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).

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.

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