Cell Cycle Control of the Cytoskeleton

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This proposal addresses the mechanisms controlling microtubule length, the size and function of the anaphase spindle, and the coordination of anaphase spindle function with other key cellular events during mitotic exit. Because the spindle is a self-organizing structure, the regulation of microtubule length is a major mechanism controlling overall spindle size. Spindle size is controlled globally by the concentration or activity of factors that promote microtubule growth or disassembly. Additionally, “measuring” mechanisms that mediate length-dependent microtubule assembly or disassembly have also been described. The best- studied length-dependent mechanism occurs through the activity of the kinesin-8 family of microtubule motors. Compromised kinesin 8 function in mammalian cells leads to high frequencies of chromosome missegregation and and the formation of abnormal nuclear structures, which are common in cancer, called micronuclei. We recently showed that micronuclei can cause “chromothripsis”, a major mutational process leading to chromosome rearrangement in cancer. In the last funding period, we defined the mechanism by which a yeast kinesin 8 selectively trims longer microtubules. In contrast to previous proposals, a combination of biochemical and single molecule imaging experiments lead to a new conformational switch model, involving kinesin 8 recognition of bent tubulin at the microtubule end, triggering microtubule disassembly. Building on a new high resolution cryo-EM structure and other data, we now propose to test this model and work out the molecular mechanism for bent tubulin recognition. Additionally, in the last funding period we have made a significant advance in understanding how anaphase spindle function is coordinated with the reassembly of the nuclear envelope around chromosomes to form daughter cell nuclei. We found that spindle microtubules block the recruitment of nuclear envelope (NE) containing nucleoporins to decondensing chromosomes, but allow other aspects of NE assembly to occur. This leads to irreversibly defective NE assembly on lagging chromosomes, explaining why the NE around micronuclei undergoes spontaneous disruption, a key step in generating chromothripsis. These findings alter the thinking on the organization of mitotic exit in metazoan cells. Rather than precise checkpoint controls, our findings indicate that chromosome segregation and NE assembly are only loosely coordinated through the timing of anaphase spindle disassembly. The absence of precise regulatory controls can explain why errors during mitotic exit are frequent, and represent a major source of catastrophic genome rearrangements. A series of cell biological experiments is proposed to address key unanswered questions, such as the mechanism by which microtubules inhibit NPC assembly. A tractable system using the fission yeast S. japonicus is described that will enable us to use powerful genetic tools for understanding NE assembly and its coordination with the completion of mitosis. RELEVANCE (See instructions): This proposal addresses centrals questions in eukaryotic cell division: the mechanisms controlling microtubule length, the size and function of the anaphase spindle, and the integration of anaphase spindle function with other key cellular events during mitotic exit. The project has broad relevance because it addresses how the size of an intracellular structure is scaled to cell and genome size. The project also has relevance to human health because it provides insight into an important mutational process generating chromosome aberrations in cancer genomes.