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Most cell types

Microtubule severing

Op18 (stathmin)


Most cell types

Binds tubulin dimers

filaments, or other microtubules, and its length controls how far apart microtubules are spaced (Figure 20-12).

The microtubule-binding domain contains several repeats of a conserved, positively charged four-residue amino acid sequence that binds the negatively charged C-terminal part of tubulin. This binding is postulated to neu tralize the charge repulsion between tubulin subunits within a microtubule, thereby stabilizing the polymer. MAP1A and MAP1B are large, filamentous molecules found in axons and dendrites of neurons as well as in nonneuronal cells. Each of these MAPs is derived from a single precursor polypeptide, which is proteolytically processed in a cell to generate one light chain and one heavy chain.

Other stabilizing MAPs include MAP2, MAP4, Tau, and CLIP170. MAP4, the most widespread of all the MAPs, is found in neuronal and non-neuronal cells. In mitosis, MAP4 regulates microtubule stability, and CLIP170 cross-links microtubules to chromosomes. MAP2 is found only in dendrites, where it forms fibrous cross-bridges between microtubules and links microtubules to intermediate filaments. Tau, which is much smaller than most other MAPs, is present in both axons and dendrites. This protein exists in several iso-

M EXPERIMENTAL FIGURE 20-12 Spacing of microtubules depends on length of projection domain in bound microtubule-associated proteins. Insect cells transfected with DNA expressing either long-armed MAP2 protein or short-armed Tau protein grow long axonlike processes. (a) Electron micrographs of cross sections through the processes induced by the expression of MAP2 (left) or Tau (right) in transfected cells. Note that the spacing between microtubules (MTs) in MAP2-containing cells is larger than in Tau-containing cells. Both cell types contain approximately the same number of microtubules, but the effect of MAP2 is to enlarge the caliber of the axonlike process. (b) Diagrams of association between microtubules and MAPs. Note the difference in the lengths of the projection arms in MAP2 and Tau. [Part (a) from J. Chen et al., 1992, Nature 360:674.]

Membrane Tau

forms derived from alternative splicing of a tau mRNA. The ability of Tau to cross-link microtubules into thick bundles may contribute to the stability of axonal microtubules. Findings from gene transfection experiments implicate Tau in ax-onal elongation. Deletion of the genes encoding Tau and MAPIB leads to more severe phenotypes of axonal dysgenesis and lethality. Furthermore, aberrant polymerization of Tau into filaments is linked to neurodegenerative diseases such as human dementia in Alzheimer's patients.

When stabilizing MAPs coat the outer wall of a micro-tubule, tubulin subunits are unable to dissociate from the ends of that microtubule. Although bound MAPs generally dampen the rate of microtubule disassembly, the assembly of microtubules is affected to varying degrees: some MAPs, such as Tau and MAP4, stabilize microtubules, whereas other MAPs do not. Because of the effect of assembly MAPs on microtubule dynamics, modulating the binding of MAPs can control the length of microtubules. In most cases, this control is accomplished by the reversible phosphorylation of the MAP projection domain. Phosphorylated MAPs are unable to bind to microtubules; thus they promote micro-tubule disassembly. MAP kinase, a key enzyme for phospho-rylating MAPs, is a participant in many signal-transduction pathways (Chapter 14), indicating that MAPs are targets of many extracellular signals. MAPs, especially MAP4, are also phosphorylated by a cyclin-dependent kinase (CDK) that plays a major role in controlling the activities of various proteins in the course of the cell cycle (Chapter 21).

A second group of MAPs directly destabilizes microtubules in many cell types. One of this group, called katanin, severs intact cytosolic microtubules by an ATP-dependent process. Internal bonds between tubulin subunits in the microtubule wall are broken, causing microtubules to fragment. This activity may release microtubules at the MTOC. Another protein, called Op18 or stathmin, increases the frequency of rapid disassembly of microtubules in the mitotic spindle. This protein may act by binding tubulin dimers, thereby reducing the pool of dimers available for polymerization. Phosphorylation inactivates Op18 and inhibits its destabilizing effect.

Colchicine and Other Drugs Disrupt Microtubule Dynamics

Some of the earliest studies of microtubules employed several drugs that inhibit mitosis, a cell process that depends on mi-crotubule assembly and disassembly. Two such drugs isolated from plants, colchicine and taxol, have proved to be very powerful tools for probing microtubule function, partly because they bind only to ap-tubulin or microtubules and not to other proteins and because their concentrations in cells can be easily controlled.

