Hailing Yang

Inhibition of Cell Migration and Cell Division Correlates with Distinct Effects of Microtubule Inhibiting Drugs

Drugs that target microtubules are thought to inhibit cell division and cell migration by suppressing dynamic instability, a “search and capture” behavior that allows microtubules to probe their environment. Here, we report that subtoxic drug concentrations are sufficient to inhibit plus-end microtubule dynamic instability and cell migration without affecting cell division or microtubule assembly. The higher drug concentrations needed to inhibit cell division act through a novel mechanism that generates microtubule fragments by stimulating microtubule minus-end detachment from their organizing centers. The frequency of microtubule detachment in untreated cells increases at prophase suggesting that it is a regulated cellular process important for spindle assembly and function. We conclude that drugs produce differential dose-dependent effects at microtubule plus and minus-ends to inhibit different microtubule-mediated functions.

Paclitaxel-Dependent Cell Lines Reveal a Novel Drug Activity

We previously described the isolation of Tax 18 and Tax 11-6, two paclitaxel-dependent cell lines that assemble low amounts of microtubule polymer and require the drug for cell division. In the present studies, fluorescence time-lapse microscopy was used to measure microtubule dynamic instability behavior in these cells. The mutations were found to cause small decreases in microtubule growth and shortening, but the changes seemed unable to explain the defects in microtubule polymer levels or cell division. Moreover, paclitaxel further suppressed microtubule dynamics at low drug concentrations that were insufficient to rescue the mutant phenotype. Wild-type (WT) cells treated with similar low drug concentrations also had highly suppressed microtubules, yet experienced no problems with cell division. Thus, the effects of paclitaxel on microtubule dynamics seemed to be unrelated to cell division in both WT and mutant cell lines. The higher drug concentrations needed to rescue the mutant phenotype instead inhibited the formation of unstable microtubule fragments that appeared at high frequency in the drug-dependent, but not WT, cell lines. Live cell imaging revealed that the fragments were generated by microtubule detachment from centrosomes, a process that was reversed by paclitaxel. We conclude that paclitaxel rescues mutant cell division by inhibiting the detachment of microtubule minus ends from centrosomes rather than by altering plus-end microtubule dynamics. Mol Cancer Ther; 9(11); 2914–23. ©2010 AACR.

The Role of Microtubules and Their Dynamics in Cell Migration

Background: Microtubule effects on cell migration are poorly understood. Results: Tubulin mutations or drug treatments that suppress microtubule dynamics impede cell locomotion. Depolymerizing microtubules does not inhibit movement, but it becomes random. Conclusion: Microtubules act to restrain cell movement and specify directionality. Significance: Drugs have dose-dependent effects on microtubule behavior, cell migration, and mitosis. Understanding these actions will lead to more effective drug use. Although microtubules have long been implicated in cell locomotion, the mechanism of their involvement remains controversial. Most studies have concluded that microtubules play a positive role by regulating actin polymerization, transporting membrane vesicles to the leading edge, and/or facilitating the turnover of adhesion plaques. Here we used wild-type and mutant CHO cell lines with alterations in tubulin to demonstrate that microtubules can also act to restrain cell motility. Tubulin mutations or low concentrations of drugs that suppress microtubule dynamics without affecting the amount of microtubule polymer inhibited the rate of migration by preventing microtubule reorganization in the trailing portion of the cells where the more dynamic microtubules are normally found. Under these conditions, cells along the edge of a wound still extended lamellipodia and elongated toward the wound but were inhibited in their ability to retract their tails, thus retarding forward progress. The idea that microtubules normally act to restrain cell locomotion was confirmed by treating cells with high concentrations of nocodazole to depolymerize the microtubule network. In the absence of microtubules, wild-type CHO and HeLa cells could still move at near normal speeds, but the movement became more random. We conclude that microtubules act both to restrain cell movement and to establish directionality.

Overexpression of Mitotic Centromere–Associated Kinesin Stimulates Microtubule Detachment and Confers Resistance to Paclitaxel

Numerous studies have implicated mutations in tubulin or the overexpression of specific tubulin genes in resistance to microtubule-targeted drugs. Much less is known about the role of accessory proteins that modulate microtubule behavior in the genesis of drug resistance. Here, we examine mitotic centromere–associated kinesin (MCAK), a member of the kinesin family of microtubule motor proteins that has the ability to stimulate microtubule depolymerization, and show that overexpressing the protein confers resistance to paclitaxel and epothilone A, but increases sensitivity to colcemid. Cells transfected with FLAG-tagged MCAK cDNA using a tet-off–regulated expression system had a disrupted microtubule cytoskeleton and were able to survive a toxic concentration of paclitaxel in the absence, but not in the presence of tetracycline, showing that drug resistance was caused by ectopic MCAK production. Moreover, a population that was heterogeneous with respect to FLAG-MCAK expression became enriched with cells that produced the ectopic protein when it was placed under paclitaxel selection. Similar to previously isolated mutants with altered tubulin, paclitaxel resistant cells resulting from MCAK overexpression were found to have decreased microtubule polymer and a seven-fold increase in the frequency of microtubule detachment from centrosomes. These data are consistent with a model for paclitaxel resistance that is based on stability of the attachment of microtubules to their nucleating centers, and they implicate MCAK in the mechanism of microtubule detachment. Mol Cancer Ther; 10(6); 929–37. ©2011 AACR.

