Characterization of HeLa cell microtubules and microtubule motor proteins

In an effort to better understand the dynamic behaviors of non-neuronal microtubules and the proteins that associate with the microtubules, I purified and characterized HeLa cell tubulin. I found that microtubules assembled with HeLa cell tubulin exhibited remarkably slow dynamic instability in comparison with dynamics of microtubules prepared from purified brain tubulin. The slow dynamic instability of HeLa cell microtubules in vitro contrasts strongly with the dynamics of microtubules in dividing cells.; HeLa cell tubulin was found to consist largely of the αβ _I-isotype of tubulin with a minor portion of αβ_IV-tubulin. To test whether the slow dynamic instability of HeLa cell microtubules was due to the high content of αβ_I tubulin, the αβ _I-tubulin was isolated from HeLa cell tubulin. I found that although microtubules assembled from αβ_I-tubulin exhibited slow dynamic instability, the microtubules were more dynamic than HeLa cell microtubules, indicating that the combination of αβ_I and αβ _IV-tubulins results in less dynamic microtubules.; The characterization of HeLa cell microtubule dynamics lead to the hypothesis that cellular factors are necessary for the rapid dynamics of microtubules in cells. To provide a better understanding of the influence of cellular factors on microtubule dynamics, I analyzed HeLa cell microtubule dynamics in the presence of MCAK in vitro, a microtubule-destabilizing factor. I found that MCAK dramatically increases the catastrophe frequency of HeLa cell microtubules, supporting the hypothesis that MCAK is a catastrophe-promoting factor in cells.; The final project of my dissertation involved examining regulation of the microtubule motor protein, heavy chain kinesin by kinesin light chains. Together with Jennifer DeLuca, a former student in the lab, two distinct kinesin complexes were purified from HeLa cells. They are a 9.5 S kinesin complex that in most preparations did not bind to or induce gliding of microtubules, and a 7.0 S kinesin which was capable of microtubule binding and gliding activity. Furthermore, I found that the microtubule binding and gliding activity of the 9.5 S kinesin complex was activated by addition of light chain antibodies, suggesting that light chains can regulate the microtubule binding activity of the heavy chains.