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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.

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Molecular Cell Biology. 4th edition.

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Section 19.1Microtubule Structures

A microtubule is a polymer of globular tubulin subunits, which are arranged in a cylindrical tube measuring 24 nm in diameter — more than twice the width of an intermediate filament and three times the width of a microfilament (Figure 19-1). Varying in length from a fraction of a micrometer to hundreds of micrometers, microtubules are much stiffer than either microfilaments or intermediate filaments because of their tubelike construction.

Figure 19-1. Electron micrograph of a negatively stained microtubule.

Figure 19-1

Electron micrograph of a negatively stained microtubule. Globular tubulin subunits, each about 8 nm long, form the walls of this cylindrical structure. [Courtesy of E. M. Mandelkow.]

Heterodimeric Tubulin Subunits Compose the Wall of a Microtubule

The building block of a microtubule is the tubulin subunit, a heterodimer of α- and β-tubulin. Both of these 55,000-MW monomers are found in all eukaryotes, and their sequences are highly conserved. Although a third tubulin, γ-tubulin, is not part of the tubulin subunit, it probably nucleates the polymerization of subunits to form αβ-microtubules. Encoded by separate genes, the three tubulins exhibit an interesting, but not yet understood, homology with a 40,000-MW bacterial GTPase, called FtsZ. This bacterial protein has structural and functional similarities with tubulin, including the ability to polymerize and a role in cell division. Perhaps the protein carrying out these ancestral functions in bacteria was modified during evolution to fulfill the diverse roles of microtubules in eukaryotes.

The interactions holding α-tubulin and β-tubulin in a heterodimeric complex are strong enough that a tubulin subunit rarely dissociates under normal conditions. Each tubulin subunit binds two molecules of GTP. One GTP-binding site, located in α-tubulin, binds GTP irreversibly and does not hydrolyze it, whereas the second site, located on β-tubulin, binds GTP reversibly and hydrolyzes it to GDP. The second site is called the exchangeable site because GDP can be displaced by GTP. The recently solved atomic structure of the tubulin subunit reveals that the nonexchangeable GTP is trapped at the interface between the α- and β-tubulin monomers, while the exchangeable GTP lies at the surface of the subunit (Figure 19-2a). As we discuss later, the guanine bound to β-tubulin regulates the addition of tubulin subunits at the ends of a microtubule.

Figure 19-2. Microtubule structure.

Figure 19-2

Microtubule structure. (a) Ribbon diagram of the dimeric tubulin subunit, showing the α-tubulin and β-tubulin monomers and their bound nonexchangeable GTP (red) and exchangeable (more...)

In a microtubule, lateral and longitudinal interactions between the tubulin subunits are responsible for maintaining the tubular form. Longitudinal contacts between the ends of adjacent subunits link the subunits head to tail into a linear protofilament. Within each protofilament, the dimeric subunits repeat every 8 nm. Through lateral interactions, protofilaments associate side by side into a sheet or cylinder — a microtubule. The exact arrangement of protofilaments in the wall of a microtubule is currently debated. In the model shown in Figure 19-2b, the heterodimers in adjacent protofilaments are staggered only slightly, forming spiraling rows of α- and β-tubulin monomers in the microtubule wall. In an alternative model, the α-tubulin and β-tubulin subunits are staggered enough to give the microtubule wall a checkerboard pattern.

Virtually every microtubule in a cell is a simple tube, a singlet microtubule, built from 13 protofilaments. In rare cases, singlet microtubules contain more or fewer protofilaments; for example, certain microtubules in the neurons of nematode worms contain 11 or 15 protofilaments. In addition to the simple singlet structure, doublet or triplet microtubules are found in specialized structures such as cilia and flagella (doublet microtubules) and centrioles and basal bodies (triplet microtubules). Each of these contains one complete 13-protofilament microtubule (the A tubule) and one or two additional tubules (B and C) consisting of 10 protofilaments (Figure 19-3).

Figure 19-3. Arrangement of protofilaments in singlet, doublet, and triplet microtubules.

Figure 19-3

Arrangement of protofilaments in singlet, doublet, and triplet microtubules. In cross section, a typical microtubule, a singlet, is a simple tube built from 13 protofilaments. In a doublet (more...)

