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The results of subsequent in vivo studies showed that individual cytosolic and mitotic microtubules display dynamic instability. In one set of experiments, fluorescent a^-tubulin subunits were microinjected into live cultured cells. The cells were chilled to depolymerize preexisting microtubules into tubulin dimers and then incubated at 37 °C to allow repoly-merization, thus incorporating the fluorescent tubulin into all the cellular microtubules. Video recordings of a small region in labeled cells showed that some microtubules became longer, others became shorter, and some appeared alternately to grow and to shrink over a period of several minutes (Figure 20-10). Because most microtubules in a cell associate by their (—) ends with MTOCs, their instability is largely limited to the (+) ends.

Two conditions influence the stability of microtubules. First, the oscillations between growth and shrinkage in vitro occur at tubulin concentrations near the Cc. As already stated, at tubulin concentrations above the Cc, the entire population of microtubules grows and, at concentrations below the Cc, all microtubules shrink. At concentrations near the Cc, however, some microtubules grow, whereas others shrink. The second condition affecting mi-crotubule stability is whether GTP or GDP occupies the exchangeable nucleotide-binding site on ^-tubulin at the (+) end of a microtubule (Figure 20-11). Because dissociation ("off" rate) of a GDP-tubulin dimer is four orders of magnitude as fast as that of a GTP-tubulin dimer, a micro-tubule is destabilized and depolymerizes rapidly if the (+) end becomes capped with subunits containing GDP-^-tubulin rather than GTP-^-tubulin. This situation can arise when a microtubule shrinks rapidly, exposing GDP-p-tubulin in the walls of the microtubule, or when a micro-tubule grows so slowly that the hydrolysis of p-tubulin-bound GTP converts it into GDP before additional subunits can be added to the (+) end of the microtubule. Before a shortening microtubule vanishes entirely, it can be "rescued " and start to grow if tubulin subunits with bound GTP add to the (+) end before the bound GTP hydrolyzes. Thus the parameters that determine the stability of a mi-crotubule are the growth rate, the shrinkage rate, the catastrophe frequency, and the rescue frequency.

Numerous Proteins Regulate Microtubule Dynamics and Cross-Linkage to Other Structures

A large number of proteins influence the assembly and stability of microtubules and their association with other cell structures (Table 20-1). These proteins are collectively called microtubule-associated proteins (MAPs) because most co-purify with microtubules isolated from cells. The results of immunofluorescence localization studies also have shown a parallel distribution of MAPs and microtubules in cells— strong evidence for their interaction in vivo.

MAPs are classified into two groups on the basis of their function. One group stabilizes microtubules. The stuc-ture of a stabilizing MAP consists of two domains—a basic microtubule-binding domain and an acidic projection domain. In the electron microscope, the projection domain appears as a filamentous arm that extends from the wall of the microtubule. This arm can bind to membranes, intermediate

Tubulin with bound GDP

Tubulin with bound GTP

Preexisting microtubule

Tubulin with bound GDP

Preexisting microtubule

Addition of GTP-bound tubulin

,r GTP cap

,r GTP cap

High concentration of GTP-bound free tubulin

Stable w

Low concentration of GTP-bound free tubulin

GDP cap

M FIGURE 20-11 Dynamic instability model of microtubule growth and shrinkage. GTP-bound ap-tubulin subunits (red) add preferentially to the (+) end of a preexisting microtubule. After incorporation of a subunit, the GTP (red dot) bound to the p-tubulin monomer is hydrolyzed to GDP Only microtubules whose (+) ends are associated with GTP-tubulin (those with a GTP cap) are stable and can serve as primers for the polymerization of additional tubulin. Microtubules with GDP-tubulin (blue) at the (+) end (those with a GDP cap) are rapidly depolymerized and may disappear within 1 minute. At high concentrations of unpolymerized GTP-tubulin, the rate of addition of tubulin is faster than the rate of hydrolysis of the GTP bound in the microtubule or the rate of dissociation of GTP-tubulin from microtubule ends; thus the microtubule grows. At low concentrations of unpolymerized GTP-tubulin, the rate of addition of tubulin is decreased; consequently, the rate of GTP hydrolysis exceeds the rate of addition of tubulin subunits and a GDP cap forms. Because the GDP cap is unstable, the microtubule end peels apart to release tubulin subunits. [See T Mitchison and M. Kirschner, 1984, Nature 312:237; M. Kirschner and T Mitchison, 1986, Cell 45:329; and R. A. Walker et al., 1988, J. Cell Biol. 107:1437.]

Low concentration of GTP-bound free tubulin

GDP cap

TABLE 20-1 Proteins That Modulate Microtubule (MT) Dynamics

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