A Study On Function Of Fission Yeast Phosphatase PP2A Participating In Spindle Assembly Checkpoint Silencing

During mitosis,the spindle assembly checkpoint acts to maintain genome stability by delaying cell division until all chromosomes have been segregated into two poles.When kinetochores are not correctly attached to the spindle or tension between sister kinetochores is not established,cells will activate the spindle assembly checkpoint network,which in turn blocks cell cycle progression.Only after all kinetochores are stably attached to the spindle and have established a bipolar orientation,the checkpoint is inactivated,which alleviates the cell cycle block and thus allows chromosome segregation and cell division to proceed.Previous studies have shown that fission yeast phosphatase PP1,Dis2,is actively involved in spindle assembly checkpoint(SAC)silencing through interaction with kinetochore protein Spc7.We repeated some similar assays by counting metaphase cells through spindle length measurement and following degradation of APC/C substrate cyclin B with fluorescent microscopy,we confirmed that Dis2 is indeed required for timely SAC silencing.We also examined the timing of SAC silencing in individual deletion mutants of PP2A subunits,we found that SAC silencing is slightly delayed in subunit B56 deletion mutant,although the defects are not as strong as in dis2+ deletion mutant.This weak effect of PP2A correlates with its low activity at anaphase.Since the activities of PP2A and PP1 fluctuate during cell cycle,we assumed that forced targeting of PP2A at kinetochores may be able to replace the functions of PP1 in SAC silencing.Interestingly,we found that overexpression of PP2A is able to partially rescue the defects of SAC silencing in dis2+ deletion mutant,and the rescuing effect increases with the overexpression strength.Based on our results,we propose that fission yeast PP1 functions in SAC silencing by being recruited to kinetochores by Spc7 and motor proteins Klp5 and Klp6.In the absence of dis2+,phosphatase PP2A can partially complement Dis2 in SAC silencing.Thus,fission yeast PP2A is only auxiliary in SAC silencing,which is very different from mammals.