Kinetochore Structure and Function

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DESCRIPTION (provided by applicant): The broad objective of our lab is to understand the key mechanical and regulatory events that specify accurate chromosome segregation in human cells. We have focused our efforts on characterizing the molecular functions of the kinetochore, a macromolecular structure that mediates the attachment between chromosomes and the spindle. Work supported over the lifetime of this grant has contributed towards addressing outstanding questions such as how kinetochores capture and maintain stable connections with microtubules, how proper attachments generate kinetochore tension and how improper attachments are detected and delays cells from prematurely exiting mitosis. These studies are of direct importance to understanding human health issues such as birth defects, infertility, and cancer. Our studies have also directly stimulated the development of new anti-cancer drugs that may increase the selectivity towards rapidly dividing cancer cells. Our most recent studies have focused on structure and function analysis of CENP-E to determine how it contributes to kinetochore:microtubule attachments and how these activities are monitored by the mitotic checkpoint protein, hBUBRI kinase. Our discovery of the Mitotic Checkpoint Complex (MCC) as the major inhibitor of the Anaphase Promoting Complex (APC) in Hela cells led to a new two-step model for how APC is inhibited by the checkpoint. Our studies of CENP-I provided in vivo evidence that supported the existence of kinetochore-dependent and -independent mechanisms that are required to inhibit the APC. We are addressing the mechanism of action of hBUBRI kinase by identifying residues that are phosphorylated in mitosis. We generated phospho-hBUBRI antibodies that differentially stain kinetochores lacking attachments or tension. Unattached kinetochores and propose to use them to examine how hBUBRI is regulated by microtubule attachments. We recently reported that CENP-F is a new microtubule binding protein that is essential for proper kinetochore attachments and also contributes towards the mitotic checkpoint. Finally, we report a new component of the inner centromere that we call CENP-J. Its characterization has revealed a role in error correction that can be attributed to its ability to recruit the microtubule depolymerase, MCAK to the inner centromere. Depletion of CENP-J cause cells to accumulate aberrant kinetochore attachments that fail to generate tension and thus delay at metaphase. Failure to correct these defective attachments by the time cells overcome the mitotic delay results in lagging chromosomes during the ensuing anaphase. We want to extend our studies to continue to address the major issues about how checkpoint proteins monitor kinetochore attachments, how the "wait anaphase" signal is generated and how errors in attachment are corrected.