Molecular Analysis of Kinetochore Function

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Project Summary/Abstract The goal of my laboratory is to define the molecular mechanisms by which accurate cell division occurs. Our efforts focus on the core cell division machinery, including the macromolecular kinetochore and the microtubule-based mitotic spindle. Despite the central importance of the spindle and the kinetochore, the molecular basis for their many activities remains incompletely understood. We seek to generate a coherent molecular model for how the multiple kinetochore components and spindle-associated proteins act individually and in an integrated manner to direct faithful chromosome segregation. For chromosome segregation to occur, kinetochores must stably associate with a single site on each chromosome, build a large macromolecular assembly, form robust interactions with the dynamic microtubule polymers from a bipolar mitotic spindle, and these activities must be precisely regulated to ensure that chromosome segregation occurs with high fidelity. In addition to the function of these molecular players in actively dividing cells, the cell division machinery must also be differentially modulated across diverse physiological conditions, for example during meiotic cell divisions or during the persistent cell cycle arrest that occurs in quiescent cells. To analyze these key questions, our work uses a combination of functional genetics approaches in human cells, cell biological studies on protein localization and dynamics, affinity purification and proteomics approaches to identify protein interactions and modifications, and biochemical reconstitutions. For our recent work, we have also implemented large-scale approaches to analyzing cellular phenotypes using Cas9-based optical screening, which has transformed our ability to systematically define the contributions of human genes to cell division and other core cellular processes Over the next 5 years, our lab will investigate the fundamental mechanisms of cell division in human cells, focusing on three interrelated goals: 1) Analyze the molecular basis for chromosome segregation, 2) Define the basis for the cellular changes that occur during non-dividing cell states, such as quiescence and senescence, and 3) Conduct large-scale cell biological and functional genetics approaches to analyze cell division.