Search

Separase regulates multiple aspects of the metaphase-to-anaphase transition. Separase cleaves cohesin to allow chromosome segregation and localizes to vesicles to promote exocytosis. The anaphase-promoting complex/cyclosome (APC/C) activates separase by ubiquitinating its inhibitory chaperone, securin, triggering its degradation. How this pathway controls the exocytic function of separase is unknown. During meiosis I, securin is degraded over several minutes, while separase rapidly relocalizes from kinetochore structures at the spindle and cortex to sites of action on chromosomes and vesicles at anaphase onset. The loss of cohesin coincides with the relocalization of separase to the chromosome midbivalent at anaphase onset. APC/C depletion prevents separase relocalization, while securin depletion causes precocious separase relocalization. Expression of non-degradable securin inhibits chromosome segregation, exocytosis, and separase localization to vesicles but not to the anaphase spindle. We conclude that APC/C-mediated securin degradation controls separase localization. This spatiotemporal regulation will impact the effective local concentration of separase for more precise targeting of substrates in anaphase., (© 2024 Sorensen Turpin et al.)

Separase cleaves cohesin to allow chromosome segregation. Separase also regulates cortical granule exocytosis and vesicle trafficking during cytokinesis, both of which involve RAB-11. We investigated whether separase regulates exocytosis through a proteolytic or non-proteolytic mechanism. In C. elegans, protease-dead separase (SEP-1 PD ::GFP) is dominant negative. Consistent with its role in cohesin cleavage, SEP-1 PD ::GFP causes chromosome segregation defects. As expected, partial depletion of cohesin rescues this defect, confirming that SEP-1 PD ::GFP acts through a substrate trapping mechanism. SEP-1 PD ::GFP causes cytokinetic defects that are synergistically exacerbated by depletion of the t-SNARE SYX-4. Furthermore, SEP-1 PD ::GFP delays furrow ingression, causes an accumulation of RAB-11 vesicles at the cleavage furrow site and delays the exocytosis of cortical granules during anaphase I. Depletion of syx-4 further enhanced RAB-11::mCherry and SEP-1 PD ::GFP plasma membrane accumulation during cytokinesis, while depletion of cohesin had no effect. In contrast, centriole disengagement appears normal in SEP-1 PD ::GFP embryos, indicating that chromosome segregation and vesicle trafficking are more sensitive to inhibition by the inactive protease. These findings suggest that separase cleaves an unknown substrate to promote the exocytosis of RAB-11 vesicles and paves the way for biochemical identification of substrates.