Molecular Mechanisms That Contribute to Spindle Assembly Checkpoint Inactivation

• Other Authors: Bokros, Michael H. (Michael Henry) (author)
• Format: Others
• Language: English; English
• Published: Florida State University
• Subjects: Cytology; Genetics; Molecular biology
• Online Access: http://purl.flvc.org/fsu/fd/2018_Su_Bokros_fsu_0071E_14734

The Spindle Assembly Checkpoint (SAC) prevents anaphase onset in response to chromosome attachment defects. Bipolar kinetochore attachment is required for SAC silencing which allows for anaphase onset and the release of the phosphatase, Cdc14. The Cdc14 early anaphase release (FEAR) pathway reverses Cdk1 activity to promote early anaphase events. Using S. cerevisiae, we discovered that the FEAR pathway inhibits SAC activity in anaphase, a process we have named SAC termination. We found that hyper-activation of FEAR allows premature SAC silencing, which depends on Cdc14-mediated dephosphorylation of a kinetochore protein Fin1, a regulator of protein phosphatase PP1. Surprisingly, in fin1∆ mutants we found dynamic kinetochore localization of SAC protein Bub1 after anaphase entry, indicating Fin1 regulates SAC localization. Fin1-PP1 promotes SAC termination through the dephosphorylation of the outer kinetochore Ndc80 as well as the removal of Ipl1 kinase. These results show that FEAR activation during early anaphase promotes SAC termination through Fin1-PP1 to ensure no SAC activation during anaphase. We also discovered that S-phase cyclin dependent kinase (Clb5/CDK) promotes the correction of erroneous attachments to promote kinetochore biorientation. We show that clb5Δ mutants are sensitive to syntelic attachments due to prolonged SAC arrest. This phenotype was partially dependent on Cnn1, as cnn1Δ partially rescued clb5Δ sensitivity to syntelic attachments. This result suggests that Clb5/Cdk1 inhibits Cnn1 during S-phase/early mitosis to promote bipolar establishment. Finally, we revealed that a group of inner kinetochore proteins, the Constitutive Centromere Associated Network (CCAN), prevents SAC silencing in the presence of tensionless attachments. The kinetochore protein Ybp2 interacts with the CCAN and we found that ybp2Δ partially suppresses the SAC silencing mutant ipl1-321, indicating that Ybp2 functions to promote SAC silencing. Surprisingly, ybp2Δ fully rescues CCAN mutant sensitivity to syntelic attachments indicating that the CCAN inhibits Ybp2 to prevent SAC silencing. Interestingly, ybp2Δ mutant cells show enhanced phosphorylation of a kinetochore protein Dsn1 further supporting the hypothesis that Ybp2 antagonizes Ipl1 kinase to promote SAC silencing. In summary, our results uncover new molecular mechanisms that suppress SAC activity which allows for efficient segregation of the genome. We uncover that the FEAR pathway promotes SAC termination by decreasing kinase activity while simultaneously increasing phosphatase activity on the kinetochore during anaphase. This ensures no SAC activity during anaphase. Additionally, we uncover that S-phase Cdk1 promotes the correction of erroneous kinetochore-microtubule attachments by inhibiting Cnn1 kinetochore function. This allows for efficient error correction before anaphase onset. Lastly, we found that a network of inner kinetochore protein complexes, CCAN, prevents SAC silencing when tensionless attachments are present, revealing how the CCAN supports accurate chromosome segregation. Taken together, the data presented here deepens our understanding of SAC regulation which ensures faithful chromosome segregation. === A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. === Summer Semester 2018. === July 11, 2018. === CCAN, Cdc14, Fin1, PP1, S-phase Cdk, Spindle Assembly Checkpoint === Includes bibliographical references. === Yanchang Wang, Professor Directing Dissertation; Hong-Guo Yu, University Representative; Akash Gunjan, Committee Member; Timothy Megraw, Committee Member.