Analysis of microtubule dynamics and chromosome movement in vitro

Mitosis is a complex process where the concerted action of several hundred proteins orchestrates the accurate segregation of duplicated genetic material to daughter cells. We have approached this complex process using in vitro biochemistry, with an emphasis on the polymerization dynamics of microtubules--polymers of tubulin essential for mitosis in all eukaryotes. In the first project, we used clarified Xenopus egg extracts and demembranated Xenopus sperm nuclei to develop an in vitro reaction for the assembly of kinetochores--nucleoprotein structures assembled at the centromere that generate a dynamic interface between chromosomes and spindle microtubules. We have demonstrated that this reaction can be used to analyze protein-protein interactions at the kinetochore by depleting a specific kinetochore component from the extract and assaying the effect on targeting of other components.; In the second project, we have used local fluorescence photoactivation to show that the bulk of microtubule disassembly occurs near the spindle poles during anaphase A in Xenopus extract spindles. This is in contrast to vertebrate somatic cells, where the majority of microtubule disassembly occurs near the kinetochore. This result highlights the importance of considering non-kinetochore mechanisms for anaphase chromosome movement in systems other than vertebrate somatic cells.; In the final project, we have identified a novel activity for internal catalytic domain kinesins, a specific subclass of the kinesin superfamily. We found that XKCM1, a member of this subclass, acts as a microtubule destabilizing factor in Xenopus extracts. Purified XKCM1 and XKIF2, a neuronal kinesin related to XKCM1, destabilize microtubules assembled from pure tubulin in vitro. We have analyzed the mechanism of microtubule destabilization by XKCM1 and XKIF2, and have found that these proteins directly target to the ends of microtubules where they cause a destabilizing conformational change. We have also characterized the role of ATP hydrolysis by the catalytic domain of these proteins in this process. In addition to being the first detailed characterization of microtubule-destabilizing proteins, this work provides clear evidence for a non-motor function for a certain subclass of kinesins.