Spindle Assembly Checkpoint Silencing

PROJECT SUMMARY Eukaryotic cell cycle is tightly controlled by an intricate sequence of events where both gene expression and protein degradation are under intense regulation. When the cell cycle is dysregulated, uncontrolled division ensues–a hallmark of cancer. Therefore, cell cycle checkpoints, such as the spindle assembly checkpoint (SAC), ensure faithful chromosome segregation and produce healthy daughter cells. During an active checkpoint, transcription is reduced and the primary ubiquitin ligase of mitosis, the Anaphase-Promoting Complex/Cyclosome (APC/C), is inhibited by the 4-subunit Mitotic Checkpoint Complex (MCC). Once the chromosomes are ready and the checkpoint is satisfied, the APC/C is freed from MCC inhibition through conformational rearrangements that permit MCC ubiquitination. The APC/C targets several well-established cell cycle regulators (e.g., Cyclin B and Securin) and chromatin regulators, including the chromatin building blocks–nucleosomes, for destruction, coupling mitotic exit and transcription. The APC/C-dependent progression through mitosis is incredibly complicated as it requires the participation of dozens of proteins, including activators, inhibitors, kinases, AAA-ATPases, and substrates. How all this regulation is successfully integrated on a single APC/C scaffold is unclear but highly significant because it is at the intersection of both protein degradation and gene expression for G1 phase. By reconstituting these processes in vitro with innovative techniques, such as time-resolved cryo-EM and mass photometry, we can better understand how polyubiquitination occurs, how specific cell cycle effectors influence the conformational states of MCC-bound APC/C, and how the nucleosome is targeted by the APC/C. The information gained from these studies will lead to an unprecedented understanding of the SAC and coordinated gene expression, which is at the heart of development, cell biology, and countless disease processes, including cancer.