Structure-Function Study of Human Spindly

• Author / Creator

  Moudgil, Devinderjit K

• Mitosis is the last and the shortest stage of the cell cycle with the ultimate goal of equal distribution of replicated genetic material into two daughter cells. Proper execution of mitosis is very important for the maintenance of genomic integrity. Failure of the accurate segregation of genetic material can lead to aneuploidy as well as cell death. Aneuploidy is a common occurrence in cancer cells and it can also lead to severe birth defects. The mitotic checkpoint is a surveillance mechanism that regulates metaphase to anaphase transition and ensures precise chromosome segregation during mitosis. The mitotic checkpoint-signaling pathway is a biochemical system that involves multi-protein networks present on kinetochores during mitosis. Therefore, understanding how these mitotic checkpoint proteins regulate the function of this checkpoint has been the focus of extensive research. How the mitotic checkpoint signal is generated is widely studied however far less is known about how the checkpoint is silenced. Here, we investigated the mitotic checkpoint function of the human Spindly protein, which recruits the dynein/dynactin complex to kinetochores, both of which are involved in checkpoint silencing. I have characterized the kinetochore localization domain of Spindly to its 294-605 C-terminal amino acids. Furthermore, I examined the underlying molecular mechanism of Spindly kinetochore localization and discovered that Spindly undergoes a posttranslational lipid modification known as farnesylation on its C-terminal cysteine residue. Inhibition of farnesyl transferase enzyme with farnesyl transferase inhibitors prevented kinetochore localization of Spindly. A key upstream regulator of Spindly kinetochore localization is an essential checkpoint component, the RZZ complex. We showed that Spindly farnesylation is essential for its interaction with the RZZ complex and hence its kinetochore localization. Farnesylation transferase inhibitor was reported to cause aberrant mitotic progression in cells over a decade ago and now my work has reinforced the importance of farnesylation for the proper execution of mitosis through Spindly kinetochore localization. I postulated that Spindly is likely the primary mitotic target of farnesyl transferase. Spindly is phosphorylated during mitosis and its phosphorylation sites are located within the kinetochore localization domain. Here I show that Spindly phosphorylation affects its kinetochore binding affinity and leads to premature transport to spindle poles. I also demonstrate that Spindly becomes a dynamic kinetochore component at metaphase kinetochores compared to prometaphase. These results indicate that Spindly phosphorylation is perhaps the regulatory mechanism for its release from kinetochores. Furthermore, I also identified p50/dynamitin as a novel interaction partner of Spindly, potentially explaining the dynein/dynactin kinetochore recruitment through Spindly. Together, these results support a model where farnesylation regulates physical association of Spindly/RZZ complex and Spindly acts as a direct linker between RZZ and the dynein/dynactin complex.

• Subjects / Keywords

  • Mitosis
  • Dynein/dynactin
  • Spindly
  • Farnesyl transferase inhibitors
  • Kinetochore,
  • Mitotic checkpoint
  • Farnesylation

• Graduation date

  Fall 2015

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3891254T

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.