A microtubule is a polar structure, its polarity arising from the head-to-tail arrangement of the α- and β-tubulin dimers in a protofilament. Because all protofilaments in a microtubule have the same orientation, one end of a microtubule is ringed by α-tubulin, while the opposite end is ringed by β-tubulin. Microtubule-assembly experiments discussed later show that microtubules, like actin microfilaments, have a (+) and a (−) end, which differ in their rates of assembly.

Microtubules Form a Diverse Array of Both Permanent and Transient Structures

As with microfilaments, there are two populations of microtubules: stable, long-lived microtubules and unstable, short-lived microtubules. Unstable microtubules are found when cell structures composed of microtubules need to assemble and disassemble quickly. For example, during mitosis, the cytosolic microtubule network characteristic of interphase cells disappears, and the tubulin from it is used to form the spindle-shaped apparatus that partitions chromosomes equally to the daughter cells (Figure 19-4a). When mitosis is complete, the spindle disassembles and the interphase microtubule network reforms.

Figure 19-4. Diverse microtubule structures in cells.

Figure 19-4

Diverse microtubule structures in cells. (a) In cultured animal cells at interphase, microtubules are arranged in long fibers, which fill the entire cytosol. In a cell undergoing mitosis (more...)

In contrast to these short-lived, transient structures, some cells, usually nonreplicating cells, contain stable microtubule- based structures. These include the axoneme in the flagellum of sperm and the marginal band of microtubules in most red blood cells and platelets. Another example occurs in nerve cells (neurons), which are long-lived and seldom need to establish new connections in an adult. Neurons, however, must maintain long processes, called axons, and do so with the aid of an internal core of stable microtubules (Figure 19-4b). The disassembly of such stable structures would have catastrophic consequences — sperm would be unable to swim, a red blood cell would lose its springlike pliability, and axons would retract.

Microtubules Assemble from Organizing Centers

In an interphase fibroblast cell, a seemingly haphazard and random network of microtubules permeates the entire cytosol. However, upon closer analysis, we can see that the cytosolic microtubules are arranged in a hub-and-spoke array that lies at the center of a cell (Figure 19-5a). The microtubule spokes radiate from a central site occupied by the centrosome, which is the primary microtubule-organizing center (MTOC) in many interphase cells. We will use the term MTOC to refer to any of the structures used by cells to nucleate and organize microtubules. In animal cells, the MTOC is a centrosome, a lattice of microtubule-associated proteins that sometimes but not always contains a pair of centrioles (Figure 19-5b). The centrioles, each a pinwheel array of triplet microtubules, lie in the center of the MTOC but do not make direct contact with the ends of the cytosolic microtubules. Centrioles are not present in the MTOCs of plants and fungi; moreover, some epithelial cells and newly fertilized eggs from animals also lack centrioles. Thus, it is the associated proteins in an MTOC that have the capacity to organize cytosolic microtubules.

Figure 19-5. Microtubule-organizing center.

Figure 19-5

Microtubule-organizing center. (a) Fluorescence micrograph of a Chinese hamster ovary cell stained with antibodies specific for tubulin and a centrosomal protein. The microtubules (green) (more...)

The mechanism whereby the MTOC organizes cytosolic microtubules was deduced from several microtubule-assembly studies. In cells treated with colcemid, a microtubuledepolymerizing drug, almost all the cytosolic microtubules, except those in the centrosome, are depolymerized. When colcemid is removed by washing the cells with a colcemid-free culture medium, tubulin repolymerizes to form new microtubules, which radiate from the MTOC (Figure 19-6). This result could arise by either of two mechanisms: the MTOC could nucleate polymerization of tubulin subunits or it could gather together the ends of microtubules that assembled independently in the cytosol. To identify the correct mechanism, centrosomes were purified and tested for their interaction with tubulin subunits or microtubules. The addition of purified centrosomes to a solution of tubulin dimers nucleated the assembly of microtubules whose (−) ends remained associated with the centrosome. In the absence of centrosomes, the concentration of dimers was too low to permit spontaneous formation of microtubules. Thus the centrosome functions to nucleate the assembly of cytosolic microtubules.

Figure 19-6. The disassembly and reassembly of microtubules in interphase cultured animal cells can be induced either by adding and subsequently removing colcemid or by cooling to 0 °C and subsequently rewarming to 37 °C.

Figure 19-6

The disassembly and reassembly of microtubules in interphase cultured animal cells can be induced either by adding and subsequently removing colcemid or by cooling to 0 °C and (more...)

