.
Globular tubulin subunits, each about 8 nm long, form the walls of this cylindrical structure. [Courtesy of E. M. Mandelkow.]
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Globular tubulin subunits, each about 8 nm long, form the walls of this cylindrical structure. [Courtesy of E. M. Mandelkow.]
). 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.
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.
(a) Ribbon diagram of the dimeric tubulin subunit, showing the α-tubulin and β-tubulin monomers and their bound nonexchangeable GTP (red) and exchangeable GDP (blue) nucleotides. An anticancer drug, taxotere (green), was used in the structural studies to stabilize the dimer structure. (b) The organization of tubulin subunits in a microtubule. The subunits are aligned end to end into a protofilament (magenta highlight). The side-by-side packing of protofilaments forms the wall of the microtubule. In this model, the protofilaments are slightly staggered so that α-tubulin in one protofilament is in contact with α-tubulin in the neighboring protofilaments. In an alternative model, the protofilaments are staggered by one-half subunit, forming a checkerboard pattern. In either structure, the microtubule displays a structural polarity in that addition of subunits occurs preferentially at one end, designated the (+) end. [Part (a) modified from E. Nogales, S. G. Wolf, and K. H. Downing, 1998, Nature391:199; courtesy of E. Nogales. Part (b) adapted from Y. H. Song and E. Mandelkow, 1993, Proc. Nat’l. Acad. Sci. USA 90:1671.]
). As we discuss later, the guanine bound to β-tubulin
regulates the addition of tubulin subunits at the ends of a microtubule.
, 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.
In cross section, a typical microtubule, a singlet, is a simple tube built from 13 protofilaments. In a doublet microtubule, an additional set of 10 protofilaments forms a second tubule (B) by fusing to the wall of a singlet (A) microtubule. Attachment of another 10 protofilaments to the B tubule of a doublet microtubule creates a C tubule and a triplet structure.
).
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.
(a) In cultured animal cells at interphase, microtubules are arranged in long fibers, which fill the entire cytosol. In a cell undergoing mitosis (inset), the network of microtubules disappears and is replaced with the spindle-shaped arrangement of microtubules in the mitotic apparatus. (b) Microtubules and intermediate filaments in a quick-frozen frog axon visualized by the deep-etching technique. There are several 24-nm-diameter microtubules running longitudinally. Thinner, 10-nmdiameter intermediate filaments also run longitudinally and form occasional connections with microtubules. [Part (a) courtesy of B. R. Brinkley and B. Scott, Baylor College of Medicine; part (b) from N. Hirokawa, 1982, J. Cell Biol. 94:129; courtesy of N. Hirokawa.]
). When mitosis is complete, the spindle disassembles
and the interphase microtubule network reforms.
). 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.
(a) Fluorescence micrograph of a Chinese hamster ovary cell stained with antibodies specific for tubulin and a centrosomal protein. The microtubules (green) are seen to radiate from a central point, the microtubule-organizing center (MTOC), near the nucleus. The MTOC (yellow) is detected with an antibody to Cep135, a protein in the pericentriolar material. (b) Electron micrograph of the MTOC in an animal cell. The pair of centrioles (red), C and C′, in the center are oriented at right angles; thus one is seen in cross section, and one longitudinally. Surrounding the centrioles is a cloud of material, the pericentriolar (PC) matrix, which contains γ-tubulin and pericentrin. Embedded within the MTOC, but not contacting the centrioles, are the (−) ends of microtubules (MT; yellow). [Part (a) courtesy of R. Kuriyama; part (b) from B. R. Brinkley, 1987, in Encyclopedia of Neuroscience, vol. II, Birkhauser Press, p. 665; courtesy of B. R. Brinkley.]
). 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.
Both the addition and the removal of tubulin occurs at the (+) ends of the microtubules.
). 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.
(a) In interphase animal cells, the (−) ends of most microtubules are proximal to the MTOC. Similarly, the microtubules in flagella and cilia have their (−) ends continuous with the basal body, which acts as the MTOC in these structures. (b) As cells enter mitosis, the microtubule network rearranges, forming a mitotic spindle. The (−) ends of all spindle microtubules point toward one of the two MTOCs, or poles, as they are called in mitotic cells. (c) In nerve cells, the (−) ends of axonal microtubules are oriented toward the base of the axon. However, dendritic microtubules have mixed polarities. (d) In plant cells, which contain numerous MTOCs, microtubules line the cell cortex. Webs of microtubules cap the growing ends of a plant cell.
. 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)
. In both cell types the polarity of the cell is linked to the
orientation of the microtubules.
(a) Electron micrograph of γ-tubulin ring complexes (γ-TuRC). The complexes, isolated from Xenopus oocytes, consist of 8 polypeptides and measure 25nm in diameter. (b) Proposed models for γ-tubulin-mediated assembly of microtubules. In alternative models, γ-TuRC is thought to nucleate microtubule assembly by presenting a row of γ-tubulin subunits (left) or forming a protofilament (right), which can directly bind αβ-tubulin subunits. [Part (a) courtesy of Y. Zheng; part (b) modified from C. Wiese and Y. Zheng, 1999, Curr. Opin. Struc. Biol. 9:250–259.]
); 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.
). 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.Tubulins belong to an ancient family of GTPases that polymerize into hollow cylindrical structures, called microtubules, 24 nm in diameter.
).Microtubules exhibit both structural and functional polarity.
Microtubule-organizing centers (MTOCs), including centrosomes and basal bodies, nucleate the assembly of cytosolic microtubules.
).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.