Colchicine and a synthetic relative, colcemid, have long been used as mitotic inhibitors. In cells exposed to high concentrations of colcemid, cytosolic microtubules depolymer-ize, leaving an MTOC. However, when plant or animal cells are exposed to low concentrations of colcemid, the micro-tubules remain and the cells become "blocked" at meta-

phase, the mitotic stage at which the duplicated chromosomes are fully condensed (see Figure 9-3). When the treated cells are washed with a colcemid-free solution, colcemid diffuses from the cell and mitosis resumes normally. Thus experimenters commonly use colcemid to accumulate metaphase cells for cytogenetic studies; removal of the colcemid leaves a population of cells whose cell cycle is in synchrony. Such synchronous populations are advantageous for studies of the cell cycle (Chapter 21).

The interface between a-tubulin and p-tubulin monomers in dimeric tubulin contains a high-affinity but reversible binding site for colchicine. Colchicine-bearing tubulin dimers, at concentrations much less than the concentration of free tubulin subunits, can add to the end of a growing mi-crotubule. However, the presence of one or two colchicine-bearing tubulins at the end of a microtubule prevents the subsequent addition or loss of other tubulin subunits. Thus colchicine "poisons" the end of a microtubule and alters the steady-state balance between assembly and disassembly. As a result of this disruption of microtubule dynamics, mitosis is inhibited in cells treated with low concentrations of colchicine.

Other drugs bind to different sites on tubulin dimers or to microtubules and therefore affect microtubule stability through different mechanisms. For example, at low concentrations, taxol binds to microtubules and stabilizes them by inhibiting their shortening.

Drugs that disturb the assembly and disassembly of microtubules have been widely used to treat various diseases. Indeed, more than 2500 years ago, the ancient Egyptians treated heart problems with colchicine. Nowadays, this drug is used primarily in the treatment of gout and certain other diseases affecting the joints and skin. Other inhibitors of microtubule dynamics, including taxol, are effective anticancer agents and are used in the treatment of ovarian cancer. I

MTOCs Orient Most Microtubules and Determine Cell Polarity

In an interphase fibroblast cell, cytosolic microtubules are arranged in a distinctive hub-and-spoke array that lies at the center of a cell (Figure 20-13a). The microtubule spokes radiate from a central site occupied by the centrosome, which is the primary microtubule-organizing center in many interphase cells. We will use the term MTOC to refer to any of the structures used by cells to nucleate and organize micro-tubules. In animal cells, the MTOC is usually a centrosome, a collection of microtubule-associated proteins that sometimes but not always contains a pair of centrioles (Figure 20-13b). 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

Are Centrioles Always Pairs
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▲ EXPERIMENTAL FIGURE 20-13 The centrosome, which functions as a microtubule-organizing center, contains a pair of orthogonal centrioles in most animal cells. (a) Micrograph showing several cells each with an MTOC identified by fluorescently labeled antibodies against PCM1, a centrosomal protein. (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 centriole is seen in cross section, and the other longitudinally. Surrounding the centrioles is a cloud of material, the pericentriolar (PC) matrix, which contains ^-tubulin and pericentrin. Embedded within the MTOC, but not contacting the centrioles, are the (—) ends of microtubules (MT; yellow). [Part (a) from A. Kubo and S. Tsukita, 2003, J. Cell Sci. 116:919. Part (b) from B. R. Brinkley, 1987, in Encyclopedia of Neuroscience, vol. 2, Birkhauser Press, p. 665; courtesy of B. R. Brinkley.]

Armelbow Hand Pressure Frozen
(b) Mitotic animal cell
(c) Nerve cell
Cilia Microtubules

▲ FIGURE 20-14 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 have their (—) ends continuous with the basal body, which acts as the MTOC for 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 all axonal microtubules are oriented toward the base of the axon, but dendritic microtubules have mixed polarities.

proteins in an MTOC that have the capacity to organize cy-tosolic microtubules.

Because microtubules assemble from the MTOC, micro-tubule polarity becomes fixed in a characteristic orientation (Figure 20-14). In most interphase animal cells, for instance, the (—) ends of microtubules are closest to the MTOC. In 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 in mitosis. The microtubules in the axon of a nerve cell, which help stabilize the long process, are all oriented in the same direction.

In contrast with 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. In both cell types, the polarity of the cell is linked to the orientation of the microtubules.

The /-Tubulin Ring Complex Nucleates Polymerization of Tubulin Subunits

The MTOC organizes cytosolic microtubules by first nucleating microtubule assembly and then anchoring and releasing microtubules. Despite its amorphous appearance, the peri-centriolar material of an MTOC is an ordered lattice that contains many proteins that are necessary for initiating the assembly of microtubules (see Figure 20-13). One of these proteins, y-tubulin, was first identified in genetic studies designed

▲ EXPERIMENTAL FIGURE 20-15 The -/-tubulin ring complex (//-TuRC) is localized to one end of the microtubule.

