allows the virus to commandeer the host cell genetic machinery. Among the human retroviruses is HTLV-III (or "HIV"), carrier of AIDS.

9.3           Virus Structure and Collective Oscillations

Virus structure includes up to three major components: genetic material (either DNA or RNA), a protein coat which surrounds the genetic material, and a membrane (Figures 9.1 thru 9.3). One example of a viral protein coat is the cylindrical lattice similar to microtubule structure found in the tobacco mosaic virus, the first virus to be isolated (by Russian botanist Dimitri Ivanovski in the late 19th century). In adenoviruses, the protein coat is an icosahedron: a 20 sided polygon with 12 vertices. Influenza virus protein coats are spherical and may include outward extruding glycoproteins surrounded by lipid membranes. Another interesting viral structure is that of bacteriophages which actively inject their DNA into host bacterial cells. The bacteriophage looks somewhat like a nanoscale lunar lander and has an icosahedron head mounted by a collar onto a cylindrical tail which in turn is supported by a base plate which contacts a bacterial cell surface. Upon an appropriate stimulus, the bacteriophage virus protein coat undergoes a collective conformational event; contraction of the entire tail assembly results in the active injection of viral DNA through the bacterial cell wall and into the bacterial cell cytoplasm. This sequence of events requires a coordinated communication and active response and may be construed as a rudimentary intelligence.

Figure 9.2: Icosahedral structure of an adenovirus protein coat. By Paul Jablonka.

Figure 9.3: Longitudinal view of the DNA path in a tobacco mosaic virus structure. The path takes about 120 complete turns. By Paul Jablonka.

How do viruses perform cognitive acts such as sensing host cell surfaces and injecting into them their nucleic acids? One possibility is by perturbations of collective vibrations of their protein shells. Michels, Schulz, Witz, Pfieffer, and Hirth (1979) measured ultrasonic absorption of protein assemblies over a range of 10⁵ to 10⁹ oscillations per second. They found the assembled structures absorb much greater ultrasonic energy than do the individual unpolymerized subunits, suggesting a collective oscillation of the protein assembly. For example, the intact icosahedral Brome mosaic virus absorbed energy with a mean frequency of 5 x 10⁷ per second. Robach, Michels, Cerf, Braunwald and Tripier-Darcy (1983) found similar results with frog virus, and determined that the displacement amplitude of the oscillations were on the order of a tenth nanometer. Most virus research has focused on the genetic capabilities of viral DNA or RNA in coopting host cell activities. The protein coats have been considered as simple environments for DNA or RNA transport, however they perform functions analogous to organized cytoplasm. For example, the glycoprotein spikes which extend outward from some viruses while remaining anchored to the protein coat interact with the host membrane and cell wall surfaces to facilitate contact and entry. This general activity involves information signaled through the protein coat enacting a mechanical conformational change. Michels, Dormoy, Cerf and Schulz (1985) used similar techniques to study two strains of tobacco mosaic virus as well as other protein assemblies including microtubules. They observed that the fluctuations depend on specific organization and complexity of the assembly, and that certain assemblies oscillate more than others. They speculate that collective oscillations of virus coats could function in communicative functions such as the injection of viral nucleic acids into host cells. Viruses perform limited functions devoted to their own replication and survival, but their collective oscillations could be a clue to the essence of living matter. Collective oscillations of cytoskeletal proteins could serve a more versatile communicative role in all eukaryotic cells.

9.4           Nature and Origin of Viruses

Deciding whether or not viruses are alive depends on a definition of life. In his book Pirates of the Cell, Scott (1985) concludes that life is a spectrum of animate to inanimate material, and that viruses are somewhere in the middle. Some vital characteristics of living things include locomotion, nutrition, growth, respiration, excretion, sensitivity and reproduction. By these criteria viruses are not alive, however "inanimate" materials like crystals do manifest life-like growth, nutrition, reproduction, and locomotion. Defining life by the apparent ability to defy the second law of thermodynamics (creating order from disorder) by assembling complex organized biological structure from the disordered nonliving world ignores the fact that the thermodynamic law remains inviolate overall. Molecular level cytoskeletal protein subunits or viral particles assemble into more highly ordered structures, however the hydrophobic exclusion of water from protein subunits counteracts the change in entropy and the net effect remains order proceeding to disorder.

There are three general theories as to the origin of viruses (Scott, 1985). The first is that viruses originated very early in evolution before the advent of eukaryotic cells. According to this view, modern viruses are direct descendants of primitive early molecules floating in the primordial soup or mud (Chapter 3). The second idea is that they are derived from parasites which invaded other cells and gradually became simpler, de-evolving to be totally concerned only with survival and multiplication. The third notion, which dominates current beliefs, is that viruses evolved from genetic material of cellular life: the "escaped gene" hypothesis.

9.5           Domesticated Viruses

The capabilities of viruses may be harnessed (Scott, 1985). Two hundred years ago Edward Jenner began to develop safe and effective anti-viral vaccines, a technique which amplifies the body's immune system. Slopek and co-workers (1983) have treated patients afflicted with drug resistant bacterial infection by using viral bacteriophages selected for their effectiveness against the resistant organism. British scientists (Williams, Smith and Huggins, 1983) have used bacteriophages to treat intestinal infections in animals and direct use of viruses to combat bacterial infections in humans have also been attempted. Bacteriophages have potential advantages over modern drugs: they are highly specific, can leave the host cells unharmed with minimal side effects, and could be produced inexpensively. However, bacteria could develop resistances to bacteriophages as they do to some drugs.

Other researchers have used exploited viruses to mass produce natural proteins in "genetic engineering." For example, the "Epstein-Barr" virus has been used to transform selected immune cells which then multiply and produce large quantities of specific antibodies useful in medicine and industry. In some cases viruses are changed into novel forms for use as live vaccines. Genes for influenza and hepatitis B organisms can be combined in a vaccine virus genome to protect us against diseases such as hepatitis B and the flu. Genes encoding proteins of parasites such as the protozoan that causes malaria are being added to suitable viral genomes (Smith, 1984). Viruses can transfer foreign genes into bacterial cells. Gene coding for any wanted protein can be linked up to the genetic material of a virus and, when the virus infects suitable bacterial cells, the foreign gene is carried in with it. Once inside, the transferred gene may then begin to direct the manufacture of plentiful supplies of the protein it codes for. Viruses are thus being turned into versatile ferrymen which can carry a whole range of proteins into humans and livestock.

Other exciting possibilities include transferring new genes into human cells. Many research groups including Richard Mulligan and cohorts at MIT (Kolata, et al., 1984) are trying to construct viruses that will carry new genes to human cells to cure genetic diseases. A number of hereditary diseases could thus be treated by genetic manipulation using bacteriophages. A first step would be having a retrovirus insert a therapeutic gene into the DNA of cells deficient in that particular gene. A promising area might be diseases of blood cells which proliferate continuously. Related ideas are to use membranes, virus protein coats, or other proteins to transport drugs to immuno-targeted intracellular destinations ("magic bullet").

Unfortunately, viruses may also be utilized as diabolical weapons, capable of infecting large populations. However like other technological "double-edged swords," their potential benefit is even greater. Nanotechnology combined with genetic engineering could lead to virus-like entities which could stalk and destroy lethal infectious agents, malignant cells, atherosclerotic plaques which obstruct blood vessels, scar tissue which limits nerve regeneration, neurofibrillary tangles associated with senile dementia, and perhaps other diseases. Future virus-like "nano-doctors" may be making cellular house calls.

10   NanoTechnology

Functional communication among biomolecules and technological devices will require dramatic advances in our abilities to fashion logic devices from matter. The attainable limit appears to be computer components precisely structured at the atomic level, on the order of 0.1-0.3 nanometers. Nanoscale fabrication of information devices capable of biological interfacing would also enable construction of valuable nanoscale robots, sensors, and machines (Schneiker, 1986). Ultimate computing may be but a single facet of a wide range of applications: "nanotechnology."

10.1     Early NanoTechnologists

The American Physical Society held its 1959 annual meeting at the California Institute of Technology. Scheduled to speak was a man who would win the 1965 Nobel physics prize for his historic work in quantum electrodynamics. Still later he would serve on a Presidential Commission and find the fault in the Challenger disaster in a disturbingly brief period of time. In 1959 however, he spoke of other work that may be even more important. In his talk entitled There's Plenty of Room at the Bottom, physicist Richard Feynman (1961) proposed a simple and straightforward strategy for constructing useful structures ranging in size down to the atomic scale! He suggested using machine tools to make many more sets of much smaller machine tools, which would in turn make many times that number of other, even smaller machine tools, and so on. At the lowest level, he noted the possibility of mechanically assembling molecules, an atom at a time.

Feynman proposed the construction of nanomachines, nanotools, tiny computers, molecular scale robots, new materials and other exotic products which would have far reaching applications and benefits. Considering the relevance of nanotechnology to living molecules, Feynman (1961) noted,

A biological system can be exceedingly small ... . Consider the possibility that we too can make a thing very small, which does what we want-that we can manufacture an object that maneuvers at that level!

Machines able to directly manipulate matter on the submicron to nanometer size scale are referred to as "Feynman Machines" (FMs). The only existing FMs currently known are biological, however computer controlled or teleoperated FMs may in the future implement a broad range of nanotech applications.

Following Feynman's lead, other scientists delved into nanotechnology. Von Hippel (1962) predicted dramatic material science possibilities if new advances in "molecular designing" and "molecular engineering" of materials could be achieved. Noting the eventual possibility for repairing human tissue (molecule by molecule if necessary) for life extension, Ettinger (1964) suggested repair machinery for modification and interaction with existing organisms; later he proposed development of nanorobotics. Ettinger envisioned nanoscale scavenger and guardian organisms designed to emulate and surpass the actions of white blood cells which might hunt down and clean out hostile or damaging invaders (Ettinger, 1972). Such nanorobots might be useful for fighting AIDS or cancer, excavating blocked blood vessels, or straightening neurofibrillary tangles in senile neurons.

Shoulders (1965) reported the actual operation of micromanipulators able to position tiny items with 10 nm accuracy while under direct observar tion by field ion microscopy. Ellis (1962). also developed similar (but much larger) micromanipulators and proposed the construction of "microteleoperators": remote controlled nanodevices! Drexler (1981, 1986) has described some advantages and hypothetical dangers of nanotechnology. Capabilities for atom-by-atom assembly and nanoengineering could lead to new materials and pathways (Feynman, 1961). One such material is "diamond-like carbon" films which are "transparent, insulating, chemically inert, have a high dielectric strength, good adhesion and are relatively hard" (Aisenberg, 1984). Drexler suggests that rotary hammers a few molecules long might be used to hit carbon atoms in graphite at just the right angle and force to create lightweight diamond films and fibers useful in a variety of material applications. Drexler warns of two Frankenstein aspects to nanotechnology: nanosensor surveillance, and uncontrolled replication of nanodevices with consumption of biosphere resources. However, the existing, dramatic developments in microsensor technology and microelectronics render worries about nanosensor surveillance superfluous (Schneiker, 1986). Schneiker also discounts Drexler's extreme "end of the world" worries about nanoreplicators, pointing out that the Feynman machine (top-down) approach to nanotechnology, microreplicators and other factors obviate the problem. Nonetheless, like genetic engineering, nuclear power, the automobile, and junk food, nanotechnology may well be a "double edged sword" which demands responsible management.

Potential benefits from nanotechnology attainable in perhaps a decade or two might include (Schneiker, 1986):

... vastly faster, much more powerful and numerous computers with extremely large capacity memories, ultrastrong composite materials, greatly improved scientific instrumentation, microscopic mobile robots, and automated flexible manufacturing systems, replicating systems, and achieving the practical miniaturization limits and maximum performance in virtually every area of technology.

Despite these lofty hopes and predictions, nanotechnology and molecular computing have remained mere dreams. Obstacles to their implementation center on the absence of available Feynman machines. A feasible solution has been advanced by a present day nanotechnologist whose contributions may eventually eclipse all others. Conrad Schneiker (1986) may have found the bridge to the nanoscale. He predicts that atomic level manipulative capabilities embodied in a 1981 invention, the scanning tunneling microscope (STM), can implement nanoscale Feynman machines (Hansma and Tersoff, 1987). Schneiker had noted that even without the STM, and before its invention, silicon micromechanics combined with other technologies could have been used. Not content with two approaches, he also proposed another route based on augmented machine tool technology originally developed for single crystal diamond machinery. But STMs are much more convenient and much less expensive.

10.2     Scanning Tunneling Microscopes (STMs)

Figure 10.1: Scanning tunneling microscopy (STM) depends on ultraprecise, but rapid movement of a sharp, single atom tip over a surface. The movement is controlled by X-Y-Z direction piezoceramic arms which move fractions of nanometers in response to voltage changes. By Paul Jablonka (Schneiker and Hameroff, 1987).

Figure 10.2: Nanoscale view of substrate to STM tip electron tunneling. By Paul Jablonka (Schneiker and Hameroff, 1987).

Table 10‑1: STM operating modes; i, v, h are tunneling current, voltage, and height (tip to sample surface distance) respectively (Schneiker, 1986).

The 1986 Nobel Prize for Physics was split between the 50 year old cornucopia of microknowledge, the electron microscope, and a brand new, unfulfilled technology, scanning tunneling microscopy (STM). The STM half of the Prize was awarded to Gerd Binnig and Heinrich Rohrer of the IBM Zurich Research Laboratories where STM was invented in 1981 (Binnig, Rohrer, Gerber and Weibel, 1982). The principle used in STM is extremely simple (Figure 10.1). Piezoceramic materials expand or shrink extremely small distances (i.e. angstroms, or tenth nanometers) in response to applied voltage. In STM, piezoceramic holders scan an ultrasharp conducting tip (such as tungsten) over a conducting or semiconducting surface (Figure 10.1). When the STM tip is within a few angstroms of a surface, a small voltage applied between the two gives rise to a tunneling current of electrons (Figures 10.2, 10.3 and 10.4). The tunneling current depends exponentially on the tip-to-substrate separation (about an order of magnitude per angstrom or tenth nanometer). Depending on the substrate, typical tunneling currents and voltages are on the order of nano-amperes and millivolts, respectively. A servo system uses a feedback control that keeps the tip to substrate separation constant by modulating the voltage across a piezoelectric positioning system. As the tip is scanned across the surface, variations in this voltage, when plotted, correspond to surface topography. Detailed surface topography maps of various materials have been obtained which demonstrate individual atoms like cobblestones. By varying and measuring different combinations of tunneling current, voltage, and distance, different types of information may be obtained about the surface being probed.

