Mechanism and Regulation of Stu2:Ndc80 Association

Proper chromosome segregation is essential for cell division, relying on the attachment of duplicated chromosomes to microtubules, which pull chromosomes to opposite poles during anaphase of cell division. The kinetochore is the protein complex that physically links chromosomes to microtubules, acting as the direct mediator of chromosome segregation. Many kinetochore proteins are necessary for this process, and their malfunction can lead to aneuploidies, often associated with cancer and other diseases. Misregulation in the timing of kinetochore and microtubule functions can disrupt chromosome segregation, so cells exert precise temporal control of these events during cell division. Despite the centrality of this process, the molecular mechanisms by which the kinetochore ensures accurate chromosome segregation are not fully understood. Furthermore, it remains unclear how cells coordinate the timely progression of multiple steps in chromosome segregation. Elucidating the kinetochore’s role in these processes and its regulatory mechanisms could clarify how aneuploidy arises and potentially inform therapeutic strategies.This dissertation addresses these key questions in the following chapters. Chapter 1 introduces the research topics and key kinetochore proteins, specifically Stu2 and the Ndc80 complex (Ndc80c). Chapter 2 presents mechanistic insights into the Stu2:Ndc80c interaction, including a crystal structure of their interaction and analysis of STU2 mutants that prevent Stu2:Ndc80c binding. I show that STU2 mutants disrupting Ndc80c association cause substantial defects in chromosome segregation, underscoring the importance of this interaction in accurate segregation. Chapter 3 explores the cell-cycle regulation of Stu2 and Ndc80c function, detailing the roles of various kinases and a phosphatase that regulate Stu2:Ndc80c binding through phosphorylation of Stu2. This kinase-phosphatase network is shown to be crucial for promoting the microtubule cytoskeleton’s function with precise timing. Finally, Chapter 4 discusses the broader implications of these findings for kinetochore research and suggests directions for future work. Appendix A contains supplementary experiments supporting Chapter 3, and additional studies on Stu2 post-translational modification and Ndc80c function are included in Appendices B and C.