Most Microtubules Have a Constant Orientation Relative to MTOCs

The MTOC, which probably is the major organizing structure in a cell, helps determine the organization of microtubule-associated structures and organelles (e.g., mitochondria, the Golgi complex, and the endoplasmic reticulum). In a nonpolarized animal cell such as a fibroblast, an MTOC is perinuclear and strikingly at the center of the cell. Although the centering mechanism is not well understood, it most likely involves microtubules that scout out the cell periphery. Because microtubules assemble from the MTOC, microtubule polarity becomes fixed in a characteristic orientation. In most animal cells, for instance, the (−) ends of microtubules are closest to the MTOC or basal body (Figure 19-7a). During mitosis, the centrosome duplicates and migrates to new positions flanking the nucleus. There the centrosome becomes the organizing center for microtubules forming the mitotic apparatus, which will separate the chromosomes into the daughter cells during mitosis( Figure 19-7b). The microtubules in the axon of a nerve cell, which help stabilize the long process, are all oriented in the same direction (Figure 19-7c)

Figure 19-7. . Orientation of cellular microtubules.

Figure 19-7

. Orientation of cellular microtubules. (a) In interphase animal cells, the (−) ends of most microtubules are proximal to the MTOC. Similarly, the microtubules in flagella and cilia (more...)

In contrast to the single perinuclear MTOC present in most interphase animal cells, plant cells, polarized epithelial cells, and embryonic cells contain hundreds of MTOCs, which are distributed throughout the cell, often near the cell cortex. In plant cells and polarized epithelial cells, a cortical array of microtubules aligns with the cell axis (Figure 19-7d). In both cell types the polarity of the cell is linked to the orientation of the microtubules.

The γ-Tubulin Ring Complex Nucleates Polymerization of Tubulin Subunits

Despite its amorphous appearance, the pericentriolar material of an MTOC is an ordered lattice that contains many proteins that are necessary for initiating the assembly of microtubules. One of these, γ-tubulin, was first identified by genetic studies designed to discover proteins that interact with β-tubulin. Subsequent studies demonstrated that γ-tubulin and the lattice protein pericentrin are part of the pericentriolar material of centrosomes (Figure 19-8a); it has also been detected in MTOCs that lack a centriole. The finding that introduction of antibodies against γ-tubulin into cells blocks microtubule assembly implicates γ-tubulin as a necessary factor in nucleating polymerization of tubulin subunits.

Figure 19-8. γ-Tubulin-mediated assembly of microtubules.

Figure 19-8

γ-Tubulin-mediated assembly of microtubules. (a) Electron micrograph of γ-tubulin ring complexes (γ-TuRC). The complexes, isolated from Xenopus oocytes, consist (more...)

Approximately 80 percent of the γ-tubulin in cells is part of a 25S complex, which has been isolated from extracts of frog oocytes and fly embryos. It is named the γ-tubulin ring complex (γ-TuRC) for its ringlike appearance in the electron microscope. In vitro experiments show that the γ-TuRC can directly nucleate microtubule assembly at subcritical tubulin concentrations, that is, at concentrations below which polymerization would not occur in the absence of the γ-TuRC (Figure 19-8b). These observations provide additional evidence that γ-tubulin plays a key role in directing microtubule assembly in vivo. Current biochemical, structural, and genetic studies are beginning to clarify the mechanism of nucleation.

SUMMARY

  •  Tubulins belong to an ancient family of GTPases that polymerize into hollow cylindrical structures, called microtubules, 24 nm in diameter.
  •  Dimeric αβ-tubulin subunits interact longitudinally to form protofilaments, which associate laterally into microtubules (see Figure 19-2b).
  •  Microtubules exhibit both structural and functional polarity.
  •  Microtubule-organizing centers (MTOCs), including centrosomes and basal bodies, nucleate the assembly of cytosolic microtubules.
  •  In most cases, the (−) end of a microtubule is adjacent to the MTOC from which it assembles and the (+) end is distal (see Figure 19-7).
  •  In some animal cells, the MTOC lies at the cell center, where it organizes cellular organelles.
  •  A γ-tubulin-containing complex is a major component of the pericentriolar material and is able to nucleate polymerization of tubulin subunits to form microtubules in vitro.

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Copyright © 2000, W. H. Freeman and Company.
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