The basic modes of operation of an STM, described by Hansma and Tersoff (1987), are summarized in Table 10.1. Here i, v, and h are the tunneling current, the voltage across the gap, and the gap size respectively. Mode I is used to measure the topography of the surface of a metal or semiconductor and is the slowest mode since the electro-mechanical servo system must follow the shape of the surface during the scanning operation.

Figure 10.3: Multi-directional movement of STM tip can be controlled by single piezo-tube scanner. By Paul Jablonka (Schneiker and Hameroff, 1987).

Figure 10.4: Schematic of STM from Tunneling Microscope Corporation (Doug Smith). T = tip, S = sample stage, PZT = piezoceramic tube, CAM = coarse adjust micrometer, FAM = fine adjust micrometer, LRS = lever reduction system. By Conrad Schneiker (Schneiker, 1986).

The scanning speed in Mode I is determined by the response of the servo system. Modes II and III are faster since the tip maintains only a constant average height above the surface. The scanning speed in these modes is determined by the response of the preamplifier only. Mode IV measures the joint density of states which, for a small tip, is a map of the local distribution of electron states of the substrate. For this mode, one "dithers" the tip-to-substrate bias voltage with a small alternating current signal and monitors i/v. Using this mode, Smith and Quate (1986) have done spatially resolved tunneling spectroscopy and observed charge density waves. STM can thus be used to identify the elements comprising specific atoms and to monitor molecular dynamics (Figure 10.5).

Figure 10.5: STM scan with t-axis showing evolution of a nanosystem over time. With permission from Ritter, Behm, Potschke and Wintterlin (1987), courtesy of Eckhart Ritter.

Adaptability and versatility have been shown by STMs operated in air, water, ionic solution, oil and high vacuum (Drake, Sonnenfeld, Schneir, Hansma, Solugh and Coleman, 1986; Miranda, Garcia, Baro, Garcia, Pena and Rohrer, 1985). Their scanning speed may be pushed into the real-time imaging domain (Bryant, Smith and Quate, 1986). One technique for machining STM tips is based on a simple ion milling process that can generate ultrasharp tips with single atom points; a similar technique can generate ultrasharp knife edges and other nanotool shapes as well (Dietrich, 1984). Dieter Pohl (1987) of IBM-Zurich considers STM one example of a group of "stylus" microscopic technologies. Tips are being developed as thermocouples of two different metals sensitive to extremely low changes of heat, and hollow pipettes able to administer or detect individual molecules in solution. Pohl refers to these STM capabilities as molecular "tasting and smelling." Another STM related function can "touch and feel." In "atomic force microscopy," a nanoscale lever positioned by STM piezo-technology is deflected by contact and/or movement of atoms, molecules or their surrounding ions. Van der Waals force induced lever deflection is monitored by an STM tip or by interferometry. Binnig, Quate, and Gerber (1986) propose to measure forces as small as 10-18 newtons using atomic force microscopy. Mechanical shape and structural dynamics can be probed without tunneling through the sample material using this mode, which may be particularly important in the study of biomolecules.

Figure 10.6: Formation of near-field optical nano-apertures. By Conrad Schneiker (Schneiker, 1986).

Another STM spinoff may lead to "nanovison." Resolution of "nearfield microscopy" depends on the diameter of the aperture through which the reflected light passes. Binnig (1985) described how to use an STM to make 20 nm holes in opaque metal films which, when used in scanning light microscopes, become capable of resolving features much smaller than the wave-length of the light. In general, a cone of light cannot be focused to a spot which is significantly smaller than the light's wavelength. However, light passed through an aperture that is many times smaller in diameter than its wavelength results in a narrow beam of light that may be scanned mechanically close to a sample being examined. STM technology is capable of creating nanoapertures, and also the scanning mechanism required for imaging an area (Figure 10.6). Pohl, Denk and Lanz (1984) described their "optical stethoscope" in which "details of 25-nm size can be recognized using 488-nm radiation." A significant advantage of the optical stethoscope concept is "that it allows samples to be nondestructively investigated in their native environments" (Lewis, Isaacson, Harootunian and Muray, 1984). Betzig and colleagues (1986) conclude that this technology will be "able to follow the temporal evolution of macromolecular assemblies in living cells ... to provide both kinetic information and high spatial resolution." Scanning nanoapertures could also use ultraviolet, X-ray, gamma ray, synchotron, or particle beams to "snoop" in the nanoscale if suitable materials and configurations are found (Schneiker, 1986).

Schneiker's breakthrough was to realize that STM tips can be used as ultraminiature, ultraprecise robot fingers that can both "see" and be used to directly manipulate individual atoms and molecules along the lines suggested by Feynman. The scaling down process proposed by Feynman (machines building smaller machines, and so on) can be reduced to just one step! According to Schneiker's concept, STMs can directly link up to the nanoscale to implement, construct, and evaluate Feynman machines and other nanotechnologies. He notes that many natural biomolecules such as enzymes, t-RNAs, antibodies, cytoskeleton, and many synthetic compounds provide a wide variety of potential finger tips to realize Feymans vision of molecular manipulation. Rudimentary nanomanipulation has already been described. Ringger and colleagues (1985) have described STM nanolithography: atomic scale surface etching. Becker, Golovchenko and Swartzentruber (1987) of Bell Labs have reported a single atom modification of the surface of a nearly perfect germanium crystal by the tungsten tip of an STM. They conclude that manipulation of atomic sized structures on surfaces will lead to "new frontiers in high density memories, new devices whose electrical properties are dominated by quantum-size effects, and modification of single, self replicating molecules,"-supporting the earlier predictions of Schneiker. In its first few years, STM has imaged single atoms, probed atomic forces, manipulated atoms, and won a Nobel prize. Eventually STMs and their spinoffs may enable researchers to alter and communicate with genes, viruses, proteins, cytoskeletal assemblies, and perhaps biomolecular consciousness.

Figure 10.7: Nanotechnology workstation is envisioned as a synergistic combination of multiple STM tips mounted in an optical microscope. By Paul Jablonka (Schneiker and Hameroff, 1987).

Figure 10.8: Multiple tip geometry for STM probing of nanoscale systems. By Conrad Schneiker (Schneiker, 1986).

Schneiker and Hameroff (1987) have proposed an integrated system of STM and other technologies to best take advantage of STM capabilities. The "nanotechnology workstation" (Figures 10.7 and 10.9) proposes to combine multiple STM tips, optical microscopy, nanoapertures, and fiber optic waveguides in a hopeful attempt at synergism. For example, the STM is extremely "near-sighted;" it can have difficulty finding its target ("a needle in a haystack"). If integrated into a high powered light microscope, the microscope can act as a coarse approach mode to bring the STM tip to the desired area. From there the STM can perform as a "cursor" mode for the microscope: able to expand the view of, and manipulate, atoms and molecules. In one configuration of the nanotech workstation, four STMs are placed symmetrically about a central STM and these each form a 45 degree angle to the sample plane; they may later be augmented with fiber optical interferometers for precise spatial coordinate determination and motion control (Figure 10.8). The central STM may be used for high speed scanning while the peripheral STMs are used as oblique STM scanners, nanoaperture scanners, electrodes, mechanical "fingers" or optical frequency antennas. In addition, four other micrometer-adjusted mini-arms can carry horizontal optical fibers: two for general illumination and two for experimental interfaces.

Reflecting optical microscope objectives may be positioned such that the optical axis is perpendicular to the plane of the opposite pair of STMs, and the focus is where their tips would meet when fully extended. The optics of these microscopes would permit them to peer around the fiber optic illumination system mounted on the forward axis of each microscope. The optical system would be achromatic, and able to work in far infrared to ultraviolet which should lead to increased optical resolution. Fluorescent coating for STM tips (excluding the last few nm) can aid in visualizing their location. The final imaging system should consist of solid state detectors whose output may be displayed on high resolution color monitors: STM modes, optical microscope, nanoaperture "nanovision," etc. (Figure 10.9 and 10.10).

Figure 10.9: Graphic displays, monitors and control module for nanotechnology workstation. By Paul Jablonka (Schneiker and Hameroff, 1987).

Figure 10.10: View of four STM tips approaching sample stage in nanotechnology workstation. By Paul Jablonka (Schneiker and Hameroff, 1987).

Figure 10.11: Molecular movement and construction with an STM. By Conrad Schneiker (Schneiker, 1986).

A system akin to the STM nanotech workstation may dynamically observe and manipulate nanoscale materials and systems, capabilities consistent with Feynman's notions for molecular machines.

10.3     STM/Feynman Machines (FMs)

STMs utilized to implement Feynman's ideas concerning atomic and molecular level fabrication and machining (Feynman machines: "FMs") have been dubbed by Schneiker as "STM/FMs."

Feynman had noted the possibility of doing chemical synthesis mechanically. In an effort to move in this direction, Schneiker (1986) proposed the following STM experiments (Figure 10.11). 1) Use an STM tip to move a pair of adatoms (or molecular fragments) on a substrate (or STM tip) together so as to induce chemical bonding. 2) Use an STM tip to cleave a chemical bond of a molecule adsorbed on a substrate. 3) Use an enzyme or synthetic catalyst adsorbed on an STM tip to repeat experiments (1) and (2).

Figure 10.12: Nanomilling/nanolithography.          By Conrad Schneiker (Schneiker, 1986).

Perhaps the first step in such ambitious proposals has already been accomplished. The Bell Labs group (Becker, Golovchenko and Swartzentruber, 1987) used an STM tip to place a single atom on a germanium surface. Further STM/FM modifications and applications elucidated by Schneiker (1986) include: 1) tip shape modifications (scalpel, chisel, cylindrical, and other configurations) for scanning, scribing, etching, milling, and polishing operations or for electrical interfaces, electrochemical synthesis or machining, 2) attached tip structures (enzymes, synthetic catalysts, shape selective crown ethers, transducer molecules, etc.) for molecular recognition with species selective (and perhaps electrostatic or electromagnetic assisted) pick, place, join, and cleave operations, or nano-environmental sensing, 3) multiple tip configurations (parallel or radial configurations) for use as ultraminiature tweezers, jigs, and arcs or interface electrodes, or to generate rapidly rotating electric fields, and 4) tip materials modification (insulating, semiconducting, ferroelectric or ferromagnetic) for electrostatic, electromagnetic, magnetic, kHz-GHz acoustic (longitudinal, transverse, or torsional) and optical modulation in mono-or multi-polar configurations (Figures 10.12 and 10.13).

Figure 10.13: Nanoscale view of STM nanomilling/nanolithography. By Paul Jablonka (Schneiker and Hameroff, 1987).

In addition to the above modifications, STM/FMs or their tips could be augmented with a wide variety of sensors and transducers; the atomic force microscope of Binnig, Quate and Gerber (1986) is an excellent example. The augmentation of STM/FMs with fiber optic interferometers (or comparable techniques) could provide extremely accurate realtime calibration of absolute and relative STM/FM tip positioning, thus overcoming the problems of electrical noise, creep, ageing and hysteresis inherent in present STM piezo-positioning systems. The technique given in Dietrich, Lanz and Moore (1984) for making tips with uniform tip-to-base conical profiles would be useful for STM/FMs using closely spaced multiple tips. Schneiker conjectures that properly configured and instrumented sets of STM/FMs can operate as machine tools with effectively perfect lead screws and bearings. STM/FMs also can be used as sub-atomic resolution proximity detectors and coordinate measuring machines on conducting surfaces (AFMs would be used for insulators) to monitor nearly perfect superaccurate nanomachining operations (limited by the graininess of atoms and other materials science considerations). Many useful macroscopic mechanical structures and mechanisms may thus be duplicated at the submicron level, and many of these mechanisms may require no lubrication due to force/area scaling and very rapid heat dissipation (Feynman, 1961). For even smaller mechanisms, a switch to Feynman's mechanical chemistry approach would be needed to build up molecular devices in a series of joining and trimming operations. Many presently existing synthetic molecules could be utilized as building blocks; the molecular gear and bearing system of Yamamoto (1985) provides an interesting example and is thought to be capable of rotating about a billion times a second.

STM/FMs may execute many complex mechanical motions for driving such nanomechanisms (Schneiker, 1986). For instance, an essentially infinite series of three dimensional tip motions (single straight lines, circles, spirals, helices, etc.) can be made at speeds presently limited mainly by mechanical resonances in the driving system of current STMs-another motivation for miniaturizing them considerably. Thus it would be possible, for example, to turn a molecular scale mechanical crank (say of 2 nm diameter) at thousands of revolutions per minute. Similar to flagella and cilia of single cell organisms, these could be used for propulsion of nanoscale robots and other uses.

The capabilities of STM/FMs have been heretofore nonexistent; therefore applications may abound in the near future as scientists in many disciplines become aware of this versatile and inexpensive technology.

10.4     Micro/Nano STM Contest

Feynman's original (1961) proposal for molecular and atomic scale machines included prizes and competition to "get kids interested in this field." He offered $1000 to the first person to a) reduce the information on the page of a book to an area 1/25,000 smaller in linear scale to be read by an electron microscope, b) construct an electric motor which is only 1/64 inch cube.

The latter prize was presented by Prof. Feynman on November 28, 1960 to William McLellan, who built an electric motor the size of a speck of dust (Gilbert, 1961). The former prize was awarded in 1985 to Tom Newman, a Stanford graduate student who used electron beam lithography to reduce a page from A Tale of Two Cities to 5.9 x 5.9 micrometers and magnified it back using an electron microscope.

