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Mechanistic Insights Into the Function of Mad1 During Mitosis and Development /


Correct alignment and segregation of chromosomes during mitosis are essential for cell viability and organismal development. Proper attachment of chromosomes to the mitotic spindle ensures correct chromosome segregation. Unattached chromosomes activate an intracellular signaling cascade, termed the spindle assembly checkpoint. The spindle assembly checkpoint generates a signal that arrests the cell cycle until all chromosomes are properly attached. This signal originates from the kinetochore, which is a macromolecular machine built on centromeric chromatin that connects chromosomes to the mitotic spindle. The central protein in the kinetochore-based spindle assembly checkpoint is Mad1. Mad1 is recruited to unattached kinetochores where it generates the signal that arrests the cell cycle. Recruitment of Mad1 to unattached kinetochore is the central reaction in spindle assembly checkpoint activation, but how Mad1 is recruited to unattached kinetochores has remained elusive. To address this question I performed a kinetochore-wide yeast two- hybrid screen and identified an interaction between Mad1 and Bub1. Using mutagenic yeast two hybrid screens I developed, I identified mutations in a small coiled coil domain in Mad1 that disrupts the Mad1-Bub1 interaction. To test the functional significance of the Mad1-Bub1 interaction in vivo, I used the nematode, Caenorhabditis elegans. I generated single-copy transgenic strains that contained Bub1-binding mutants of Mad1 and determined that the Mad1-Bub1 interaction was required for recruitment of Mad1 to unattached kinetochores and spindle assembly checkpoint activation. In summary, my work revealed that the central reaction in spindle assembly checkpoint activation is recruitment of Mad1 directly by Bub1 to unattached kinetochores. In C. elegans, Mad1 is required for organismal viability. It was assumed that this was because of Mad1's central role in the spindle assembly checkpoint. Using the Bub1-binding mutants of Mad1, I determined that Mad1's checkpoint function was not essential for organismal viability. This data suggested that Mad1 performed an unknown yet essential function during development. I explored possible developmental functions of Mad1, and this is presented in detail in Chapter 3. Overall, my work determined that Bub1 recruits Mad1 to kinetochores to activate the spindle assembly checkpoint and discovered that Mad1 is required for development, independent of its checkpoint function

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