Year-end Sale % OFF 
Abstract
ABSTRACTChemically induced dimerisation (CID) uses small molecules to control specific protein–protein interactions. We employed CID dependent on the plant hormone abscisic acid (ABA) to reconstitute spindle checkpoint signalling in fission yeast. The spindle checkpoint signal usually originates at unattached or inappropriately attached kinetochores. These are complex, multiprotein structures with several important functions. To bypass kinetochore complexity, we took a reductionist approach to studying checkpoint signalling. We generated a synthetic checkpoint arrest ectopically by inducing heterodimerisation of the checkpoint proteins Mph1 (the fission yeast homologue of Mps1) and Spc7 (the fission yeast homologue of KNL1). These proteins were engineered such that they cannot localise to kinetochores, and only form a complex in the presence of ABA. Using this novel assay we were able to checkpoint arrest a synchronous population of cells within 30 min of ABA addition. This assay allows detailed genetic dissection of checkpoint activation and, importantly, also provides a valuable tool for studying checkpoint silencing. To analyse silencing of the checkpoint and the ensuing mitotic exit, we simply washed out the ABA from arrested fission yeast cells. We show here that silencing is critically dependent on protein phosphatase 1 (PP1) recruitment to Mph1-Spc7 signalling platforms.
Highlights
Spindle checkpoint signalling was initially reconstituted in Xenopus egg extracts (Kulukian et al, 2009; Minshull et al, 1994) and most recently using recombinant complexes of human checkpoint proteins (Faesen et al, 2017)
We found that the kinetics of release was critically dependent on recruitment of protein phosphatase 1 (PP1) to the Mph1-Spc7 signalling platform
Addition of abscisic acid (ABA) to SynCheckABA strains induces the heterodimerisation of Mph1-ABI and Spc7-PYL fusion proteins and this is sufficient to generate an activated signalling scaffold and metaphase arrest within minutes
Summary
Spindle checkpoint signalling was initially reconstituted in Xenopus egg extracts (Kulukian et al, 2009; Minshull et al, 1994) and most recently using recombinant complexes of human checkpoint proteins (Faesen et al, 2017) Major advantages of such in vitro assays are that complex systems can be simplified through biochemical fractionation and manipulated through immunodepletion. Yeast genetics has driven the identification of most of the molecular components of this pathway, such as the mitotic arrest deficient (Mad) and budding uninhibited by benzimidazoles (Bub) proteins (Hoyt et al, 1991; Li and Murray, 1991) and their Cdc effector (Hwang et al, 1998; Kim et al, 1998).
Sign-in/Register to view highlights & summary 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.