Motivated by a desire to accelerate development of STM derived technology as a bridge to more powerful Feynman Machines, Schneiker (1985, 1986) has announced a series of construction challenges and prizes (Hansma and Tersoff, 1987). The challenges are to construct, operate, and publicly demonstrate STMs (including the active/moving part of the mechanical positioning and scanning systems) which can be controlled from the outside, and which (not counting lead-in wires, fiber optics, etc.) are of the following sizes or smaller: (1 mm)³, (100 µm)³, (10µm)³, (1 µm)³, (100 nm)^s, and (10 nm)³. The imaging capability should be comparable to that indicated by the picture on page 482, vol. 56 of the physics journal Helvetica Physica Acta, 1983. The two imaging tests will be: a) imaging a (10 nm)² square highly ordered pyrolytic graphite surface, and b) imaging the entire conical surface of another atomically sharp (nonrotating) tungsten STM tip (forming a 60° cone or less), extending 2 nm from that tip. Successive scans must be made to demonstrate that your device doesn't modify these test items. An additional pair of challenges are 1: to use an STM/FM to a) mechanically synthesize 10 molecules of either of the proposed spherical C₆₀ or C₁₈₀ molecules known as "Buckminster Fullerene" (Schneiker, 1986) and to b) demonstrate conclusively that the synthesis was successful, and 2: to use an STM/FM to a) mechanically synthesize a 10 nm x 10 nm layer of graphite, and to b) demonstrate conclusively that the synthesis was successful. The current prizes being offered, one per category, are $1000, with a maximum of one prize being paid out per year. Potential winners should contact the author. Additional sponsors and prizes are solicited. And as Feynman originally advised:

... have some fun! Lets have a competition between laboratories.

10.5     STM/FMs and Molecular Computing

The ability to use multi-tip STM/FMs for mechanical chemistry and as electrical interfaces might solve two formidable problems in the development of molecular computing devices as envisioned by Carter (1983): 1) the synthesis of prototype devices and 2) making individual, reliable, electrical connections to them for testing.

Arrays of nanoscale electrodes or STM tips comprising sub-micron scale assemblies may be extremely useful if their position can be precisely and rapidly regulated. Schneiker (1987) has suggested that optimal utilization of piezo-ceramic materials may be used for parallel scanning of nanoscale arrays. The piezo-ceramic "biomorph-bender" described by Dieter Pohl and colleagues (1986) 'may be used to manipulate such arrays for fast parallel reading and writing of nanoscale array chips for mass storage and processing of information (Figures 10.14 and 10.15, Schneiker, 1987).

STM/FMs could be used for more conventional ultraprecise circuit or component trimming and repair operations. The extremely accurate positioning systems of STM/FM could be exploited for minimal scale wirebonding systems. Other STM derived applications could include inspection of, and electrical and mechanical interfacing to, superlattice quantum well devices or "quantum dot" devices on the scale of cubic nanomenters. In 1959 Feynman noted that using his strategy it should eventually be possible to make what are now called superlattices and that they would probably have some very interesting properties (Feynman, 1961). Even though STM/FMs may not ultimately be used to mass produce such devices, they could be used to construct prototypes, help characterize test devices, and be used to optimize them. STM tip induced sputtering (Binnig, Gerber, Rohrer and Weibel, 1985) might be used to etch or mill ultrafine conductors for such devices. The availability of even small numbers of such extremely high performance devices would have great instrumentation value as interfaces and transducers (Figures 10.16 thru 10.19).

Computing components each consisting of a few atoms might use quantized energy levels or spin effects (Feynman, 1960); if such devices could be designed and assembled, they could be thousands of times faster than conventional devices used in today's computers. Other aspects of quantum computers are discussed in Maddox (1985), and other interesting references may be found in Schneiker (1986). Efficiently interfacing these and other such devices to the outside world in a manner that can effectively utilize their potential computing bandwidth presents some interesting problems in clocking, input/output, and other areas. Schneiker claims that the optical electronics technology suggested by Javan (1985, 1986) might be useful in these and other nanoapplications as well. Using micro and nano-antennas coupled to laser radiation, intense, localized high frequency electric fields modulated by the laser beam's intensity, phase, and polarization could control and power arrays of STM/FM constructed nanoscale devices. This same idea could be used to control, power, and communicate with swarms of mobile nanorobots.

Figure 10.14: Computer mass storage for fast parallel reading and writing "nano-film" coated chips. Ultimately, electrodes could be replaced with micro-STMs (Figure 10.15) for accessing individual molecular devices. A simple system might also be configured for highly parallel nanolithography. By Conrad Schneiker (Schneiker, 1986).

Figure 10.15: A micro-STM formed on a silicon substrate; thousands of these structures may be placed on a single silicon chip. By Conrad Schneiker (Schneiker, 1986).

Figure 10.16: Three STM tips in configuration for tunnel modulation experiment. By Paul Jablonka (Schneiker and Hameroff, 1987).

Figure 10.17: Electrochemical tunnel switch/atomic memory device. By Conrad Schneiker (Schneiker, 1986).

During recent talks on his quantum computing ideas, Feynman briefly speculated on a simple possibility for making nanocomputer components: use STM tips to make tiny holes in very thin metal sheets, thus forming grids for tunneling "nanovacuum tubes," perhaps around 3 to 10 nm in size, or smaller (Feynman, 1985, 1986). Analogous, but much larger (down to 100 nm scale) devices with calculated picosecond range switching speeds have been proposed by Shoulders (1965). Although he considered even smaller and faster devices, limitations of electron beam micromachining technology at that time prevented further size reduction (Shoulders, 1986). STM/FMs could solve that problem and many others. Indeed, the Naval Research Laboratory is now studying subpicosecond (thousandth nanosecond) submicron vacuum tubes (Robinson, 1986).

Figure 10.18: STM-induced/detected molecular conformation change. By Paul Jablonka (Schneiker and Hameroff, 1987).

10.6     STM/FMs and Biomedical Applications

Nondestructive STM interactions with biological material have immense potential, assuming certain technical obstacles are overcome. Potential problems include the following:

1. Biomolecules, cells, and tissues are not conductors. Electron tunneling through these materials, if it occurs, may be damaging. Nevertheless, several groups including IBM Zurich have succeeded in imaging biomaterials in air such as protein coated DNA and virus structures. The tunneling is thought to occur from tip onto biomolecular surface followed by "low resistance electron transport to the conducting substrate" (Travaglini, Rohrer, Amrein and Gross, 1986). Simultaneous optical microscopy, as can occur with the nanotech workstation, may help this situation since photons can lower tunneling barriers. Appropriate choice of optical microscopy wavelengths may thus facilitate STM imaging by permitting non damaging tunneling currents.

An alternative approach is to utilize an atomic force microscope (AFM) mode of operation for biological materials. In this case a lever arrangement adapted to, and mounted on, an STM which trails along the surface, or is held steady to observe mechanical dynamics of the material (i.e. protein conformational change). The movement of the lever is monitored by the STM, so that mapping and dynamics can be observed without direct tunneling through the biomaterial. Nanoscale thermocouple and pipette STM tips as described by IBM's Pohl (1987) may also be useful for biological materials.

2. Biomaterials should be studied in a stable environment as much like the aqueous medium within cytoplasm as possible. Temperature, pH, ionic concentrations, availability of high energy phosphate groups and numerous other parameters need to be closely regulated. Researchers at University of California-Santa Barbara have demonstrated that STM imaging can occur at ionic liquid-solid interfaces. By insulating STM probes to very near their tips, significant leakage of current to the ionic aqueous environment is apparently avoided (Sonnenfeld and Hansma, 1986). Thus STM should be applicable to characterization of living biomolecules under stable physiological conditions.

Figure 10.19: STM tunneling switches modulated at optical frequencies with lasers. By Conrad Schneiker (Schneiker, 1986).

3. A third problem has been the "near sightedness" of the STM. Hansma's Santa Barbara group and the IBM-Zurich team had difficulty in locating DNA molecules on the background substrate with the STM. This "needle in a haystack" problem is potentially solved by a nanotechnology workstation configuration in which the coarse approach of the STM is made simpler by visual observation. Further, immunofluorescence techniques may be utilized to identify specific biomolecular targets and the STM probes themselves. Electophoretic fields applied to the sample stage may attract and immobilize charged biomolecules rendering them easier to locate and study (Lindsay, 1987).

Potential applications of STM to biology and medicine include repetitive imaging and structural mapping of living material, nondestructive study of dynamical structural changes such as protein receptors (via alterations in the AFM mode), detection of possible propagating phonons or solitons (using the multiple STM configurations, one tip can perturb and another detect perturbation at a second position on a macromolecule or polymer), spectroscopic analysis (i.e. DNA base pair reading, cytoskeletal lattice communication), and real time imaging (via high speed scanning) of living structures can expand the horizons of experimental biosciences. Further, a capability for nanoscale manipulation of biomaterials and organelles offers a host of imaginative possibilities. For instance, a few dozen multitip STM/FMs might be developed to directly extract, manipulate, analyze, and modify DNA or other cellular components, thereby greatly assisting some subfields of genetic engineering and medical research. Synthesis or modulation of important biological molecules which are "topologically complex" (e.g. receptors, enzymes, antibodies, cytoskeleton) which may be difficult and relatively expensive using present day synthetic chemistry, could ultimately be feasible with STM/FMs. Feynman (1961) commented that great progress may be expected in biology when "you can simply look at, and work with individual molecules." STM/FMs could help materialize some even grander biomedical applications when used in combination with genetic engineering: to create, modify, or program micro-organisms for therapeutic uses.

Ettinger (1972):

If we can design sufficiently complex behavior patterns into microscopically small organisms, there are obvious and endless possibilities, some of the most important in the medical area. Perhaps we can create guardian and scavenger organisms in the blood, superior to the leukocytes and other agents of our human heritage, that will efficiently hunt down and clean out a wide variety of hostile or damaging invaders.

Asimov (1981) suggests that we:

Consider the bacteria. These are tiny living things made up of single cells far smaller than the cells in plants and animals ... . [We] can, by properly designing these tiniest slaves of ours, use them to reshape the world itself and build it close to our hearts' desire ... .

White (1969) proposed a similar notion for programmable cell repair machines:

appropriate genetic information can be introduced by means of artificially constructed virus particles into a congenitally defective cell for remedy (and) ... repair. The repair program must use (the cells own protein synthesis and metabolic pathways to diagnose and repair any damage.

Approaches to biomedical nanotechnology relying solely on genetic/protein engineering and self assembly (Asimov, 1981; Aridane, 1983; Drexler, 1981, 1986) are severely constrained by the lack of direct control and observation: the potential attributes of STM/FMs. Hybrids and components of organisms, cytoskeletal structures, viruses, and synthetic structures may evolve, facilitated by STM/FM capabilities, to fabricate, examine, and modify nanostructures. Ettinger (1972) anticipated "nanominiaturized robots to serve us." Feynman (1961) reported that a friend, Albert R. Hibbs suggested:

a very interesting possibility for relatively small machines. He says that, although it is a very wild idea, it would be interesting in surgery if you could swallow the surgeon. You put the mechanical surgeon inside the blood vessel ... . Other small machines might be permanently incorporated inside the body ... .

Feinberg (1985) sees

... the construction of nanosensors that could be implanted into the human body. They could ... [monitor] various physiological functions from subcellular molecules up through tissues and organs ... essential in determining some of the mechanisms involved in growth and aging.

Feinberg (1985) also proposed the use of laser energy to power and communicate with implanted sensors, and to direct activities of nanorobots.

... using coherent, short-wavelength radiation, we too will be able to match the accomplishments of the cells that compose us, and do molecular engineering.

These molecular structures will be much more complex than anything that human technology has thus far achieved ... . It may be possible to extend some of the methods using ... shortwavelength coherent radiation ... to nanofabrication as well as to seeing what we are doing in the nanoworld, since microholography can be used to demagnify patterns as well as magnify them.

There exist rationale and applicability for mobile nanodevices whose missions might be to stalk and destroy lethal viruses or malignancies, excavate clogged blood vessels, correct genetic expression and differentiation, repair or remove the processes of ageing including senile tangles of cytoskeletal proteins, and perhaps augment natural capabilities. Virtually every disease might be amenable to intracellular house calls by such structures, if they ever exist. Such nanodevices might be patterned after, or directly utilize, cytoskeletal elements (centrioles, microtubules, etc.), viruses (bacteriophages) programmed by specifically engineered genes, and/or controlled by telemetry. Synthetic materials including nano STM/FMs composed of piezo-materials may be incorporated.

Ellis (1962) discussed the possibility of microteleoperators (and suggested that protein enzymes may function in this manner). Nanoantennas have been proposed by Marks (1985) who described two orthogonal layers (about 200 nanometers in length) of metallic dipole antenna arrays which can convert photons to direct current with 75 percent efficiency. Javan (1985, 1986) has studied metal whiskers which can form tunnel junctions and may be suitable as nanoantennas. Feinberg's (1985) notion of communication by short wavelength coherent radiation, and Pohl's nanoaperture concept could be utilized for remote nanovision feedback and observation of nanoscale activities (Schneiker, 1986).

STM/FMs combined with genetic engineering and immunological techniques could be used to create, observe, and evaluate such mobile nanodevices whose capabilities would be optimized as replicating automata.

10.7     Replicating Automata

Ideas of automatic machines and robots have existed for centuries, but not till the work of John von Neumann in the mid twentieth century was there mathematical proof of the possible existence of self-replicating automata in a real physical sense. Assuming the proper material could be found, and using relatively simple neighbor logic, subunits could spontaneously assemble into complex structures, dynamically disassemble, multiply, or rearrange into successive configurations. Freeman Dyson (1979), Schneiker (1986) and others have considered the profound usefulness of such hypothetical robotic replicators in uses and sizes ranging from outer space exploration to household furniture to nanoscale "Hibb's machines," or nanodoctors. Dyson (1979) foresaw self-replicators which, after being launched from earth to an asteroid or planet, could mine, their own raw materials and form symbiotic relationships with other "life" forms. Shoulders (1961) extended the concept of replicators to the nanoscale where their existence could have wide ranges of scientific and medical applications.

