UC San Diego

During mitosis, chromosomes must connect to the microtubule mitotic spindle and divide equally into two new daughter cells. The molecular machinery that accomplishes this task is the kinetochore, which is built on the centromeres of chromosomes and attaches to the mitotic spindle. My dissertation work has focused on 1) How the centromere is specified, 2) How kinetochores control the amount of time a cell spends in mitosis, and 3) How the essential protein bub-1 is recruited to kinetochores. Centromeres are specified by the presence of the histone like protein CENP-A. However, in C. elegans the chaperone proteins that recruit CENP-A to chromatin are missing. We discovered that the N-terminal tail of CENP-A functions as a chaperone to promote its binding to KNL-2/MIS18BP1 and centromere deposition. Once the kinetochore is built upon the centromeres of chromosomes, it optimizes the amount of time the cell spends in mitosis to promote timely anaphase onset. Kinetochores control mitotic timing in part by regulating flux and de-phosphorylation of the APC/C co-activator CDC-20. Kinetochore localization of CDC-20 is mediated by the ABBA motif of BUB-1. We discovered that a PLK-1 docking site on BUB-1 was also required for CDC-20 recruitment, and that PLK-1 phosphorylates the ABBA motif of BUB-1 to promote CDC-20 kinetochore recruitment and proper mitotic timing. In order to regulate mitotic timing and execute its other functions, BUB-1 must localize to the kinetochore. The current model in the field is that BUB-1 binds to BUB-3 to bind with p-MELT motifs on KNL-1. However, BUB-1 and BUB-3 depletions have significantly different phenotypes in early embryos, and perturbing MELT phosphorylation or phospho-recognition does not prevent BUB-1 kinetochore recruitment. Instead, we discovered that the N-terminal TPR domain of BUB-1 is necessary for BUB-1 kinetochore recruitment, and sufficient to localize to kinetochores on its own.