Abstract
Separase is best known for its function in sister chromatid separation at the metaphase-anaphase transition. It also has a role in centriole disengagement in late mitosis/G1. To gain insight into the activity of separase at centrosomes, we developed two separase activity sensors: mCherry-Scc1(142-467)-ΔNLS-eGFP-PACT and mCherry-kendrin(2059-2398)-eGFP-PACT. Both localize to the centrosomes and enabled us to monitor local separase activity at the centrosome in real time. Both centrosomal sensors were cleaved by separase before anaphase onset, earlier than the corresponding H2B-mCherry-Scc1(142-467)-eGFP sensor at chromosomes. This indicates that substrate cleavage by separase is not synchronous in the cells. Depletion of the proteins astrin or Aki1, which have been described as inhibitors of centrosomal separase, did not led to a significant activation of separase at centrosomes, emphasizing the importance of direct separase activity measurements at the centrosomes. Inhibition of polo-like kinase Plk1, on the other hand, decreased the separase activity towards the Scc1 but not the kendrin reporter. Together these findings indicate that Plk1 regulates separase activity at the level of substrate affinity at centrosomes and may explain in part the role of Plk1 in centriole disengagement.
Highlights
Centrosomes are the main microtubule organizing centers of animal cells that consist of the organizing centrioles and pericentriolar material
Separase localizes to centrosomes during mitosis where it regulates the centriole disengagement [12,30]. It remains to be established how this centrosomal separase activity is regulated. To address this important question, we have developed two distinct ‘‘separase sensors’’ that measure separase activity at the centrosomes of individual cells in real time
The sensors contained the separase cleavage sites (SCS) of either Scc1 (142–467 aa; cleavage sites at R172 - cleavage site 1, R450 and R460 - cleavage site 2) or kendrin (2059–2398 aa; cleavage site at R2231), the two known centrosomal separase substrates [26,31]. mCherry was fused to the N-terminus and eGFP to the C-terminus of each SCS element
Summary
Centrosomes are the main microtubule organizing centers of animal cells that consist of the organizing centrioles and pericentriolar material. Depletion of either astrin or Aki1 induces multipolar spindles in mitosis with disengaged centrioles, which would be consistent with premature separase activation [14,15]. Depletion of Sgo1 promotes centriole disengagement in human cells in a manner that requires Plk1 activity [18].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