Implementation of real-world, useful replicators faces many obstacles. A NASA study group (Bekey and Naugle, 1980) found that von Neumann's famous proof made major assumptions and avoided significant problems. Schneiker (1987) has summarized an approach to simplifying the development of replicative automata by minimizing the different types of materials/parts needed, the number of scale-dependent factors, part tolerance and wear, and assembly complexity. One other requirement is to increase reliability (i.e. by intrinsic error detection, repair, regeneration). Presently, these traits are fulfilled only by computer simulations or purely biological structures such as cytoskeletal proteins and viruses.

Schneiker (1986) notes that simple microreplicators, augmented with STM/FMs could mass produce nanotechnology products in virtually unlimited quantities. Nanotechnology applied to new superconductive materials (and vice versa) may help to implement useful replicative micro-automata which in turn could turn out nanodevices in vast quantities. Until recently superconductivity has been thought limited to near absolute zero temperatures, however some materials have been shown to have superconductive transitions at much warmer, easily attainable temperatures (Robinson, 1987).

The dramatic increase in superconducting transition temperatures in certain materials, and the availability of lossless current, magnets, and motors may herald a wave of scientific, technological, and economic advances. Schneiker contends that among these will be the facilitation of STM technology (superconductive tunneling), Josephson junctions, magnetic field sensors, as well as superconducting replicators. Superconducting materials offer scaling benefits, design simplicity, low power needs, and high component density. Superconducting magnets and coils could form switchable "glue" or "clamps" to hold subassemblies, generate switchable magnetic field patterns, direct assembly, positioning, and dynamic functions, drive rotary/linear motors or form superconducting channels or guideways for material transport (Schneiker, 1987).

Collective intelligence occurring in parallel replicating automata has been studied by Christopher Langton (1986) of Los Alamos National Laboratories. Langton is the organizer of a Los Alamos conference on "Artificial Life," the study of computer systems that exhibit behavior characteristic of natural life. By exploring models of cellular automata and self-replicators, Langton (1987) hopes to "extract the molecular logic of living systems."

Langton (1987) states.

Microelectronic technology and genetic engineering will soon give us the capability to create new life forms in silico as well as in vitro. This capacity will present humanity with the most far-reaching technical, theoretical, and ethical challenges it has ever confronted.

11   The Future of Consciousness

Nanotechnology may enable the dream of Mind/Tech merger to materialize. At long last, debates about the nature of consciousness will move from the domain of philosophy to large scale experiments. The visions of consciousness interfacing with, or existing within, computers or mind piloted robots expressed by Moravec, Margulis, Sagan and Max Headroom could be realized. Symbiotic association of replicative nanodevices and cytoskeletal networks within living cells could not only counter disease processes, but lead to exchange of information encoded in the collective dynamic patterns of cytoskeletal subunit states. If these are indeed the roots of consciousness, a science fiction-like deciphering and transfer of mind content may become possible. One possible scenario could utilize a small window in a specific brain region. Hippocampal temporal lobe, a site where memories enter and where electromagnetic radiation from outside the skull penetrates most readily and harmlessly, is one possible area where information distributed throughout the brain may perhaps be accessed and manipulated. Techniques such as laser interferometry, electroacoustical probes scanned over brain surfaces, or replicative nanoprobes immunotargeted to key hippocampal tubulins, MAPs, and other cytoskeletal components might be developed to perceive and transmit the content of consciousness.

What technological device would be capable of receiving and housing the information emanating from some 10¹⁵ tubulin subunits changing state some 10⁹ times per second? One possibility is a customized array of nanoscale automata, perhaps utilizing superconducting materials. Another possibility is a genetically engineered array of some 10¹⁵ tubulin subunits (or many more) assembled into parallel tensegrity arrays of interconnected microtubules, and other cytoskeletal structures. Current and near future genetic engineering capabilities should enable isolation of genes responsible for a specific individual's brain cytoskeletal proteins, and reconstitution in an appropriate medium. Thus the two evident sources of mind content (heredity and experience) may be eventually reunited in an artificial consciousness environment. A polymerized cytoskeletal array would be highly unstable and dependent on biochemical, hormonal, and pharmacological maintenance of its medium. Precise monitoring and control of cytoskeletal consciousness environments may become an important new branch of anesthesiology. Polymerization of cell-free cytoskeletal lattices would be limited in size (and potential intellect) due to gravitational collapse. Possible remedies might include hybridizing the cytoskeletal array by metal deposition, symbiosis with synthetic nanoreplicators, or placement of the cytoskeletal array in a zero gravity environment. Perhaps future consciousness vaults will be constructed in orbiting space stations or satellites. People with terminal illnesses may choose to deposit their mind in such a place, where their consciousness can exist indefinitely, and (because of enhanced cooperative resonance) in a far greater magnitude. Perhaps many minds can comingle in a single large array, obviating loneliness, but raising new sociopolitical issues. Entertainment, earth communication, and biochemical mood and maintenance can be supplied by robotics, perhaps leading to the next symbiosis-robotic space voyagers (shaped like centrioles?) whose intelligence is derived from cytoskeletal consciousness.

Yes, this is science fiction. Will it become reality like so much previous science fiction has? Probably not precisely as suggested; but if past events are valid indicators, the future of consciousness may be even more outrageous.

Figure 11.1: Multiple STM tip probing microtubule. By Paul Jablonka (Schneiker and Hameroff, 1987).

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Feenstra, R. M., W. A. Thompson and A. P. Fein (1986). Real-Space Observation of pi-Bonded Chains and Surface Disorder on Si(111) 2x1. Phys. Rev. Lett., 56(6), 10 February, 608-611.

Feuchtwang, T. E., P. H. Cutler and N. M. Miskovsky (1983). A Theory of Vacuum Tunneling Microscopy. Physics Letters, 99A(4), November, 167-171.

Fink, H. W. (1986). Mono-Atomic Tips for Scanning Tunneling Microscopy. IBM J. Res. Develop., 30(5), Sept., 460-465.

Flores, F. and N. Garcia (1984). Voltage Drop in the Experiments of Scanning Tunneling Microscopy for Si. Phys. Rev. B, 30(4), 15 August, 2289-2291.

Garcia, R., J. J. Saenz, J. M. Solar and N. Garcia (1986). Distance-Voltage Characteristics in Scanning Tunneling Microscopy. J. Phys., 19, L131-L134.

Garcia, N. (1986). Theory of Scanning Tunneling Microscopy and Spectroscopy: Resolution, Image and Field States, and Thin Oxide Layers. IBM J. Res. Develop., 30(5), Sept., 533-542.

Garcia, N. and F. Flores (1984). Theoretical Studies for Scanning Tunneling Microscopy. Physica 127B, 137-142.

Garcia, N., A. M. Baro, R. Miranda, H. Rohrer, C. Gerber, R. Garcia Cantu and J. L. Pena (1985). Surface Roughness Standards, Obtained with the Scanning Tunneling Microscope Operated at Atmospheric Air Pressure. Metrologia, 21, 135-138.

Garcia, N., B. Reihl, K. H. Frank and A. R. Williams (1985). Image States: Binding Energies, Effective Masses, and Surface Corrugation. Phys. Rev. Lett., 54(6), 11 Feb., 591-594.

Garcia, N., C. Ocal and F. Flores (1983). Model Theory for Scanning Tunneling Microscopy: Application to Au(110)(1x2). Phys. Rev. Lett., 50(25), 20 June, 2002-2005.

Garcia, R. (1986). Tunneling Current Through Localized Surface States Via Inelastic Processes. In Press.

Garcia, R., J. J. Saenz and N. Garcia (1986). Conductivity and Structure of Thin Oxide Layers Grown on a Metal Substrate: Scanning-Tunneling Microscopy in NiO on Ni (100). Physical Review B, 33(6), March, 4439-4442.

Gerber, C., G. Binnig, H. Fuchs, O. Marti and H. Rohrer (1985). Scanning Tunneling Microscope Combined With a Scanning Electron Microscope. Rev. Sci. Instrum., 57(2), 221-223.

Gimzewski, J. K. and A. Humbert (1986). Scanning Tunneling Microscopy of Surface Microstructure on Rough Surfaces. IBM J. Res. Develop., 30(5), Sept., 472-477.

Gimzewski, J. K., A. Humbert, D. W. Pohl and S. Veprek (1986). Scanning Tunneling Microscopy of Nanocrystalline Silicon Surfaces. Surface Sci., 168, 795-800.

Gimzewski, J. K., A. Humbert, J. G. Bednorz and B. Reihl (1985). Silver Films Condensed at 300 and 90 K: Scanning Tunneling Microscopy of Their Surface Topography. Phys. Rev. Lett., 55(9), 26 August, 951-954.

Golovchenko, J. A. (1986). The Tunneling Microscope: A New Look at the Atomic World. Science, 232, 4 April, 48-53.

Gomer, R. (1986). Extensions of the Field Emission Fluctuation Method for the Determination of Surface Diffusion Coefficients. In Press: Applied Physics.

Gomer, R. (1986). Possible Mechanisms of Atom Transfer in Scanning Tunneling Microscopy. IBM J. Res. Develop., 30(4), July, 428-430.

Gomez, J., L. Vazquez, A. M. Baro, N. Garcia, C. L. Perdriel, W. E. Triaca and A. J. Arvia (1986). Scanning Tunneling Microscopy and Electrochemistry: Surface Topography of (100)-Type Electrofaceted Platinum. In press.

Hamers, R. J., R. M. Tromp and J. E. Demuth (1986). Scanning Tunneling Microscopy of Si(001). Phys. Rev. B, 34(8), Oct., 5343-5357.

Hansma, P. K. and J. Tersoff (1987). Scanning Tunneling Microscopy. J. Appl. Phys., 61(2), 15 Jan., R1-R23.

Hosler, W., R. J. Behm, E. Ritter (1986). Defects on the Pt (100) Surface and Their Influence on Surface Reactions-A Scanning Tunneling Microscopy Study. IBM J. Res. Develop., 30(4), July, 403-410.

Hsue-Yang Liu, Fu-Ren F. Fan, Charles W. Lin and A. J. Bard (1986). Scanning Electrochemical and Tunneling Ultramicroelectrode Microscope for High-Resolution Examination of Electrode Surfaces in Solution. J. Am. Chem. Soc., 108, 3838-3839.

Humbert, A., J. K. Gimzewski and B. Reihl (1985). Postannealing of Coldly Condensed Ag Films: Influence of Pyridine Preadsorption. Phys. Rev. B, 32(6), 15 Sept., 4252-4253.

IBM (1985a). Fast Scan Piezo Drive. IBM Technical Disclosure Bulletin, 27(10b), March, 5976-5977.

IBM (1985b). Magnetostrictive Positioner. IBM Technical Disclosure Bulletin, 27(11), April, 63-73.

IBM (1985c). Replacement Mechanism for the Tips of Scanning Electron, Tunneling and Optical Microscopes. IBM Technical Disclosure Bulletin, 28(1), June, 435-436.

IBM (1985d). Piezo-electrostatic Rotary Drive. IBM Technical Disclosure Bulletin, 28(2), July, 504-505.

Kaiser, W. J., R. C. Jaklevic (1986). Spectroscopy of Electronic States of Metals with a Scanning Tunneling Microscope. IBM J. Res. Develop., 30(4), July, 411-416.

Kuk, Y. and P. J. Silverman (1986). Role of Tip Structure in Scanning Tunneling Microscopy. Appl. Phys. Lett., 48(23), June, 1597-1599.

Lang, N. D. (1985) Vacuum Tunneling Current from an Absorbed Atom. Phys. Rev. Lett., 55(2), 8 July, 230-233.

Lang, N. D. (1986a). The Theory of Single-Atom Imaging in the Scanning Tunneling Microscope. Phys. Rev. Lett., 56(11), 17 March, 1164-1167.

Lang, N. D. (1986b). Electronic Structure and Tunneling Current for Chemisorbed Atoms. IBM J. Res. Develop., 30(4), July, 374-379.

Lang, N. D. (1986c). Spectroscopy of Single Atoms in the Scanning Tunneling Microscope. Phys. Rev. Lett., 34(8), 15 Oct., 5947-5950.

Lang, N. D. (1987). Apparent Size of an Atom in the Scanning Tunneling Microscope as a Function of Bias. Phys. Rev. Lett., 58(1), 5 Jan., 45-48.

Liu, H.-Y., F: R. Fan, C. W. Lin and A. J. Bard (1986). Scanning Electrochemical and Tunneling Ultramicroelectrode Microscope for High-Resolution Examination of Electrode Surfaces in Solution. J. Am. Chem. Soc., 108(13), 3838-3839.

Louis, E., F. Flores and P. M. Echenique (1986). Current Saturation Through Image Surface States in Scanning Tunneling Microscopy. Solid State Comm., 59(7), 453-455.

Mamn, H. J., D. W. Abraham, E. Ganz and J. Clarke (1985). 2 Dimensional, Remote Micro positioner for a Scanning Tunneling Microscope. Rev. Sci. Instrum., 56(11), November, 2168-2170.

McCord, M. A., and R. F. W. Pease (1985). High Resolution, Low-Voltage Probes From a Field Emission Source Close to the Target Plane. J. Vac. Sci. Technol. B, 3(1), Jan./Feb., 198-201.

McCord, M.A. and R. F. W. Pease (1986). Lithography with the Scanning Tunneling Microscope. J. Vac. Sci. Technol. B, 4(1), Jan/Feb., 86-88.

Miranda, R., N. Garcia, A. Baro, R. Garcia, J. L. Pena and H. Rohrer (1985) Technological Applications of Scanning Tunneling Microscopy at Atmospheric Pressure. Appl. Phys. Lett., 47(4), 15 August, 367-369.

Moreland, J., S. Alexander, M. Cox, R. Sonnenfeld and P. K. Hansma (1983). Squeezable Electron Tunneling Junctions. Appl. Phys. Lett., 43(4), 15 August, 387-388.

