Identification and characterisation of spindle checkpoint silencing factors in Schizosaccharomyces pombe

Abstract

In cell division, the spindle-assembly checkpoint (SAC) is an important mechanism which ensures proper segregation of chromosomes into daughter cells by delaying anaphase onset until all chromosomes are correctly attached to the mitotic spindle via their kinetochores. This reduces the risk of aneuploidy, which is associated with severe consequences such as birth defects and cancer. Once all kinetochores have been properly attached the SAC is rapidly silenced, allowing the cell to progress through anaphase.

Several SAC silencing factors have been identified to date but the mechanisms by which silencing occurs remain unclear. This project aims to improve our understanding of SAC silencing mechanisms by identifying factors involved in this process and characterising their functions.

High-throughput genetic screening was carried out in fission yeast (Schizosaccharomyces pombe) to identify silencing defective mutants. In designing this genetic screen, we aimed to improve upon previous screens by avoiding false positives due to mutations that lead to prolonged mitotic arrest for reasons unrelated to checkpoint silencing defects, e.g. disruption of kinetochore function. To achieve this, an ectopic synthetic checkpoint mechanism developed as part of previous work in the lab was used to spatially separate checkpoint activation from the kinetochore (Yuan et al, 2016).

This screening approach has produced a list of candidates. Assays to confirm and characterise the checkpoint silencing roles of a subset of these factors have been carried out. These factors were selected on the basis of strength of phenotype in the screen and include SWI/SNF component Sol1 and golgi-associated protein Grh1, among others.

Additionally, work was carried out to characterise a previously identified checkpoint silencing factor, Protein Phosphatase 1 (PP1) Dis2 in a synthetic ectopic checkpoint arrest (SynCheckABA). This work illustrated the suitability of this synthetic system as a tool for further study of SAC silencing (Amin et al, 2019).