Mitotic Centromere-associated Kinesin (MCAK) Mediates Paclitaxel Resistance

Background: Mutations causing paclitaxel resistance stimulate microtubule detachment from centrosomes. Results: Depletion of mitotic centromere-associated kinesin (MCAK) reverses microtubule detachment and paclitaxel resistance. Conclusion: MCAK plays a pivotal role in the mechanism of microtubule detachment and drug resistance. Significance: The ability of MCAK to reverse paclitaxel resistance identifies modulators of microtubule detachment as important new drug targets. Paclitaxel has powerful anticancer activity, but some tumors are inherently resistant to the drug, whereas others are initially sensitive but acquire resistance during treatment. To deal with this problem, it will be necessary to understand the mechanisms of drug action and resistance. Recent studies indicate that paclitaxel blocks cell division by inhibiting the detachment of microtubules from centrosomes. Here, we demonstrate that mitotic centromere-associated kinesin (MCAK), a kinesin-related protein that destabilizes microtubules, plays an important role in microtubule detachment. Depletion of MCAK altered mitotic spindle morphology, increased the frequency of lagging chromosomes, and inhibited the proliferation of WT CHO cells, confirming that it is an essential protein for cell division. In contrast, MCAK depletion rescued the proliferation of mutant paclitaxel-dependent cell lines that are unable to divide because of defective spindle function resulting from altered α-tubulin or class III β-tubulin overexpression. In concert with the correction of mitotic defects, loss of MCAK reversed an aberrantly high frequency of microtubule detachment in the mutant cells and increased their sensitivity to paclitaxel. The results indicate that MCAK affects cell sensitivity to mitotic inhibitors by modulating the frequency of microtubule detachment, and they demonstrate that changes in a microtubule-interacting protein can reverse the effects of mutant tubulin expression.

Class V β-tubulin alters dynamic instability and stimulates microtubule detachment from centrosomes

The need for multiple tubulin genes in vertebrate organisms is poorly understood. This article shows that a minor, ubiquitious β-tubulin isotype strongly influences microtubule plasticity by altering dynamic behavior and the stability of microtubule attachment to centrosomes.

Heightened sensitivity to paclitaxel in class IVa β -tubulin -transfected cells is lost as expression increases

Stably transfected Chinese hamster ovary cell lines expressing increasing levels of β4a, a class IV neuronal-specific β-tubulin, were compared for effects on microtubule organization, assembly, and sensitivity to antimitotic drugs. It was found that β4a reduced microtubule assembly in proportion to its abundance and thereby caused supersensitivity to microtubule disruptive drugs such as colcemid, vinblastine, and nocodazole. However, the response to paclitaxel was more complex. Low expression of β4a caused supersensitivity to paclitaxel, whereas higher expression resulted in the loss of supersensitivity. The results suggest that β4a may possess an enhanced ability to bind paclitaxel that increases sensitivity to the drug and acts substoichiometrically. At high levels of β4a expression, however, microtubule disruptive effects counteract the assembly promoting pressure exerted by paclitaxel binding, and drug supersensitivity is lost. β4a-Tubulin differs from the more ubiquitous β4b isotype at relatively few amino acid residues, yet β4b expression has little effect on microtubule assembly or drug response. To determine which amino acids mediate the effects of β4a expression, β4a and β4b were altered by site-directed mutagenesis and expressed in Chinese hamster ovary cells. The introduction of N332S or N335S mutations into β4b-tubulin was sufficient to confer microtubule disruption and increased colcemid sensitivity. On the other hand, mutation of Ala115 to serine in β4a-tubulin almost completely reversed heightened sensitivity to paclitaxel, but introduction of an S115A mutation into β4b had no effect, suggesting that a complex interaction of multiple amino acids are necessary to produce this phenotype.

Tubulin isotype specificity and identification of the epitope for antibody Tub 2.1.

Tub 2.1, a monoclonal antibody commonly used to measure cellular tubulin content, is widely believed to recognize all beta-tubulin isotypes. Though it has been used for more than two decades, the epitope for this antibody is not well established. We report for the first time that contrary to common belief, this antibody does not react with all isotypes of beta-tubulin. Of the seven vertebrate beta-tubulins, the more divergent class V and VI isotypes are not recognized by this antibody. Among the isotypes that do react, binding is similar for beta2, beta3, beta4a and beta4b but lower for beta1, the most abundant isotype. Expression of chimeric tubulins verified that the epitope for Tub 2.1 is near the C-terminal end of beta-tubulin. Site-directed mutagenesis of this region in nonreactive beta5 to match the sequence of beta4b resulted in strong reaction to Tub 2.1 and narrowed the epitope to amino acids 431-436.

Detection and quantification of microtubule detachment from centrosomes and spindle poles.

Microtubule detachment from microtubule organizing centers is an important cellular process required for normal cell proliferation. When cells enter mitosis, microtubule turnover increases along with a concurrent increase in microtubule detachment. MCAK, a kinesin-related protein whose abundance is highest during the early stages of mitosis, has been shown to regulate microtubule detachment. Abnormal increases or decreases in the frequency of detachment interfere with spindle function and inhibit cell division. It has been shown that drugs able to promote microtubule assembly (e.g., paclitaxel, epothilones) prevent cell division by suppressing microtubule detachment from centrosomes. Conversely, cytotoxic concentrations of microtubule destabilizing drugs (e.g., vinblastine, nocodazole), tubulin mutations that cause paclitaxel resistance, and specific β-tubulin isotypes increase the frequency of microtubule detachment. In this chapter, we describe a method to calculate the frequency of microtubule detachment by transfecting cells with EGFP-MAP4 and directly observing detachment by live cell imaging.