Moreland, J. and J. W. Ekin (1985). Electron Tunneling into Superconducting Filaments using Mechanically Adjustable Barriers. Appl. Phys. Lett., 47(2), July, 175-177.

Moreland, J. and P. K. Hansma (1984). Electromagnetic Squeezer for Compressing Squeezable Electron Tunneling. Rev. Sci. Instrum., 55(3), March, 399-403.

Morita, S. K. Itaya and N. Mikoshiba (1986). Images of Barrier Layer of Anodic Aluminum Oxide in Air Obtained with Scanning Tunneling Microscope. Japanese J. of Applied Physics, 25(9), Sept., L743-L745.

Morita, S., T. Okada, Y. Ishigame and N. Mikoshiba (1986). Scanning Tunneling Microscopy of Metal Surfaces in Air. In Press; presented at Scanning Tunneling Microscopy '86, Santiago de Compostela, (Spain), July 14-18,1-8.

Morita, S., T. Okada, Y. Ishigame, C. Sato and N. Mikoshiba (1986). Voltage-Dependence of Scanning Tunneling Microscopy on Titanium Surface in Air. Japanese J. of Applied Physics, 25(6), L516-L518.

NASA (1986). Electrochemical Process Makes Fine Needles. NASA Tech Briefs, May/June, 135.

Newmark, P. and L. Garwin (1986). Electron Microscopy Acclaimed. Nature, 323, 23 Oct., 663.

Park, S. L. and C. F. Quate (1986). Tunneling Microscopy of Graphite in Air. Appl. Phys. Lett., 48(2), 13 January, 112-114.

Pashley, M. D., J. B. Pethica and J. Coombs (1985). Scanning Tunneling Microscope Studies. Surface Science, 152/153, 27-32.

Persson, B. N. J., and J. E. Demuth (1986). Inelastic Electron Tunneling from a Metal Tip. Solid State Communications, 57(9), 769-772.

Pethica, J. B. and M. D. Pashley (1983). Scanning Tunneling Microscopy. Nature, 305, October, 666.

Pohl, D. W. (1986). Some Design Critieria in Scanning Tunneling Microscopy. IBM J. Res. Develop., 30(4), July, 417-427.

Pohl, D. W., J. K. Gimzewski, A. Humbert and S. Veprek (1985). Surface Topography Studies of Nanocrystalline Si by STM. Proc. SPIE, 565, Micron and Submicron Integrated Circuit Metrology, 98-101.

Quate, C. F. (1986). Vacuum Tunneling: A New Technique for Microscopy. Physics Today, Aug., 26-33.

Ringger, M., B. W. Corb, H: R. Hidber, R. Schlogl, R. Wiesendanger, A. Stemmer, L. Rosenthaler, A. J. Brunner, P. C. Oelhafen, H: J. Guntherodt (1986). STM Activity at the University of Basel. IBM J. Res. Develop., 30(5), Sept., 500-508.

Ringger, M., H. R. Hidber, R. Schlogl, P. Oelhafen and H. J. Guntherodt (1985). Nanometer Lithography With the Scanning Tunneling Microscope. Appl. Phys. Lett., 46(9), 1 May, 832-834.

Ringger, M., H. R. Hidber, R. Schlogl, P. Oelhafen, H. J. Guntherodt, K. Wandelt and G. Ertl (1985). The Surface Topography of a Pd(100) Single Crystal and Glassy Pd81Si19 Studied by Scanning Tunneling Microscopy. In: M. A. Van Hove and S. Y. Tong, The Structure of Surfaces, Springer-Verlag, 48-53.

Ritter, E., R. J. Behm, G. Potschke and J. Wintterlin (1987). Direct Observation of a Nucleation and Growth Process on an Atomic Scale. In Press.

Robinson, A. L. (1983a). IBM Images Surfaces by Electron Tunneling. Science, 220, 1 April, 43-44.

Robinson, A. L. (1985a). A Spatially Resolved Surface Spectroscopy. Science, 229, 1074-1076.

Scheinfein, M. and M. Isaacson (1986). Electronic and Chemical Analysis of Fluoride Interface Structures at Subnanometer Spatial Resolution. J. Vac. Sci. Technol. B, 4(1), Jan/Feb., 326-332.

Schneiker, C. (1986). NanoTech: Science, Engineering and Related Topics. Department of Anesthesiology Advanced Biotechnology Laboratory, University of Arizona, Tucson, Arizona 85724 USA.

Schneiker, C. and S. R. Hameroff (1987). NanoTechnology Workstation Based on Scanning Tunneling/Optical Microscopy: Applications to Molecular Scale Devices. To be published in Proceedings of the Third International Workshop on Molecular Electronic Devices (Elsevier North-Holland).

Schneir, J., R. Sonnenfeld and P. K. Hansma (1986). Tunneling Microscopy Study of the Graphite Surface in Air and Water. Phys. Rev. B, 34(8), 15 Oct., 4979-4984.

Schroer, P. H, and J. Becker (1986). Computer Automation for Scanning Tunneling Microscopy. IBM J. Res. Develop., 30(5), Sept., 543-552.

Selloni, A., P. Carnevali, E. Tosatti and C. D. Chen (1985). Voltage-Dependent Scanning-Tunneling Microscopy of a Crystal Surface: Graphite. Physical Review B, 31(4), 15 February, 2602-2605.

Smith, D. P. E. and G. Binnig (1986). Ultrasmall Scanning Tunneling Microscope for Use in a Liquid-Helium Storage Dewar. Rev. Sci. Instrum., 57(10), Oct., 2360-2361.

Smith, D. P. E., G. Binnig and C. F. Quate (1986). Atomic Point-Contact Imaging. Appl. Phys. Lett., 49(18), 3 Nov., 1166-1168.

Smith, D. P. E., A. Bryant, C. F. Quate, J. P. Rabe, C. Gerber and J. D. Swalen (1986). Images of a Lipid Bilayer at Molecular Resolution by Scanning Tunneling Microscopy. Submitted to Proc. Nat. Acad. Sci. USA.

Smith, D., P. E. Scott and A. Elrod (1985) Magnetically Driven Micropositioners. Rev. Sci. Instru., 56(10), October, 1970-1971.

Smith, D. P. E. and C. F. Quate (1986). Material Presented at the STM 86 Meeting in Santiago de Campostela, Spain, August, 1986.

Soler, J. M., A. M. Baro and N. Garcia (1986). Interatomic Forces in Scanning Tunneling Microscopy: Giant Corrugations of the Graphite Surface. Phys. Rev. Lett., 57(4), 28 July, 444-447.

Sonnenfeld, R., J. Moreland and P. K. Hansma (1985). Contactless Tunneling to Semiconductors. J. Appl. Phys., 58(1), July, 392-396.

Sonnenfeld, R., and P. K. Hansma (1986). Atomic-Resolution Microscopy in Water. Science, 232, 11 April, 211-213.

Sonnenfeld, R., and B. C. Schardt (1986). Tunneling Microscopy in an Electrochemical Cell: Images of Ag Plating. Appl. Phys. Lett., 49(18), 3 Nov., 1172-1174.

Stoll, E. (1984). Resolution of the Scanning Tunnel Microscope. Surf. Sci. Lett., 143, L411-L416.

Stoll, E. (1986). Information and Image-Processing of Scanning Tunneling Microscope Data. In: W. F. Fagan, ed., Optics in Engineering Measurement, Proc. SPIE Vol. 599, 442-450.

Stoll, E., A. Baratoff, A. Selloni and P. Carnevali (1984). Current Distribution in the Scanning Vacuum Tunnel Microscope: A Free-Electron Model. J. Phys. C, 17, 3073-3086.

Stroscio, J. A., R. M. Feenstra and A. P. Fein (1986). Electronic Structure of the Si(111)2x1 Surface by Scanning-Tunneling Microscopy. Phys. Rev. Lett., 57(20), 17 Nov., 2579-2582.

Tersoff, J. (1985). Theory of the Scanning Tunneling Microscope. In: M. A. Van Hove and S. Y. Tong, eds., The Structure of Surfaces, Springer-Verlag, 54.

Tersoff, J. and D. R. Hamann (1983). Theory and Application for the Scanning Tunneling Microscope. Phys. Rev. Lett., 50(25), 20 June, 19982001.

Tersoff, J. and D. R. Hamann (1985). Theory of the Scanning Tunneling Microscope. Phys. Rev. B, 31(2), 15 January, 805-813.

Thompson, D. E. (1984). Atom Detection Improves, On The Surface. Science News, 126, 18 Aug., 101.

Thompson, D. E. (1985). Electron Tunneling for Ultrafine Detail. Science News, 127(14), 215.

Tokumoto, H., et al. (1986). Observation of Atomic Image of 2H-NbSe2 Surface by Scanning Tunneling Microscope. Japanese J. Appl. Phys., 25(8), Aug., L621-L623.

Travaglini, G., H. Rohrer, M. Amrein and H. Gross (1986). Scanning Tunneling Microscopy on Biological Matter. (Material presented at STM 86 Meeting in Santiago de Campostela, Spain, August, 1986).

Tromp, R. M., R. J. Hamers, and J. E. Demuth (1985). Si(001) Dimer Structure Observed with Scanning Tunneling Microscopy. Phys. Rev. Lett., 55(12), 16 Sept., 1303-1306.

Tromp, R. M., R. J. Hamers and J. E. Demuth (1986). Quantum States and Atomic Structure of Silicon Surfaces. Science, 234, 17 Oct., 304-309.

Van de Walle, G. F. A., H. van Kempen and P. Wyder (1986). Tip Structure Determination by Scanning Tunneling Microscopy. Surf. Science, 167, L219-L224.

Van de Walle, G. F. A., H. van Kempen and P. Wyder (1987). Scanning Tunneling Microscopy on Photoconductive Semi-insulating GaAs. Appl. Phys. Lett., 50(1), 5 Jan., 22-24.

Van Kempen, H. (1986). Construction and Applications of the Scanning Tunneling Microscope. In press. Presented at 5th Phefferkorn Conf. on Physical Aspects of Materials Characteristics.

Van Kempen, H. and G. F. A. Van de Walle (1986). Applications of a High-Stability Scanning Tunneling Microscope. IBM J. Res. Develop., 30(5), Sept., 509-514.

Van Loenen, E. J., J. E. Demuth, R. M. Tromp and R. J. Hamers (1987). Local electron States and Surface Geometry of Si(111)-(√3x√3) Ag. Phys. Rev. Lett., 58(4), 26 Jan., 373-376.

Vieira, S. (1986). The Behavior and Calibration of Some Piezoelectric Ceramics Used in the STM. IBM J. Res. Develop., 30(5), Sept., 553-556.

West, P., J. Kramar, D. V. Baxter, R. J. Cave and J. D. Baldeschwieler (1986). Chemical Applications of Scanning Tunneling Microscopy. IBM J. Res. Develop., 30(5), Sept., 484-491.

Wilson, R. J. and S. Chiang (1987). Structure of the Ag/Si(111) Surface by Scanning Tunneling Microscopy. Phys. Rev. Lett., 58(4), 26 Jan., 369-372.

Young, R. D., J. Ward and F. Scire (1971). Observation of Metal-Vacuum-Metal Tunneling, Field Emission, and the Transition Region. Phys. Rev. Lett., 27, 922-nnn.

Young, R., J. Ward and F. Scire (1972). The Topografiner: An Instrument for Measuring Surface Microtopography. Rev. Sci. Instr., 43(7), July, 999-1011.

12.2     Near-Field Scanning Optical Microscopes

Betzig, E., A. Lewis, A. Harootunian, N. Isaacson and E. Kratschmer (1986). Near-Field Scanning Optical Microscopy (NSOM), Development and Biological Applications. Biophysics Journal, 49, January, 269-279.

Binnig, G. K., C. Gerber, H. Rohrer and E. Weibel (1985a). Nano-Aperture. IBM Tech. Disclosure Bul., 27(8), Jan., 4893.

De Brabander, R. Nuydens, G. Geuens, M. Moeremans, J. De Mey and C. Hopkins (1988). Nanovid Ultramicroscopy: A New Non-Destructive Apporach Providing New Insights in Subcellular Motility. In: M. De Bravander and J. De Mey, eds., Microtubules and Microtubule Inhibitors 1985. Elsevier Science Publishers, North Holland, 187-196.

Durig, U., D. Pohl and F. Rohner (1986). Near-Field Optical Scanning Microscopy with Tunnel-Distance Regulation. IBM J. Res. Develop., 30(5), Sept., 478-483.

Harootunian, A., E. Betzig, M. Isaacson and A. Lewis (1986). Super-Resolution Fluorescence Near-Field Scanning Optical Microscopy. Appl. Phys. Lett., 49(11), 15 Sept., 674-676.

IBM (1985e). Optical Microscope Beyond the Defraction Limit. IBM Technical Disclosure Bulletin, 28(6), November, 2691-2693.

Lewis, A., M. Isaacson, A. Harootunian and A. Muray (1984). Development of a 500 Å  Spatial Resolution Light Microscope. Ultramicroscopy, 13, 227-232.

McCutchen, C. W. (1967). Superresolution in Microscopy and the Abbe Resolution Limit. J. Optical Society of America, 57(10), October, 1190-1192.

Pohl, D. W., W. Denk and M. Lanz (1984). Optical Stethoscopy: Image Recording with Resolution. Appl. Phys. Lett., 44(7), 1 April, 651-653.

Pohl, D. W., W. Denk and U. Duerig (1985). Optical Stethoscopy: Imaging with λ /20 Resolution. Proc. of the SPIE, 565, Micron and Submicron Integrated Circuit Metrology, August, 56-61.

12.3     Other STM-Related Instruments

Binnig, G., C. F. Quate and C. Gerber (1986). Atomic Force Microscope. Phys. Rev. Lett., 56(9), 3 March, 930-933.

Muralt, P. (1986). GaAs pn Junction Studied by Scanning Tunneling Potentiometry. Appl. Phys. Lett., 49(21), 24 Nov., 1441-1443.

