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

Recruitment of Mad1-Mad2 complexes to unattached kinetochores is a central event in spindle checkpoint signaling. Despite its importance, the mechanism that recruits Mad1-Mad2 to kinetochores is unclear. In this paper, we show that MAD-1 interacts with BUB-1 in Caenorhabditis elegans. Mutagenesis identified specific residues in a segment of the MAD-1 coiled coil that mediate the BUB-1 interaction. In addition to unattached kinetochores, MAD-1 localized between separating meiotic chromosomes and to the nuclear periphery. Mutations in the MAD-1 coiled coil that selectively disrupt interaction with BUB-1 eliminated MAD-1 localization to unattached kinetochores and between meiotic chromosomes, both of which require BUB-1, and abrogated checkpoint signaling. The identified MAD-1 coiled-coil segment interacted with a C-terminal region of BUB-1 that contains its kinase domain, and mutations in this region prevented MAD-1 kinetochore targeting independently of kinase activity. These results delineate an interaction between BUB-1 and MAD-1 that targets MAD-1-MAD-2 complexes to kinetochores and is essential for spindle checkpoint signaling.

In the eukaryotic cell cycle, a threshold level of cyclin B accumulation triggers the G2-to-M transition, and subsequent cyclin B destruction triggers mitotic exit. The anaphase-promoting complex/cyclosome (APC/C) is the E3 ubiquitin ligase that, together with its co-activator Cdc20, targets cyclin B for destruction during mitotic exit. Here, we show that two pathways act in concert to protect cyclin B from Cdc20-activated APC/C in G2, in order to enable cyclin B accumulation and the G2-to-M transition. The first pathway involves the Mad1-Mad2 spindle checkpoint complex, acting in a distinct manner from checkpoint signaling after mitotic entry but employing a common molecular mechanism-the promotion of Mad2-Cdc20 complex formation. The second pathway involves cyclin-dependent kinase phosphorylation of Cdc20, which is known to reduce Cdc20's affinity for the APC/C. Cooperation of these two mechanisms, which target distinct APC/C binding interfaces of Cdc20, enables cyclin B accumulation and the G2-to-M transition.

The conserved Bub1/Bub3 complex is recruited to the kinetochore region of mitotic chromosomes, where it initiates spindle checkpoint signaling and promotes chromosome alignment. Here we show that, in contrast to the expectation for a checkpoint pathway component, the BUB-1/BUB-3 complex promotes timely anaphase onset in Caenorhabditis elegans embryos. This activity of BUB-1/BUB-3 was independent of spindle checkpoint signaling but required kinetochore localization. BUB-1/BUB-3 inhibition equivalently delayed separase activation and other events occurring during mitotic exit. The anaphase promotion function required BUB-1's kinase domain, but not its kinase activity, and this function was independent of the role of BUB-1/BUB-3 in chromosome alignment. These results reveal an unexpected role for the BUB-1/BUB-3 complex in promoting anaphase onset that is distinct from its well-studied functions in checkpoint signaling and chromosome alignment, and suggest a new mechanism contributing to the coordination of the metaphase-to-anaphase transition.