Mechanisms of mitotic spindle function in Saccharomyces cerevisiae

Cell division is an essential process for multicellular organisms. The fundamental goal of mitosis is to duplicate and segregate the genetic material of one cell, to ultimately generate two cells genetically identical to each other and to the parent cell. The mitotic spindle is a microtubule-based structure that attaches to, and segregates replicated chromosomes to the two daughter cells. To accomplish such a crucial, yet intricate task, the mitotic spindle must be correctly assembled, positioned, and disassembled. Historically, research has mostly focused on addressing the first problem. However, recent studies have highlighted the importance of proper spindle positioning and disassembly. In this dissertation we attempt to address these last two problems. We focused on understanding how the activities of a group of microtubule-associated proteins are coordinated to regulate microtubule function during spindle positioning and disassembly, to finally ensure successful cell division./nThe work presented in Chapters 2 and 3 of this dissertation attempts to shed light on the process of spindle disassembly. The Aurora B protein kinase, or Ipl1 in budding yeast, is one of the main regulators of spindle disassembly. Once chromosome segregation is completed, and just before the onset of spindle disassembly, the Ipl1/Aurora B kinase concentrates at the spindle midzone, the region of spindle breakage. However, we do not fully understand how it is targeted to the midzone, or its precise role there. Chapter 2 addresses these two questions. Combining live cell microscopy with yeast genetics and biochemistry we identified a kinesin-5 (Kip1) as the main kinesin responsible for the midzone localization of Ipl1/Aurora B. Kip1 alone is sufficient to recruit Ipl1/Aurora B to microtubules, and it is able to transport Ipl1/Aurora B to the microtubule end. We also found that cells rely on cytokinesis to physically break the spindle if the Ipl1/Aurora B kinase cannot concentrate at the midzone. Once at the midzone, we know that Ipl1/Aurora B phosphorylates targets to destabilize the spindle and allow its breakage. She1 is a microtubule-associated protein that is activated during late anaphase by Ipl1/Aurora B-mediated phosphorylation. However, we do not understand its precise role during spindle disassembly. Chapter 3 investigates this question, and addresses whether She1 promotes spindle disassembly by directly destabilizing spindle microtubules./nThe mitotic spindle needs to be positioned perpendicular to the division plane for proper chromosome segregation. Chapter 4 attempts to gain some insight into this process, and focuses on understanding how the activity of two key antagonistic kinesins, present at the same time, on the same set of microtubules, is coordinated to achieve precise spindle positioning./nTogether, these studies have shed light on the mechanistic nuances of how protein function is coordinated to position and disassemble the mitotic spindle, and they present a novel model for how the Ipl1/Aurora B kinase is recruited to the spindle midzone in late anaphase, a process conserved across eukaryotes.

Advisor: Barnes, Georjana
Drubin, David G