Muralt, P. and D. W. Pohl (1986). Scanning Tunneling Potentiometry. Appl. Phys. Lett., 48(8), 24 February, 514-516.

Muralt, P., D. W. Pohl and W. Denk (1986). Wide-Range, Low-Operating-Voltage, Bimorph STM: Application as Potentiometer. IBM J. Res. Develop., 30(5), Sept., 443-450.

Smith, D. P. E., G. Binning and C. F. Quate (1986). Atomic Point-Contact Imaging. Appl. Phys. Lett., 49(18), 3 November, 1166-1168.

Williams, C. C. and H. K. Wickramasinghe (1986). Scanning Thermal Profiler. Appl. Phys. Lett., 49(23), 8 Dec., 1587-1589.

12.4     Replicating Systems References

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Arbib, M. A. (1967a). Some Comments on Self-Reproducing Automata. Systems and Computer Science. J. F. Hart and Satoru Takasu, eds., Univ. of Toronto Press, 42-59.

Arbib, M. A. (1967b). Automata Theory and Development: Part 1. J. Theor. Biol., 14(2), 131-156.

Arbib, M. A. (1969). Machines Which Compute and Construct. In chapter 10 of: Theories of Abstract Automata. Prentice Hall, Englewood Cliffs, N. J.

Berry, A. (1975). The Vision of John von Neumann. In chapter 11 of The Next Ten Thousand Years, A Vision of Man's Future in the Universe. Burks, A. W. (1975). Logic, Biology and Automata-Some Historical Reflections. Int. J. Man-Machine Studies, 7(3), 297-312.

Burks, A. W., ed. (1970). Essays on Cellular Automata. Chicago, Univ. of Illinois Press.

Cairns-Smith, A. G. (1971). The Life Puzzle. Toronto: Univ. of Toronto Press.

Cairns-Smith, A. G. (1981). Beginnings of Organic Evolution. In The Study of Time IV, J. T. Fraser, N. Lawrence, D. Park, eds., 15-33. New York, Springer-Verlag.

Cairns-Smith, A. G. (1982). Genetic Takeover. Cambridge, Cambridge University Press.

Cairns-Smith, A. G. (1985). The First Organisms. Scientific American, June.

Cairns-Smith, A. G. and H. Hartman, eds. (1986). Clay Minerals and the Origin of Life. Cambridge, Cambridge Univ. Press.

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Cliff, R. A. (1985). An Hierarchical System Architecture for Automated Design, Fabrication, and Repair. In: Space Manufacturing 4, J. Grey and L. A. Hamdan, eds., 121-126.

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Dyson, F. (1981). Disturbing the Universe. New York: Harper Colophon.

Eigen, M. (1971). Selforganization of Matter and the Evolution of Biological Macromolecules. Naturwissenschaften, 58, 465-523.

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Fox, S. W. (1973). The Rapid Evolution of Complex Systems from Simple Beginnings. Proc. 3rd Int. Conf. from Physics to Theoretical Biology, Versailles. Basel: Karger, 133-144.

Fox, S. W. (1980). The Assembly and Properties of Protobiological Structures: The Beginnings of Cellular Peptide Synthesis. BioSystems, 12(3), 155-166.

Fox, S. W. (1984). Self-Sequencing of Amino Acids and Origins of Polyfunctional Protocells. In: Proceedings of the 7th International Conference on the Origins of Life, July, 485-488.

Freitas, Jr., R. A. (1981). A Self-Replicating, Growing Lunar Factory. In: Space Manufacturing 4, Proceedings of the Fifth Princeton/HIAA Conference, J. Grey and L. A. Hamdan, eds., 109-119.

Freitas, Jr., R. A. and W. P. Gilbreath (1982). Chapter 5: Replicating Systems Concepts: Self-Replicating Lunar Factory and Demonstration. In: Advanced Automation for Space Missions, NASA Conference Publication 2255₎189-335.

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Katzir, A. and F. Vranty (1984) Use of PVC for Replicating Submicron Features for Microelectronic Devices. Rev. Sci. Inst., 55(4), April, 633-634.

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Moore, E. F. (1956). Artificial Living Plants. Scientific American, 195(4), 118-126.

Moore, E. F. (1962). Machine Models of Self-Reproduction. 17-33. Moore, E. F. (1964). Mathematics in the Biological Sciences. Scientific American, 211(3), 148-164.

Morowitz, H. J. (1959). A Model of Reproduction. American Scientist, 47,261-263.

Morrison, P. (1964). A Thermodynamic Characterization of Self-Reproduction. Reviews of Modern Physics, April, 517-524.

Myhill, J. (1964). The Abstract Theory of Self-reproduction. In: Views on General Systems Theory, M. D. Mesarovic, ed., 106-118. New York, John Wiley and Sons.

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Penrose, L. S. (1959). Automatic Mechanical Self-Reproduction. New Biology, Penguin Books, 92-117.

Penrose, L. S. and R. Penrose (1957). A Self-Reproducing Analogue. Letters to the Editors, Nature, June, 1183.

Penrose, L. S. (1959). Self-Reproducing Machines. Scientific American, 200(6), 105-114.

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Schuster, P. (1979). A Mathematical Model of the Hypercycle. Proc. of the International Symposium on Synergetics, September 24-29, 170-178.

Schuster, P. (1981). Self-Organization and Integration of Information Stored in Selfreplicative Units. Self-Organizing Systems, Verlag, New York, 47-61.

Schuster, P. and K. Sigmund (1979). Self-Organization of Biological Macromolecules and Evolutionary Stable Strategies. Proc. of the International Symposium on Synergetics, Sept. 24-29, 156-169.

Schwartz, A. W., and L. E. Orgel (1985). Template-Directed Synthesis of Novel, Nucleic Acid-Like Structures. Science, 228, 3 May, 585-587.

Vitanyi, P. M. B. (1974). Genetics of Reproducing Automata. Proceedings of the 1974 Conference on Biologically Motivated Automata Theory, June, 166-171.

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Weiss, A. (1981). Replication and Evolution in Inorganic Systems. Angew Chem. Int. Ed. Engl., 20, 850-860.

12.5     Collective Computing References

Carpenter, G. A. and S. Grossberg (1986). A Massively Parallel Architecture for a Self-Organizing Neural Pattern Recognition Machine. In press: Computer Vision, Graphics and Image Processing.

Dammasch, I. E., G. P. Wagner, and J. R. Wolff (1986). Self-Stabilization of Neuronal Networks. Biol. Cybern., 54, 211-222.

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Hogg, T. and Huberman B. A. (1984). Understanding Biological Computation: Reliable Learning and Recognition. Proc. Natl. Acad. Sci., 81, Nov., 6871-6875.

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Hopfield, J. J. (1984a). Collective Processing and Neural States. In: Modeling and Analysis in Biomedicine, C. Nicolini, ed., 371-389.

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12.6     Quantum Computing References

Albert, D. Z. (1983). On Quantum-Mechanical Automata. Physics Letters, 98A(5,6), 24 Oct., 249-252.

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Bergquist, J. C., R. G. Hulet, W. M. Itano and D. J. Wineland (1986). Observation of Quantum Jumps in a Single Atom. Phys. Rev. Lett., 57(14), 6 Oct., 1699-1702.

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Carter, F. L. (1984b). The Molecular Device Computer: Point of Departure for Large Scale Cellular Automata. Physica, 10D, 175-194.

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Ceperley D. and B. Alder (1986). Quantum Monte Carlo. Science, 231, February, 555-560.

Cook, Richard J. and H. J. Kimble (1985). Possibility of Direct Observation of Quantum Jumps. Phys. Rev. Lett., 54(10), 11 March, 1023-1026.

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Davydov, A. S. (1985). Solitons in Molecular Systems. D. Reidel Pub. Co., Boston.

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Feynman, R. P. (1985a). Quantum Mechanical Computers. Optics News, Feb., 11-20.

Feynman, R. P. (1985c). Quantum Mechanical Computing. A talk for a meeting of the Cal. Tech. Society of Physics Students on March 12.

Feynman, R. P. (1986a). Quantum Mechanical Computers. Foundations of Physics, 16(6), 507-531.

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Index

2,5 hexane dione, 125

acetylcholine, 63, 64, 125, 130, 155

acoustic soliton, 142

acoustical perception, 159

acrylamide, 125

actin, 36, 60, 63, 75, 83, 84, 87, 90, 97, 103, 104, 105, 106, 108, 109, 113, 114, 116, 117, 118, 119, 120, 121, 122, 126, 127, 130, 147, 160, 170, 171, 182

actin capping protein, 108

actin filament, 83, 84, 87, 90, 97, 103, 104, 105, 109, 113, 119, 121, 122, 126, 160, 170, 171, 182

actin-binding protein, 108

action potential, 44, 61, 63, 64, 66, 78, 135, 146, 153, 174, 177, 183

Adey, W. R., 6, 45, 61, 78, 79, 158

ADP, 90, 135

AIDS, 185, 190

Albrecht-Buehler, 55, 97, 111, 114, 115, 126, 166, 167, 169

allosteric protein, 131, 132, 134, 135

alpha actinin, 108, 119

alpha helix, 103, 130, 140, 141

alpha tubulin, 85, 123, 176

alternative life forms, 48

aluminum, 125

alveolar foam theory, 83

amide, 129, 140, 141

amino acid, 7, 47, 48, 49, 75, 85, 86, 87, 129, 130, 131, 134, 135, 137, 144, 161, 182

amoeba, 8, 22, 36, 45, 84, 87, 122

amoeboid, 36, 109, 111, 114, 115, 123, 124

amoeboid locomotion, 109, 124

amphetamines, 156

analog, 12, 20, 21, 26, 34, 41, 44, 61, 64, 66, 108, 159, 160, 163

analog functions, 34, 41, 159

analog texture, 160

anesthesia, 74, 125, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156

anesthetic, 36, 125, 134, 148, 149, 150, 151, 153, 154, 155

anharmonicity, 141, 142, 143

aperture, 198

aplysia, 74

arbiters, 13, 102

Aristotle, 121, 122

artificial intelligence, 8, 9, 10, 16, 33, 41, 64, 69, 163

assemblies, 7, 17, 19, 41, 51, 60, 69, 80, 87, 127, 129, 130, 131, 133, 139, 145, 158, 164, 166, 167, 169, 184, 187, 198, 199, 206

assembly, 12, 35, 59, 65, 76, 84, 87, 88, 89, 90, 91, 93, 94, 95, 97, 105, 113, 114, 115, 123, 128, 134, 138, 139, 156, 160, 164, 170, 171, 184, 186, 187, 191, 213, 215

associative memory, 12, 37, 60, 71, 75, 163

Atema, J., 88, 158, 159, 161

atomic force microscopy, 197

ATP, 50, 51, 76, 90, 98, 116, 117, 118, 119, 129, 130, 133, 134, 135, 140, 142, 144, 146, 154, 158, 159, 166, 167, 182

ATP hydrolysis, 76, 98, 116, 129, 130, 135, 140, 158

attractor, 152

automata, 10, 12, 26, 27, 28, 29, 30, 31, 32, 38, 45, 70, 160, 176, 177, 184, 214, 215, 217

autonomic nervous system, 59, 60

axon, 7, 44, 60, 61, 62, 64, 65, 68, 74, 76, 104, 106, 125, 146

axoplasm, 76, 104, 106, 108, 184

axoplasmic transport, 45, 46, 60, 68, 75, 76, 87, 91, 97, 99, 104, 106, 107, 109, 111, 116, 117, 119, 122, 124, 153, 177

backstroke hypothesis, 117

bacteriophage, 186

Barnett, M. P., 104, 163, 164

basal bodies, 51, 52, 55, 84, 89, 118, 168

behavior, 11, 23, 26, 29, 30, 32, 33, 34, 35, 36, 38, 43, 56, 65, 66, 67, 71, 78, 84, 107, 114, 115, 116, 124, 131, 132, 133, 139, 140, 141, 145, 147, 155, 158, 159, 160, 167, 177, 213, 215

behaviorism, 39

bending sidearm, 99, 112

Bernard, C., 124, 125, 154

beta, 7, 85, 86, 91, 92, 123, 125, 130, 131, 150, 169, 176, 177, 179, 180

beta tubulin, 7, 85, 86, 91, 123, 169, 176, 177

beta-pleated sheet, 130

Binnig, G., 193, 197, 198, 204, 206

bioelectromagnetic field, 37

bioenergetic, 136

biosensors, 150

blood brain barrier, 125

Bornens, M., 6, 55, 97, 166, 167, 168, 169

bouton, 62, 108

brain, 5, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 20, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 51, 58, 59, 60, 61, 62, 63, 64, 65, 67, 68, 69, 70, 71, 72, 74, 75, 77, 78, 86, 93, 98, 99, 102, 122, 123, 125, 127, 133, 147, 148, 149, 150, 153, 154, 155, 156, 157, 158, 160, 166, 174, 177, 183, 217

brain stem, 39, 60

brain/mind, 8, 10, 13, 14, 32, 33, 34, 35, 36, 38, 39, 44, 45, 69, 70, 123, 150, 156

brain/mind duality, 38, 39

brain/mind metaphor, 33

brain/mind/computer triangle, 34, 36

Butschli, 83

Cairns-Smith, A. G., 35, 47, 48

calcium, 24, 25, 61, 63, 74, 84, 87, 88, 90, 91, 99, 104, 106, 107, 108, 109, 110, 116, 119, 125, 126, 127, 134, 144, 145, 153, 155, 157, 160, 161, 174, 181, 182, 183

calcium ion, 24, 63, 74, 84, 90, 91, 99, 104, 106, 107, 108, 116, 119, 125, 134, 153, 155, 157, 160, 174

