PT Unknown
AU Alex, A
TI Mechanistic insights into the mitotic checkpoint through biochemical characterisation and in vivo method development
PD 08
PY 2021
DI 10.17185/duepublico/74833
LA en
AB During mitosis, the accurate segregation of chromosomes to daughter cells is ensured by the Spindle Assembly Checkpoint (SAC). Incorrect attachments of spindle microtubules with kinetochores triggers the SAC signaling and results in the assembly of an effector complex called Mitotic Checkpoint Complex (MCC). Recent studies have identified that the SAC signaling kinetochores provide a catalytic platform for the MCC assembly. To ensure the timely activation and inactivation of SAC signaling, MCC disassembly occurs in parallel with its assembly. TRIP13, a AAA+ ATPase along with its cofactor p31COMET catalyzes the MCC disassembly mainly in cells. My PhD work aim to provide some mechanistic insights into the regulation of both MCC assembly and disassembly processes. During this work, I established electroporation (EP) as a method to deliver recombinant proteins into mammalian cells for the purpose of functional studies using kinetochore and SAC proteins. Our data provides a better comprehension of the spatial distribution of MCC in SAC signaling cells. MCC disassembly was characterized in vitro using biochemical assays. I demonstrated that CDK1 phosphorylation of p31COMET impaired its interaction with TRIP13. Additionally, this study showed for the first time that MAD1:C-MAD2 complex is a TRIP13 pseudo-substrate and the interaction between this substrate-enzyme pair is negatively regulated by the CDK1 phosphorylation of p31COMET . In vitro experiments showed that the previously identified MCC assembly catalysts displace p31COMET from MAD1:C-MAD2 complex in order to favor O-MAD2 dimerization. In a cellular context, this would help to increase the p31COMET accumulation in cytosol for MCC disassembly during checkpoint. Collectively, my PhD work demonstrates that MCC disassembly is regulated in unison with the number of SAC signaling kinetochores. This proposed mechanism of regulation would ensure that SAC signaling is both adaptive and robust.
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