| During mitosis, microtubule dynamics must be regulated in order to form the mitotic spindle. Some factors regulate global microtubule dynamics, affecting the entire population of microtubules. Others are involved in locally stabilizing microtubules around the chromosomes. The goal of my thesis research has been to identify and characterize proteins involved in both of these types of microtubule regulation.; First, I chose to elucidate the role chromosomes play in spindle assembly. Chromatin had been shown to stabilize microtubules during spindle assembly and some factors that modulate this stabilization had been identified. However, little was known about how the organization of the chromosome itself could affect its ability to promote spindle assembly. Therefore, we disrupted chromosome structure by inhibiting the condensin complex, which is required for chromosome condensation in Xenopus egg extracts. Immunodepletion of condensin inhibited microtubule growth and organization around chromosomes, reducing the percentage of sperm nuclei capable of forming spindles, and causing dramatic defects in anaphase chromosome segregation. Although the motor CENP-E was recruited to kinetochores pulled poleward during anaphase, the disorganized chromosome mass was not resolved. Inhibition of condensin function during anaphase also inhibited chromosome segregation, indicating its continuous requirement. Spindle assembly around DNA-coated beads in the absence of kinetochores was also impaired upon condensin inhibition. These results support an important role for condensin in establishing chromosomal architecture necessary for proper spindle assembly and chromosome segregation.; A second goal of my thesis work was to identify novel proteins involved in regulating microtubule dynamics using a chemical genetic approach. We screened a library of purine derivatives to identify inhibitors of spindle assembly in Xenopus egg extracts. Out of a collection of approximately 1500 compounds, we identified 17 that caused microtubule destabilization, shrinking spindles to a small fraction of their normal size without targeting tubulin directly. Affinity chromatography with one compound, termed Diminutol, revealed a potential target as NQO1, an NADP-dependent oxidoreductase. A role for NQO1 in influencing global microtubule polymerization was confirmed through inhibition studies using known inhibitors and immunodepletion. Therefore, we identified a novel factor required for microtubule morphogenesis and cell division. |