calcium ions, 24, 63, 74, 84, 91, 99, 107, 108, 119, 155, 160, 174

calmodulin, 108

campaniform sensilla, 159

cancer, 5, 96, 114, 121, 124, 185, 190

carbon disulfide, 125

cardiovascular collapse, 148, 150

Cc, 90, 91, 93, 95, 99

cell assembly, 41

cell body, 60, 61, 62, 64, 76, 115, 116, 122, 123, 159

cell movement, 51, 93, 113, 126, 161

cellular automata, 10, 22, 26, 27, 29, 30, 31, 38, 45, 48, 60, 176, 215

central nervous system, 59, 60, 125

centriole, 53, 56, 93, 95, 97, 122, 158, 166, 167, 169, 171

centrosome, 54, 85, 96, 114

chaos, 25, 49, 70, 126

chloroform, 125, 154

chromophore, 157

cilia, 51, 55, 84, 87, 89, 98, 117, 118, 119, 128, 135, 158, 159, 160, 205

ciliary, 36, 99, 111, 112, 117, 119, 126, 159, 177

clay, 35, 47, 48

clear zone, 86

cleavage furrow, 96, 120

Clegg, J., 6, 110, 111, 137, 138, 174

cognition, 5, 14, 39, 40, 45, 157, 160

coherence, 17, 25, 41, 43, 122, 147, 155, 157, 177

coherent, 7, 12, 19, 24, 25, 42, 43, 45, 60, 72, 90, 92, 115, 117, 133, 144, 145, 146, 147, 157, 169, 174, 175, 176, 178, 179, 181, 182, 183, 214

coherent light, 25, 42

coherent protein excitation, 144

coherent reference, 43, 72

coherent wave interference, 43, 183

colchicine, 76, 122, 124, 125, 157, 159

collective, 6, 7, 10, 11, 12, 14, 15, 17, 20, 24, 25, 26, 29, 35, 36, 38, 41, 45, 50, 53, 57, 59, 69, 70, 77, 78, 80, 87, 92, 98, 111, 112, 122, 123, 127, 128, 130, 132, 133, 139, 144, 145, 146, 147, 148, 153, 155, 156, 157, 158, 159, 167, 177, 182, 183, 185, 186, 187, 217

collective cooperativity, 158

collective effect, 6, 10, 11, 12, 20, 24, 29, 45, 111, 132, 144, 145, 148, 156, 157, 177

collective emergence, 36

collective vibrations, 187

communicative resonance, 169

compression, 121, 170

computer, 5, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 20, 21, 24, 29, 31, 33, 34, 35, 36, 37, 41, 45, 51, 57, 64, 65, 74, 77, 111, 132, 141, 144, 146, 150, 160, 163, 176, 177, 179, 183, 190, 215

conditioned stimulus, 68

conformation, 21, 27, 75, 79, 129, 130, 131, 132, 133, 134, 140, 144, 153, 159, 161, 164, 165, 175, 176, 211

conformational change, 21, 79, 88, 91, 116, 129, 133, 135, 141, 148, 154, 155, 157, 159, 164, 182, 187, 211

conformational gradients, 164

conformational state, 10, 11, 13, 19, 21, 79, 92, 118, 119, 129, 130, 132, 133, 134, 135, 143, 144, 153, 155, 157, 164, 166, 167, 169, 176, 177

conformational switching, 131

conformon, 136

connectionism, 12, 45, 64, 67, 68, 93

connectionist, 15, 33, 41, 68, 69, 70, 71, 72, 77

Conrad, M., 21, 22, 160

consciousness, 5, 7, 8, 10, 11, 14, 15, 17, 25, 32, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 53, 55, 58, 60, 69, 72, 75, 77, 78, 108, 125, 127, 134, 143, 148, 150, 153, 154, 156, 157, 177, 183, 199, 217

consolidation, 73, 151, 153

contractile bending, 119

contractile ring, 96, 105, 120

convergence, 24, 64

critical concentration, 90, 95

crosslinking, 84, 106

crystal, 25, 47, 48, 86, 98, 103, 126, 140, 141, 172, 191, 199

crystal genes, 48

cytochalasin B, 122

cytomatrix, 103, 105, 108, 109, 110, 111, 119, 120, 127

cytomusculature, 106, 127, 170

cytoplasm, 11, 13, 16, 22, 24, 35, 43, 50, 51, 52, 54, 56, 63, 67, 80, 83, 84, 86, 87, 89, 91, 93, 97, 99, 105, 106, 107, 108, 109, 110, 112, 113, 114, 115, 119, 120, 121, 122, 123, 126, 127, 143, 146, 160, 161, 164, 166, 167, 170, 174, 175, 183, 186, 187, 212

cytoplasmic activity, 87

cytoplasmic ground substance, 59, 86, 105

cytoplasmic intelligence, 115, 126

cytoplasmic probing, 112, 157

cytoplasmic solid state, 105

cytoskeleton, 5, 8, 11, 12, 14, 15, 16, 17, 20, 24, 25, 26, 34, 35, 36, 37, 41, 42, 43, 45, 46, 51, 52, 55, 59, 60, 63, 64, 69, 75, 76, 79, 80, 83, 84, 87, 90, 92, 93, 94, 97, 99, 103, 107, 108, 110, 111, 114, 121, 122, 123, 124, 125, 127, 128, 137, 143, 144, 147, 153, 154, 156, 157, 158, 159, 160, 164, 166, 167, 170, 172, 175, 176, 181, 183, 198, 213

dalton, 129, 154

Darwin, 36, 37, 70, 77

Davydov, A. S., 20, 130, 136, 140, 141, 142, 143, 146, 147, 155, 181

DeBrabander, M., 6, 8, 55, 81, 82, 86, 89, 91, 92, 94, 95, 96, 97, 107, 114, 115, 117, 160, 161, 162

Debye layer, 87, 145

Del Giudice, E., 87, 146, 166, 174, 181, 182

dementia, 125, 189

dendrite, 45, 61, 64, 76, 106

dendritic, 16, 26, 34, 41, 42, 43, 44, 46, 60, 61, 64, 67, 68, 69, 73, 75, 76, 87, 105, 106, 107, 123, 150, 159, 183

dendritic spine, 16, 45, 46, 64, 68, 73, 75, 76, 87, 105, 107

deoxyhemoglobin, 133

depolarization, 44, 61, 64, 143, 157, 160, 183

descriptive patterns, 157

desmin, 104

deterministic chaos, 17, 29, 150

detyrosination, 86, 161, 165

development, 35, 37, 38, 49, 59, 66, 68, 69, 75, 99, 102, 121, 122, 123, 163, 190, 205, 215

developmental disability, 124

dielectric, 144, 174, 191

dielectric waveguides, 174

differentiation, 51, 53, 93, 102, 121, 122, 123, 158, 214

digital, 21, 26, 44, 61, 65, 66, 72, 163

dimensionality, 150

dimer, 7, 85, 90, 91, 92, 139, 154, 160, 164, 166, 169, 172, 176, 177, 178, 182

dipole, 7, 21, 85, 110, 131, 134, 136, 139, 140, 144, 145, 150, 153, 176, 177, 178, 179, 214

dipole coupling, 85, 136

dipole interactions, 176, 178, 179

dipole moment, 21, 139, 140, 144, 145

dipole oscillation, 21, 110, 131, 134, 144, 153, 176, 178

dipole-dipole attraction, 139

disassembly, 89, 90, 91, 92, 94, 114, 120

distributed, 14, 17, 24, 25, 39, 43, 59, 65, 71, 72, 77, 79, 109, 153, 156, 177, 217

distributed memory, 25, 72

distributedness, 43, 93

divergence, 64

DNA, 5, 22, 47, 48, 49, 50, 51, 52, 73, 84, 86, 122, 123, 126, 127, 129, 139, 161, 185, 186, 187, 189, 211, 212, 213

drug, 43, 91, 115, 124, 156, 157, 188

dynamic instability, 89, 92, 94, 171

dynein, 11, 76, 87, 98, 116, 117, 118, 119, 124, 166

EEG, 17, 26, 45, 78, 79, 149, 150, 151, 152

electret, 19, 139, 140, 144, 146, 147, 155, 158, 174, 177, 182

electric fields, 79, 144, 145, 158, 203, 206

electroencephalography, 45, 150

electron microscope, 84, 103, 105, 113, 193, 205

electron mobility, 155, 156

electron volt, 140, 146

electrosoliton, 142

embryological development, 68, 88, 108, 122, 171

emergent evolution, 38, 45

encephalization, 59

engram, 43, 71, 72

entropy, 137, 188

enzyme, 110, 117, 125, 130, 131, 134, 137, 140, 144, 185, 203

epigenesis, 121, 122

ether, 148, 149

eukaryote, 50, 58

evolution, 5, 7, 8, 9, 12, 20, 25, 35, 36, 37, 38, 45, 49, 51, 54, 56, 57, 59, 66, 70, 93, 97, 145, 172, 188, 197, 198

excitatory, 61, 62, 63, 64, 65, 71, 74, 149, 155

excitement phase, 149

extremal computer, 21

face centered cubic packing, 172

Feynman machines, 7, 191, 198, 203

Feynman, R., 7, 21, 190, 191, 198, 203, 204, 205, 206, 210, 213, 214

fibrillar theory, 80

filamin, 108, 109

filopodia, 92, 113, 114, 115, 120, 122, 123, 171

filter, 72

fimbrin, 108

firefly, 154

firefly luciferase, 155

flagella, 51, 63, 84, 87, 89, 98, 111, 117, 118, 119, 159, 160, 172, 205

fluorescence resonance transfer, 157

focusing, 64, 75, 87, 146, 147

fodrin, 83, 108

fractal, 17, 18, 19, 25, 30, 43, 45, 170, 183

free energy, 135, 137

frequency domain, 43

Frohlich, H., 6, 19, 20, 134, 143, 144, 145, 146, 147, 155, 166, 176, 179, 181

Fuller, B., 121, 170

Galvani, L., 66

ganglion, 59

gap junction, 62

gated potential, 61, 135

Gazzaniga, M., 77

GDP, 90, 91, 92, 135

gel, 25, 36, 60, 83, 84, 91, 104, 105, 106, 108, 109, 120, 126, 127, 160, 174, 175, 182, 183

gelsolin, 108

gene, 86, 188, 189

genetic code, 127

geodesic dome, 119

gestalt, 70

glial filament, 104

glutamated, 82, 86, 161, 162

glutaraldehyde, 84

glycoprotein spikes, 187

Golgi, 67, 76, 108, 114, 115

Golgi stain, 67

gout, 124

gradionators, 165

gradions, 165, 166

grain, 26, 67, 77, 78, 79, 129, 141, 166, 183

grain of the engram, 79, 129, 166, 183

graininess, 78, 204

gray matter, 61, 65

ground substance, 84, 87, 105, 108, 160

growth cone, 68, 106, 122

GTP, 90, 91, 92, 133, 134, 135, 146, 157, 164, 172

Guedel, 148, 149, 150

gyroscopic centrioles, 166

habituation, 74

halothane, 125, 153

Hameroff, S., 6, 40, 42, 44, 147, 149, 150, 151, 152, 155, 174, 176, 179, 180, 181, 192, 193, 195, 199, 200, 201, 202, 204, 209, 211, 218

Hansma, P., 191, 194, 197, 205, 212

Heidemann, S., 120, 170

helpless spectator theory, 37, 38

hemoglobin, 129, 131, 132, 133

heterosynaptic potentiation, 74, 75

hexagonal packing, 172, 178

hexanedione, 125

hierarchical, 14, 16, 39, 59, 70, 115, 129, 171

high voltage electron microscope, 105

hippocampus, 75, 78, 174

hologram, 24, 25, 42, 43, 72, 183

holographic imagery, 157

homunculi, 65

Hopfield net, 70

Hopfield, J., 10, 15, 41, 69, 70

hydrogen bond, 83, 130, 131, 132, 134, 137, 141

hydrolysis, 21, 90, 92, 99, 116, 119, 135, 140, 146

hydrophobic, 85, 87, 88, 130, 133, 134, 136, 137, 139, 144, 153, 154, 155, 184, 188

hydrophobic forces, 85, 87

hydrophobic interaction, 88, 130, 133, 136, 137, 184

hydrophobic pockets, 153, 154, 155

icosahedral, 119, 120, 187

IDPN, 125

immotile cilia syndrome, 124

Indian rope trick, 38, 122, 147, 158, 171, 174

induction effect, 139

inference engine, 77

infoplasm, 108, 111

information, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 21, 22, 24, 25, 26, 30, 31, 32, 33, 34, 35, 37, 38, 39, 42, 43, 44, 45, 46, 47, 48, 51, 53, 55, 56, 58, 59, 60, 61, 64, 65, 66, 67, 68, 70, 71, 72, 73, 76, 77, 78, 79, 84, 86, 87, 88, 93, 98, 103, 105, 108, 109, 115, 122, 126, 127, 129, 130, 131, 132, 135, 136, 137, 140, 141, 143, 145, 147, 151, 152, 157, 158, 159, 160, 163, 164, 165, 166, 167, 169, 171, 173, 175, 176, 177, 181, 182, 187, 190, 194, 198, 205, 206, 213, 217

information strings, 164

inhibitory, 36, 61, 62, 63, 64, 65, 149, 155

instantaneous dipole, 139, 155

integration, 40, 61, 64, 65, 66, 78, 108, 171

intelligence, 5, 7, 8, 9, 41, 47, 53, 55, 56, 65, 79, 93, 105, 116, 122, 123, 127, 157, 167, 176, 183, 186, 215, 217

interference patterns, 24, 25, 42, 71

intermediate filament, 83, 84, 103, 114, 125

interphase, 94, 96, 162

ion channel, 45, 46, 49, 61, 64, 76, 129, 135, 144, 154, 155, 158

ion milling, 197

isozyme, 165

Jarosch, R., 121, 170, 171

keratin, 104, 139

kilodalton, 99, 129, 131

kinesin, 76, 98, 117

kink, 29, 177, 181

kinocilium, 158

Kirschner, M., 92, 93, 171

Koruga, D., 6, 86, 98, 103, 172, 173

lamellipodia, 87, 105, 112, 113, 114, 115, 120, 122, 123, 171

Lashley, K., 14, 43, 71, 72

lattice, 21, 25, 26, 31, 35, 44, 48, 51, 83, 84, 90, 91, 92, 96, 97, 99, 103, 104, 105, 107, 111, 117, 119, 127, 136, 137, 138, 142, 145, 158, 165, 166, 167, 170, 173, 174, 176, 177, 178, 179, 181, 182, 183, 186, 213

laughing gas, 148

learning, 16, 24, 26, 34, 35, 36, 37, 41, 45, 57, 60, 64, 68, 69, 70, 71, 72, 74, 75, 76, 99, 107, 127

levels, 5, 8, 14, 15, 31, 41, 45, 64, 65, 66, 77, 99, 108, 111, 123, 127, 129, 133, 136, 144, 148, 149, 183, 206

