Mechanisms of mitotic regulation

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Project Summary/Abstract: Mechanisms of mitotic regulation Defects in the segregation of chromosomes in mitosis underlie aneuploidy and other structural rearrangements found in human cancers. The Stukenberg lab has studied mitotic regulation for 22 years and recently shown human breast tumors dysregulate the transcription of a large numbers of mitotic regulators to generate highly aneuploid tumors. Many of the most dysregulated transcripts were the Aurora B kinase, its complex members and a signaling network that controls multiple mitotic events from the inner centromere of every chromosome. The next phase of the Stukenberg lab will build on two recent breakthroughs that have enabled new lines of experimentation to elucidate how these proteins ensure accurate chromosome segregation and how dysregulation by tumors lowers this fidelity. First, we recently demonstrated that the Chromosome Passenger Complex containing Aurora B kinase undergoes liquid-liquid phase separation in vitro and in vivo. The insight that the kinase exists in a condensate enabled the reconstitution of a number of reactions in vitro. For example, Aurora B kinase activates in condensates under physiological concentrations and conditions. It has also elucidated surprising mechanisms for how Aurora phosphorylates kinetochores and activates downstream kinases. Condensates also activate a microtubule bundling activity that is not seen with soluble proteins. In fact, CPC rapidly generates microtubule parallel bundled structures that emanate from coacervates with the opposite polarity of centrosomes representing a novel microtubule activity. Second, we have identified new functions of the CPC in prophase including the generation of R-loops in the centromere. This new activity was found by first purifying the complex from mitotic chromosomes where we identify 32 regulators of R-loops. R-loops both direct the localization of the CPC and are required for cohesion of sister chromatids. The study of Aurora B regulation of R-loops has suggested another exciting line of investigation of Aurora B in controlling mitotic transcription. To increase the rigor and power of our experimentation the Stukenberg lab has developed tools to combine in vitro reconstitution, in silico modeling and replacement of key point mutants in cell assays. The result of these studies will provide important insight into how dysregulation of the chromosome passenger complex drive aneuploidy in cancer cells and how to better combine Aurora inhibitors to effectively treat cancer.