(a) A fluorescence micrograph (left) and an electron micrograph (right) of microtubules stained with gold-labeled antibodies to y-tubulin or XGRIP a microtubule-binding protein. Both proteins are components of the y-TuRC. The labeled proteins are localized to one end of the microtubules. (b) A model of the y-TuRC. This complex is thought to nucleate microtubule assembly by to discover proteins that interact with p-tubulin. The results of subsequent studies demonstrated that y-tubulin and the lattice protein pericentrin are part of the pericentriolar material of centrosomes; these proteins have also been detected in MTOCs that lack a centriole. The finding that the introduction of antibodies against y-tubulin into cells blocks microtubule assembly implicates y-tubulin as a necessary factor in nucleating the polymerization of tubulin subunits.

Approximately 80 percent of the y-tubulin in cells is part of a 25S complex, which has been isolated from extracts of frog oocytes and fly embryos. Named the y-tubulin ring complex (y-TuRC) for its ringlike appearance in the electron microscope, the complex comprises eight polypeptides and measures 25 nm in diameter. Findings from in vitro experiments show that the y-TuRC can directly nucleate microtubule assembly at subcritical tubulin concentrations—that is, at concentrations below which polymerization would not take place in the absence of the y-TuRC. To investigate how y-TuRC associates with microtubules, scientists performed immunola-beling experiments with the use of gold-conjugated antibodies specific for y-TuRC components, either y-tubulin or XGRIP. The results of these studies reveal that complexes are localized to one end of a microtubule and are not present along the sides (Figure 20-15a). This location is consistent with a role for presenting a row of y-tubulin subunits, which can directly bind ap-tubulin subunits. This model is supported by plotting the positions of gold-labeled antibodies to either y-tubulin (red) or XGRIP109 (yellow) from several experiments onto a microtubule end. [Parts (a) and (b, right) from T J. Keating and G. G. Borisy, 2000, Nature Cell Biol. 2:352; courtesy of T J. Keating and G. G. Borisy. Part (b, left) modified from C. Wiese and Y Zheng, 1999, Curr. Opin. Struc. Biol. 9:250.]

7-TuRC in nucleating microtubule assembly. A model of 7-TuRC based on electron microscopy shows 7-tubulin in contact with the (—) end of a microtubule (Figure 20-15b).

Cytoplasmic Organelles and Vesicles Are Organized by Microtubules

Fluorescence microscopy reveals that membrane-limited organelles such as the endoplasmic reticulum (ER), Golgi, endosomes, and mitochondria are associated with micro-tubules. For instance, in cultured fibroblasts stained with anti-tubulin antibodies and DiOC6, a fluorescent dye specific for the ER, the anastomosing ER network in the cytosol is seen to colocalize with microtubules (Figure 20-16). If cells are treated with a microtubule-depolymerizing drug, the ER

Dioc6 Fluorescent Dye

▲ EXPERIMENTAL FIGURE 20-16 Fluorescence microscopy reveals colocalization of endoplasmic reticulum membranes and cytosolic microtubules. DiOC6, an ER-binding fluorescent dye (green), and fluorescently-labeled anti-tubulin antibodies (red) were used to stain a cultured frog fibroblast. The alignment of the ER network and microtubules in many but not all regions of the cytoplasm is evident because the cell has sparse microtubules. [Courtesy of M. Terasaki.]

loses its networklike organization. After the drug has been washed from the cells, tubular fingers of ER grow as new microtubules assemble. In cell-free systems, the ER can be reconstituted with microtubules and an ER-rich cell extract. Even under this cell-free regime, ER membranes elongate along microtubules. This close association between the ER and intact microtubules suggests that proteins bind ER membranes to microtubules.

The role of microtubules in organizing the Golgi complex also has been studied extensively. In interphase fibroblasts, the Golgi complex is concentrated near the MTOC. In mitosis (or after the depolymerization of microtubules by col-cemid), the Golgi complex breaks into small vesicles that are dispersed throughout the cytosol. When the cytosolic micro-tubules re-form during interphase (or after removal of the colcemid), the Golgi vesicles move along these microtubule tracks toward the MTOC, where they reaggregate to form large membrane complexes.

These observations were among the first to suggest that microtubules play a role in the intracellular transport of membrane-limited organelles and vesicles. Other examples of such transport and the motor proteins that power them are described in Section 20.2.

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