Microtubule Structure and Function

Microtubules (MT) are hollow cylinders about 25 nanometers in diameter whose walls are polymerized arrays of protein subunits. Their lengths may range from tens of nanometers during early assembly, to possibly meters in nerve axons within large animals. The protein subunits assemble in longitudinal strings called protofilaments; thirteen parallel protofilaments laterally align to form the hollow "tubules." The subunits are "barbell shaped" proteins ("dimers") which in turn consist of two globular proteins ("monomers") known as alpha and beta tubulin (Figure 5.4). Alpha and beta tubulin monomers are similar molecules with identical orientation within protofilaments and tubule walls. Each monomer consists of about 500 amino acids, weighs about 55 kilodaltons (Chapter 6), and has a local polarity or charge orientation. MT which grow from cell centers have a plus end (beta tubulin) which extends outward from the cell center ("centrosome") into the cell periphery. The minus end (alpha tubulin) remains anchored to a microtubule organizing center (MTOC) within the centrosome. Each dimer, as well as each MT, appears to have an electrical polarity or dipole, with the negative end oriented towards the alpha monomer and cell center, and the positive end towards the beta monomer and cell periphery.

Figure 5.4: Microtubules are cylinders whose walls are 13 protofilaments, each a string of 8 nm tubulin dimers. Alpha and beta tubulin monomers form the dimers. Each dimer has 6 neighbors. By Fred Anderson.

The dimers are held together by relatively weak Van der Waals hydrophobic forces such as dipole coupling (Chapter 6). Dimer neighbors form hexagonal lattices with a "leftward" tilt and several helical patterns may be discerned in the relations among dimers. The crystal-like symmetry packing of tubulin in microtubules has been evaluated by Djuro Koruga (1986) of the Molecular Machines Research Unit at the University of Belgrade in Yugoslavia (Chapter 8).

MT from different life forms have marked similarities, but subtle differences. Comparison of MT from nerve cells of earthworms and mammals shows that the more primitive worm MT are more variable in geometric structure with MT ranging from 9 to 11 protofilaments, whereas mammalian MT generally have 13. Tubulins from among different species including mammals and plants bind to common antibodies and tubulins from different species may coassemble into hybrid MT. Despite these common traits, the diversity of tubulin gene expression has proved far greater than imagined years ago. Analysis of tubulin by amino acid sequencing and advanced electrophoretic techniques have shown that multiple, different alpha and beta tubulins exist concurrently, with the greatest diversity shown by beta tubulin. For example, Lee (1986) and colleagues at St. Louis University have shown that as many as 11 different tubulin forms exist in rat thyroid microtubules and 17 different forms exist in rat brain microtubules. Thus alpha and beta tubulin are families of "isozymes," each of which may have specific functions or binding of microtubule associated proteins (MAPs). Another tubulin variable, detyrosination, occurs in the cytoplasm subsequent to DNA transcription. Detyrosination is the removal of the terminal amino acid, tyrosine, from the polypeptide chain which comprises beta tubulin. Removal of tyrosine exposes an acidic amino acid, glutamate. Local factors in the cytoplasm independent of genetic programming determine whether or not individual tubulin subunits are "tyrosinated" or "glutamated." Marc DeBrabander (1986) and collaborators at Janssen Pharmaceutica in Belgium have been able to identify specific tubulin subunits within assembled microtubules which are either tyrosinated or glutamated. Their elegant studies show heterogeneous patterns of tyrosinated and glutamated tubulin which could indicate an information representation coupled to specific MT functions by the action of MAPs (Figure 5.5).

Figure 5.5: Microtubule double labeled with immunogold tubulin antibody. Large circles, 10 nanometer gold particles, label glutamated tubulin; small circles, 5 nanometer gold particles, label tyrosinated tubulin. With permission from Geuens et al (1986), courtesy of Marc DeBrabander and Janssen Pharmaceutica Research Laboratories.

Figure 5.5: Microtubule double labeled with immunogold tubulin antibody. Large circles, 10 nanometer gold particles, label glutamated tubulin; small circles, 5 nanometer gold particles, label tyrosinated tubulin. With permission from Geuens et al (1986), courtesy of Marc DeBrabander and Janssen Pharmaceutica Research Laboratories.

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.

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