E7389, which is in phase I and II clinical trials, is a synthetic macrocyclic ketone analogue of the marine sponge natural product halichondrin B. Whereas its mechanism of action has not been fully elucidated, its main target seems to be tubulin and/or the microtubules responsible for the construction and proper function of the mitotic spindle. Like most microtubule-targeted antitumor drugs, it inhibits tumor cell proliferation in association with G 2 -M arrest. It binds to tubulin and inhibits microtubule polymerization. We examined the mechanism of action of E7389 with purified microtubules and in living cells and found that, unlike antimitotic drugs including vinblastine and paclitaxel that suppress both the shortening and growth phases of microtubule dynamic instability, E7389 seems to work by an end-poisoning mechanism that results predominantly in inhibition of microtubule growth, but not shortening, in association with sequestration of tubulin into aggregates. In living MCF7 cells at the concentration that half-maximally blocked cell proliferation and mitosis (1 nmol/L), E7389 did not affect the shortening events of microtubule dynamic instability nor the catastrophe or rescue frequencies, but it significantly suppressed the rate and extent of microtubule growth. Vinblastine, but not E7389, inhibited the dilution-induced microtubule disassembly rate. The results suggest that, at its lowest effective concentrations, E7389 may suppress mitosis by directly binding to microtubule ends as unliganded E7389 or by competition of E7389-induced tubulin aggregates with unliganded soluble tubulin for addition to growing microtubule ends. The result is formation of abnormal mitotic spindles that cannot pass the metaphase/ anaphase checkpoint. [Mol Cancer Ther 2005;4(7): 1086 -95]

The spindle assembly checkpoint monitors microtubule attachment to kinetochores and tension across sister kinetochores to ensure accurate division of chromosomes between daughter cells. Cytoplasmic dynein functions in the checkpoint, apparently by moving critical checkpoint components off kinetochores. The dynein subunit required for this function is unknown. Here we show that human cells depleted of dynein light intermediate chain 1 (LIC1) delay in metaphase with increased interkinetochore distances; dynein remains intact, localised and functional. The checkpoint proteins Mad1/2 and Zw10 localise to kinetochores under full tension, whereas BubR1 is diminished at kinetochores. Metaphase delay and increased interkinetochore distances are suppressed by depletion of Mad1, Mad2 or BubR1 or by re-expression of wtLIC1 or a Cdk1 site phosphomimetic LIC1 mutant, but not Cdk1-phosphorylation-deficient LIC1. When the checkpoint is activated by microtubule depolymerisation, Mad1/2 and BubR1 localise to kinetochores. We conclude that a Cdk1 phosphorylated form of LIC1 is required to remove Mad1/2 and Zw10 but not BubR1 from kinetochores during spindle assembly checkpoint silencing.

Stathmin is a ubiquitous microtubule destabilizing protein that is believed to play an important role linking cell signaling to the regulation of microtubule dynamics. Here we show that stathmin strongly destabilizes microtubule minus ends in vitro at steady state, conditions in which the soluble tubulin and microtubule levels remain constant. Stathmin increased the minus end catastrophe frequency ϳ13-fold at a stathmin:tubulin molar ratio of 1:5. Stathmin steady-state catastrophe-promoting activity was considerably stronger at the minus ends than at the plus ends. Consistent with its ability to destabilize minus ends, stathmin strongly increased the treadmilling rate of bovine brain microtubules. By immunofluorescence microscopy, we also found that stathmin binds to purified microtubules along their lengths in vitro. Co-sedimentation of purified microtubules polymerized in the presence of a 1:5 initial molar ratio of stathmin to tubulin yielded a binding stoichiometry of 1 mol of stathmin per ϳ14.7 mol of tubulin in the microtubules. The results firmly establish that stathmin can increase the steady-state catastrophe frequency by a direct action on microtubules, and furthermore, they indicate that an important regulatory action of stathmin in cells may be to destabilize microtubule minus ends.

The EB1+TIP protein family and its binding partners track growing plus ends of microtubules in cells and are thought to regulate their dynamics. Here we determined the effects of EB1 and the N-terminal CAP-Gly domain (p150n) of one of its major binding partners, p150Glued, both separately and together, on the dynamic instability parameters at plus ends of purified steady-state microtubules. With EB1 alone, the shortening rate, the extent of shortening, and the catastrophe frequency were suppressed in the absence of significant effects on the growth rate or rescue frequency. The effects of EB1 on dynamics were significantly different when p150n was added together with EB1. The rate and extent of shortening and the catastrophe frequency were suppressed 3-4 times more strongly than with EB1 alone. In addition, the EB1-p150n complex increased the rescue frequency and the mean length the microtubules grew, parameters that were not significantly affected by EB1 alone. Similarly, deletion of EB1's C-terminal tail, which is a crucial binding region for p150n, significantly increased the ability of EB1 to suppress shortening dynamics. EB1 by itself bound along the length of the microtubules with 1 mol of EB1 dimer bound per approximately 12 mol of tubulin dimer. Approximately twice the amount of EB1 was recruited to the microtubules in the presence of p150n. Our results indicate that inactivation of EB1's flexible C-terminal tail significantly changes EB1's ability to modulate microtubule dynamics. They further suggest that p150Glued may activate and thereby facilitate the recruitment of EB1 to the tips of microtubules to regulate their dynamics.

