Regulated reconstitution of spindle checkpoint arrest and silencing through chemically induced dimerisation  in vivo .
Chemically 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.
Amin P, Soper Ní Chafraidh S, Leontiou I, Hardwick KG