Microtubules (MTs), key components of the cytoskeleton, are dynamic polymers of tubulin that form a well-organized network of polarized tube filaments. MT dynamics are highly regulated both spacially and temporally by several MT-related proteins, themselves regulated by several kinases and phosphatases via signaling cascades, and also by coordinated interactions with actin cytoskeleton and adhesion sites. Regulation of MT dynamics is crucial for mitosis, cell migration, cell signaling and trafficking. MT-targeted drugs (MTDs), which constitute a major anticancer drug family with antimitotic and antiangiogenic properties, inhibit tumor progression mainly by altering MT dynamics in both cancer and endothelial cells. Identification of proteins regulating the MT network will lead to a better understanding of tumor progression regulators and will be helpful in improving cancer therapy.

We have previously reported that anti-tubulin agents induce the release of cytochrome c from isolated mitochondria. In this study, we show that tubulin is present in mitochondria isolated from different human cancerous and non-cancerous cell lines. The absence of polymerized microtubules and cytosolic proteins was checked to ensure that this tubulin is an inherent component of the mitochondria. In addition, a salt wash did not release the tubulin from the mitochondria. By using electron microscopy, we then showed that tubulin is localized in the mitochondrial membranes. As compared with cellular tubulin, mitochondrial tubulin is enriched in acetylated and tyrosinated ␣-tubulin and is also enriched in the class III ␤-tubulin isotype but contains very little of the class IV ␤-tubulin isotype. The mitochondrial tubulin is likely to be organized in ␣/␤ dimers and represents 2.2 ؎ 0.5% of total cellular tubulin. Lastly, we showed by immunoprecipitation experiments that the mitochondrial tubulin is specifically associated with the voltage-dependent anion channel, the main component of the permeability transition pore. Thus, tubulin is an inherent component of mitochondrial membranes, and it could play a role in apoptosis via interaction with the permeability transition pore.

Microtubule dynamics are crucial for mitotic spindle assembly and chromosome movement. Suppression of dynamics by Taxol appears responsible for the drug's potent ability to inhibit mitosis and cell proliferation. Although Taxol is an important chemotherapeutic agent, development of resistance limits its efficacy. To examine the role of microtubule dynamics in Taxol resistance, we measured the dynamic instability of individual rhodamine-labeled microtubules in Taxol-sensitive and -resistant living human cancer cells. Taxol-resistant A549-T12 and -T24 cell lines were selected from a human lung carcinoma cell line, A549. They are, respectively, 9-and 17-fold resistant to Taxol and require low concentrations of Taxol for proliferation. We found that microtubule dynamic instability was significantly increased in the Taxol-resistant cells. For example, with A549-T12 cells in the absence of added Taxol, microtubule dynamicity increased 57% as compared with A549 cells. The length and rate of shortening excursions increased 75 and 59%, respectively. These parameters were further increased in A549-T24 cells, with overall dynamicity increasing by 167% compared with parental cells. Thus, the decreased Taxol-sensitivity of these cells can be explained by their increased microtubule dynamics. When grown without Taxol, A549-T12 cells were blocked at the metaphase͞anaphase transition and displayed abnormal mitotic spindles with uncongressed chromosomes. In the presence of 2-12 nM Taxol, the cells grew normally, suggesting that mitotic block resulted from excessive microtubule dynamics. These results indicate that microtubule dynamics play an important role in Taxol resistance, and that both excessively rapid dynamics and suppressed dynamics impair mitotic spindle function and inhibit proliferation.