Stathmin is an important regulator of microtubule polymerization and dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass through sequestration of soluble tubulin into an assemblyincompetent T2S complex (two ␣:␤ tubulin dimers per molecule of stathmin), and by increasing the switching frequency (catastrophe frequency) from growth to shortening at plus and minus ends by binding directly to the microtubules. (1-5, 8 -11), and kinases associated with cell cycle regulation such as cyclin-dependent kinase 1 (3,(12)(13)(14). Phosphorylation of stathmin is required for cell cycle progression through mitosis and for proper assembly/function of the mitotic spindle (3, 13-16). Inhibition of stathmin phosphorylation produces strong mitotic phenotypes characterized by disassembly and disorganization of mitotic spindles and abnormal chromosome distributions (3,(13)(14).Stathmin is known to destabilize microtubules in two ways. One is by binding to soluble tubulin and forming a stable complex that cannot polymerize into microtubules, consisting of one molecule of stathmin and two molecules of tubulin (T2S complex) (17-24). Addition of stathmin to microtubules in equilibrium with soluble tubulin results in sequestration of the tubulin and a reduction in the level of microtubule polymer (17-18, 22, 25-28). In addition to reducing the amount of assembled polymer, tubulin sequestration by stathmin has been shown to increase the switching frequency at microtubule plus ends from growth to shortening (called the catastrophe frequency) as the microtubules relax to a new steady state (17,29). The second way is by binding directly to microtubules (27)(28)(29)(30). The direct binding of stathmin to microtubules increases the catastrophe frequency at both ends of the microtubules and considerably more strongly at minus ends than at plus ends (27). Consistent with its strong catastrophe-promoting activity at minus ends, stathmin increases the treadmilling rate of steady-state microtubules in vitro (27). These results have led to the suggestion that stathmin might be an important cellular regulator of minus-end microtubule dynamics (27).Phosphorylation of stathmin diminishes its ability to regulate microtubule polymerization (3,14,(25)(26). Phosphorylation of Ser 16 or Ser 63 appears to be more critical than phosphorylation of Ser 25 and Ser 38 for the ability of stathmin to bind to soluble tubulin and to inhibit microtubule assembly in vitro (3,25). Inhibition of stathmin phosphorylation induces defects in spindle assembly and organization (3,14) suggesting that not only soluble tubulin-microtubule levels are regulated by phosphorylation of stathmin, but the dynamics of microtubules could also be regulated in a phosphorylation-dependent manner.It is not known how phosphorylation at any of the four serine residues of stathmin affects its ability to regulate microtubule dynamics and, specifically, its ability to increase the catastrophe frequency at plus and minus ends due to it...

Tubulin, a ubiquitous protein of eukaryotic cytoskeleton, is a building block unit of microtubule. Although several cellular processes are known to be mediated through the tubulin-microtubule system, the participation of tubulin or microtubule in protein folding pathway has not yet been reported. Here we show that goat brain tubulin has some functions and features similar to many known molecular chaperones. Substoichiometric amounts of tubulin can suppress the non-thermal and thermal aggregation of a number of unrelated proteins such as insulin, equine liver alcohol dehydrogenase, and soluble eye lens proteins containing ␤-and ␥-crystallins. This chaperone-like activity of tubulin becomes more pronounced as temperature increases. Aging of tubulin solution at 37°C also enhances its chaperone-like activity. Tubulin loses its chaperone-like activity upon removal of its flexible hydrophilic C-terminal tail. These results suggest that both electrostatic and hydrophobic interactions are important in substrate binding by tubulin and that the negatively charged C-terminal tails play a crucial role for its chaperone-like activity.

SCG10 (superior cervical ganglia neural-specific 10 protein) is a neuron specific member of the stathmin family of microtubule regulatory proteins that like stathmin can bind to soluble tubulin and depolymerize microtubules. The direct actions of SCG10 on microtubules themselves and on their dynamics have not been investigated previously. Here, we analyzed the effects of SCG10 on the dynamic instability behavior of microtubules in vitro, both at steady state and early during microtubule polymerization. In contrast to stathmin, whose major action on dynamics is to destabilize microtubules by increasing the switching frequency from growth to shortening (the catastrophe frequency) at microtubule ends, SCG10 stabilized the plus ends both at steady state and early during polymerization by increasing the rate and extent of growth. For example, early during polymerization at high initial tubulin concentrations (20 microM), a low molar ratio of SCG10 to tubulin of 1:30 increased the growth rate by approximately 50%. In contrast to its effects at plus ends, SCG10 destabilized minus ends by increasing the shortening rate, the length shortened during shortening events, and the catastrophe frequency. Consistent with its ability to modulate microtubule dynamics at steady state, SCG10 bound to purified microtubules along their lengths. The dual activity of SCG10 at opposite microtubule ends may be important for its role in regulating growth cone microtubule dynamics. SCG10's ability to promote plus end growth may facilitate microtubule extension into filopodia, and its ability to destabilize minus ends could provide soluble tubulin for net plus end elongation.

Several sulfonamides have antitumor activities and are currently undergoing clinical evaluation for the treatment of cancer. In this study, we have elucidated the antiproliferative mechanism of action of five indole sulfonamides. The indole sulfonamides inhibited the polymerization of microtubule protein into microtubules in vitro. In addition, three representative derivatives, ER-68378 (2), ER-68384 (4) and ER-68394 (5), suppressed the dynamic instability behavior at the plus ends of individual steady-state microtubules in vitro. The analogues inhibited HeLa cell proliferation with half-maximal inhibitory concentrations in the range of 6-17 microM. The compounds blocked cell cycle progression at mitosis. At their lowest effective antimitotic concentrations, they depolymerized the spindle microtubules and disorganized the chromosomes but did not affect the microtubules in interphase cells. However, at relatively high concentrations, interphase microtubules were also depolymerized by these sulfonamides. Furthermore, all five compounds were found to induce apoptosis in the cells in association with the phosphorylation of bcl-2. The results suggest that the indole sulfonamides inhibit cell proliferation at mitosis by perturbing the assembly dynamics of spindle microtubules and that they can kill cancer cells by inducing apoptosis through the bcl-2-dependent pathway.