Liberman, E., 21, 22, 160

life, 8, 26, 29, 33, 35, 36, 37, 47, 48, 49, 50, 51, 53, 57, 58, 71, 80, 86, 121, 124, 167, 184, 187, 188, 190, 215, 216

ligand, 132, 134, 135, 153, 165, 177

lipid, 154, 155, 185, 186

London dispersion force, 139

long term potentiation, 74, 75

LSD, 156

LTP, 75

luminescence, 154

MacLuhan, M., 126

macron, 25, 26

magnetic fields, 158

malignant, 114, 124, 189

McCulloch-Pitts neuron, 66

mechanical distortion, 158

mechanochemical engines, 160

mechanoenzyme, 108

Mechano-lonic Transducers, 159

meiosis, 121

membrane, 7, 16, 22, 34, 45, 46, 49, 50, 54, 55, 61, 62, 63, 64, 68, 75, 76, 79, 83, 89, 91, 96, 107, 108, 112, 113, 116, 120, 121, 126, 128, 129, 135, 136, 144, 146, 150, 153, 154, 155, 156, 157, 158, 159, 160, 164, 172, 186, 187

memory, 9, 14, 16, 21, 24, 25, 33, 36, 37, 39, 43, 45, 65, 67, 70, 71, 72, 73, 74, 75, 76, 77, 78, 104, 107, 109, 127, 148, 151, 152, 161, 163, 164, 177, 180, 210

metaphysical imposition, 35, 37, 38

metastable state, 19, 144, 145

methylmercury, 125

Meyer, 154

microelectrode, 66

microfrontier, 108, 111

microholography, 214

micromanipulators, 190

microteleoperators, 191, 214

microtrabecular lattice (MTL), 103, 107

microtubule, 7, 8, 11, 13, 14, 24, 51, 53, 54, 55, 56, 75, 76, 85, 86, 87, 89, 90, 92, 94, 95, 97, 103, 104, 115, 117, 121, 125, 128, 139, 145, 153, 159, 161, 162, 163, 165, 166, 169, 171, 172, 173, 176, 182, 186, 218

microtubule bridges, 165

microtubule organizing center (MTOC), 55, 85

mind, 5, 14, 33, 34, 35, 36, 38, 39, 57, 58, 69, 77, 217

mineral fiber asbestos, 125

miniature endplate potential, 63

Mitchison, T., 92, 93, 171

mitochondria, 50, 51, 62, 76, 87, 115, 133

mitotic apparatus, 84

mitotic spindle, 5, 54, 83, 89, 120, 124

module, 77, 201

molecular computing, 20, 21, 191, 205

molecular orbitals, 156, 164

monomer, 85, 98, 169, 172, 177, 178

Moran, 88, 159

morphogenesis, 25, 108

motility, 36, 50, 51, 99, 111, 113, 121, 126

movement, 20, 29, 35, 36, 37, 41, 51, 52, 76, 80, 87, 91, 97, 98, 109, 111, 112, 113, 115, 116, 117, 119, 122, 135, 136, 144, 149, 158, 160, 166, 179, 181, 192, 195, 197, 202, 211

muscle, 20, 22, 61, 63, 65, 68, 80, 83, 98, 104, 108, 111, 112, 116, 119, 120, 123, 126, 129, 130, 133, 134, 135, 140, 141, 148, 149, 160

myelin, 45, 60, 62, 64

myosin, 63, 98, 106, 108, 112, 114, 116, 117, 119, 120, 123, 126, 127, 129, 130, 135, 140, 141, 143, 147, 160

myosin head, 112, 116, 119, 140, 141, 143

nano-antennas, 206

nanoapertures, 198, 200

nanodevices, 191, 214, 215, 217

nanolithography, 20, 199, 203, 204, 207

nanomachines, 190

nanometer, 5, 7, 43, 82, 86, 103, 107, 125, 133, 144, 161, 162, 174, 182, 187, 190, 194

nanoscale, 5, 7, 8, 9, 20, 21, 25, 32, 37, 97, 105, 116, 127, 143, 150, 184, 186, 190, 191, 197, 198, 200, 203, 205, 206, 213, 214, 215, 217

nanoscale robots, 190, 205

nanosecond, 5, 7, 19, 43, 127, 133, 136, 144, 176, 177, 178, 179, 210

nanotechnology, 6, 20, 37, 46, 57, 58, 126, 127, 142, 150, 190, 191, 200, 201, 202, 212, 213, 215

nanotechnology workstation, 200, 201, 202, 212

nanovision, 201, 214

NASA, 215

negative entropy, 88, 184

nerve conduction, 153

nerve impulse, 61, 66, 67

network, 11, 12, 35, 41, 44, 45, 55, 67, 69, 70, 72, 74, 78, 79, 80, 81, 87, 94, 99, 106, 108, 120, 167, 170, 183

neural architecture, 65

neural net, 9, 10, 15, 16, 17, 33, 34, 37, 41, 45, 64, 67, 68, 69, 70, 71, 72, 76, 77, 127, 183

neurofibrillary tangle, 125, 189, 190

neurofilament, 105, 123, 125, 163, 164

neuron, 11, 12, 14, 33, 34, 39, 41, 44, 45, 60, 61, 64, 65, 66, 67, 68, 72, 75, 76, 77, 78, 87, 123, 127, 159

neuron growth cone, 87

neurotransmitter, 46, 62, 63, 64, 68, 74, 76, 87, 106, 107, 153, 154, 185

Nirenberg, M., 127

nmemonic trace, 71

nonlinear, 20, 22, 30, 36, 38, 41, 45, 49, 51, 57, 131, 133, 140, 141, 145, 155, 167

nuclei, 8, 39, 50, 61, 65, 80, 87, 115, 119

olive oil, 154

order and chaos, 150

ordered water, 60, 110, 111, 127, 138, 145, 166, 167, 174, 181, 183

osmium tetroxide, 84

Overton, 154

oxygen, 38, 50, 57, 125, 129, 130, 131, 132, 133, 140, 148, 154

oxyhemoglobin, 133

pain, 124, 148, 149

parallelism, 11, 12, 22, 45, 60, 93

Pavlov, 68

peptide, 111, 129, 140, 141

perceptron, 69

pericentriolar substance, 93

perikaryon, 60, 62

peripheral nervous system, 60, 66

phase space, 70, 150, 151

phonon, 136

phosphorylation, 21, 134, 135

piezoceramic, 20, 192, 193, 196

piezoelectricity, 139

Pihlaja, 131, 164, 165, 166, 173

pineal gland, 39

Pohl, D., 197, 198, 206, 212, 214

poison, 124

polarity, 59, 85, 89, 92, 115, 122, 161, 171, 177, 181, 182

polarizability, 139

polypeptide, 86, 129, 130, 131, 141, 144

preformation theory, 122

prerepresentation, 70

primary structure, 129

primordial soup, 47, 49, 188

prions, 36, 48, 184

profilin, 108

prokaryote, 58

prophase, 94, 96

proprioception, 159

protein, 7, 8, 17, 21, 35, 36, 45, 48, 49, 51, 58, 61, 62, 75, 76, 79, 83, 84, 87, 92, 97, 98, 99, 105, 108, 110, 112, 115, 116, 117, 118, 127, 129, 130, 131, 132, 133, 134, 135, 136, 138, 139, 140, 141, 144, 147, 148, 153, 154, 155, 156, 157, 160, 164, 166, 167, 169, 172, 184, 186, 187, 188, 189, 211, 213, 214

protein assembly, 187

protein coat, 186, 187, 189, 211

protein conformational changes, 154

protein structure, 36, 76, 130, 138, 141

proteinoids, 48, 184

protoplasm, 35, 45, 50, 80

protoplasmic consciousness, 36

psychon, 72

pulse logic, 66, 67, 68

pyroelectric, 140

quantum field theory, 146, 174

quantum vibrational energy, 136

quantum well devices, 20, 206

Quate, C., 196, 197, 204

quaternary structure, 130

Ramon y Cajal, 67

reaction-diffusion pattern, 24

recall, 43, 71, 72, 73, 75, 151, 152, 153

receptor, 61, 63, 64, 68, 106, 107, 130, 144, 153, 156, 159, 168

receptor potentials, 61, 64

reflex center, 65, 66, 68

replicators, 6, 7, 215

representation, 17, 21, 22, 67, 68, 71, 72, 75, 77, 79, 86, 97, 109, 157, 160, 164

resonance, 45, 48, 72, 92, 129, 135, 136, 137, 143, 144, 145, 147, 157, 169, 177, 217

respiratory arrest, 150

reticular activating system (RAS), 39

reticular theory, 80

retroviruses, 185

RNA, 5, 47, 48, 49, 51, 52, 109, 185, 186, 187

Rohrer, H., 193, 197, 206, 211

Roth, 131, 164, 165, 166, 173

sarcode, 80

satellite, 56, 60, 167

satellite cell, 60

satiety center, 65

scanning tunneling microscopy (STM), 20, 193

Schneiker, C., 6, 13, 14, 16, 18, 19, 28, 30, 31, 190, 191, 192, 193, 195, 196, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 214, 215, 218

Schwann cell, 60

Scott, A. C., 6, 44, 61, 141, 187, 188

second law of thermodynamics, 49, 88, 137, 184, 188

secondary structure, 130

Seifriz, 83

selectionist, 69, 70, 71

self, 8, 9, 14, 20, 30, 49, 52, 60, 77, 88, 121, 141, 146, 147, 163, 170, 174, 181, 184, 199, 213, 215

self-focusing, 146, 147, 174

self-replicating automata, 215

semiconductor, 20, 77, 194

senility, 125

sensory transduction, 159

serotonin, 62, 156

Sherrington, 65, 66

Shigenaka, 164, 165, 166, 173

short term, 24, 37, 73, 76, 109, 152

signal detector, 55, 97, 167

signal transduction, 157, 161

slime mold, 36, 119, 125, 154

Smith, S., 6, 35, 48, 154, 174, 176, 179, 180, 181, 188, 196, 197

smooth endoplasmic reticulum, 105

Snow, 148, 150

sol, 24, 25, 36, 60, 84, 91, 104, 106, 108, 109, 127, 157, 160, 174, 175, 182, 183

sol-gel field, 157

sol-gel transformation, 24

solid state, 35, 87, 110, 111, 201

solitary wave, 141, 143

soliton, 11, 20, 21, 118, 119, 141, 142, 143, 147, 155, 182

spin glass, 70

spinal cord, 39, 59, 60

spirochete, 50, 52

stage of analgesia, 148

stage of delirium, 148, 149

stage of respiratory paralysis, 148

stereotyped, 68

STM, 20, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 208, 209, 210, 211, 212, 213, 214, 215, 218

STM/FMs, 203, 204, 205, 206, 210, 211, 213, 214, 215

superconductivity, 11, 17, 19, 87, 144, 145, 166, 177, 182, 215

superlattices, 206

super-sensitivity, 68

supersound acoustic soliton, 142

surgical procedures, 148

surgical stage, 148

symbiosis, 8, 50, 57, 58, 217

symbols, 12, 16, 21, 22, 68

synapses, 16, 34, 37, 40, 41, 42, 44, 45, 61, 62, 63, 64, 65, 66, 68, 69, 73, 74, 75, 76, 77, 78, 105, 107, 116, 122, 123, 125, 153, 183

synaptic plasticity, 17, 41, 45, 60, 68, 75, 87, 158

synaptic potentials, 61, 150

synaptic terminal, 61, 106

Szent-Gyorgyi, 135

tabula rasa, 69, 70

talin, 108

tau, 99, 102, 123

tensegrity, 60, 112, 121, 127, 170, 182, 217

tensegrity mast, 170

tertiary structure, 130

theoretical models, 157

thermodynamic, 21, 188

thick filament, 116

threshold, 41, 44, 64, 70, 76, 141, 144, 145, 146

tobacco mosaic virus, 138, 139, 184, 186, 187

toxin, 124, 125

transient redundancy, 69

transition temperatures, 215

treadmilling, 91, 92, 94

trophism, 45, 68, 121

troponin, 108, 116

tubulin, 7, 75, 81, 82, 85, 86, 87, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 102, 118, 119, 121, 123, 129, 130, 139, 140, 144, 145, 154, 157, 158, 159, 161, 162, 164, 165, 169, 172, 175, 176, 177, 178, 179, 182, 217

tyrosinated, 82, 86, 161, 162

tyrosinated/glutamated, 161

ultrasharp knife edges, 197

ultrasharp tips, 197

ultrasonic energy, 187

unitary mechanism, 154

universe, 10, 21, 24, 31, 43, 47, 56, 59, 130

urate, 124

vaccines, 188

Van der Waals force, 130, 131, 136, 138, 139, 153, 154, 169, 197

van Leeuwenhoek, 80, 111

Varela, 88, 159

vesicle, 63, 107

vicinal water, 110, 111, 137, 138

villin, 108

vimentin, 104, 125

vinblastine, 124, 159, 164

vincristine, 124

vinculin, 108

viral infection, 124

virus, 7, 131, 138, 167, 184, 185, 186, 187, 188, 189, 211, 213

von Neumann, J., 7, 9, 12, 21, 29, 184, 215

Wallace, 37, 70, 77

Watt, R., 6, 150, 151, 152, 155, 174, 175, 176, 179, 180, 181

white matter, 60

Winfree, A., 6, 22, 23, 24

word processors, 164

zinc tubulin sheets, 91