When a cell divides, it must accurately replicate its genetic material and then faithfully segregate this material into the resulting daughter cells. My research addresses the latter half of this problem, focusing on how the cell regulates the function of the mitotic spindle, an elegant microtubule-based machine that attaches to replicated DNA and pulls it apart during mitosis. Here, I present two studies that investigate how the cell disassembles the mitotic spindle at the end of mitosis and how the cell positions the mitotic spindle prior to mitotic completion.

I combined genetic analysis with live-cell fluorescence microscopy to identify the subprocesses driving spindle disassembly as well as the proteins that perform these subprocesses. Our results suggest that mechanistically distinct pathways largely governed by the anaphase-promoting complex, Aurora B kinase, and kinesin-8 cooperate to drive spindle disassembly in budding yeast. We also describe the roles of novel disassembly factors such as the spindle protein She1 and the 7-protein Alternative Replication Factor C complex. Together, these pathways disengage the mitotic spindle halves, inhibit spindle microtubule growth, and promote sustained spindle microtubule depolymerization. Strikingly, combined inhibition of pairs of disassembly pathways yielded cells with hyper-stable spindle remnants, which caused dramatic defects in cell cycle progression, thus establishing that regulated and rapid spindle disassembly is crucial for cell proliferation.

To better understand the mechanisms of spindle positioning, I examined how the dynein-driven spindle-positioning pathway in budding yeast is silenced. My work suggests that dynein activity is regulated through interaction with the multi-subunit dynactin complex at anaphase and identifies a new cellular factor, She1, which controls this interaction. Dynactin is a well-known dynein activator, and, in budding yeast, the complete complex is required for dynein-dependent spindle movement. I found that localization of the dynactin complex is cell cycle-regulated, such that dynactin is recruited to astral microtubules, via interaction with dynein, primarily during anaphase. Additionally, we discovered that the protein She1 is a cell cycle-regulated inhibitor of dynein activity. Without She1, dynein activity extends beyond anaphase and, as a result, mis-positions the mitotic spindle. Strikingly, loss of She1 also permits recruitment of the dynactin complex to astral microtubules throughout the cell cycle. These results suggest that in wild-type cells, She1 restricts dynein activity to anaphase by preventing the interaction between dynein and the complete dynactin complex.

Mitosis is the process by which eukaryotic cells segregate their chromosomes before division. A critical stage of mitosis is anaphase, when the microtubule-based spindle segregates chromosomes into mother and daughter cells prior to cytokinesis. My dissertation research aimed to provide a better understanding of the regulation of anaphase progression and spindle function during mitosis in Saccharomyces cerevisiae.

My research focused on two microtubule-associated proteins, Slk19 (CENP-F homolog) and Stu1 (CLASP homolog). These proteins play fundamental roles in anaphase progression, Stu1 in microtubule spindle stability and Slk19 in spindle midzone organization and Cdc14 phosphatase regulation. I found that Stu1 and Slk19 physically interact and that Slk19 regulates Stu1 localization during anaphase. In addition to its interaction with Slk19, I identified a number of other physical and genetic interactors of Stu1 through mass spectrometry, yeast two-hybrid and synthetic genetic analyses. These interactors provide insight into the role of Stu1 at kinetochores. I also investigated Stu1 function through a protein truncation analysis and purification of full-length Stu1 protein. My truncation analysis revealed that the Stu1 C-terminus is dispensable for viability but is necessary for proper protein localization. The N-terminus, however, is essential for viability. My attempts to purify Stu1 from insect cells were partially successful; the protein is extremely sensitive to proteolytic degradation, and under conditions that limit proteolysis, the protein appears to aggregate or oligomerize in solution.

I also investigated the role of Slk19 sumoylation in anaphase progression. I determined that the Cdc14 early anaphase release (FEAR) network protein Slk19 is sumoylated in vivo and that sumoylation is important for restricting Cdc14 phosphatase localization to the nucleus at the end of anaphase. A slk19 sumoylation mutant causes premature Cdc14 movement from the nucleus to the bud neck, which affects mitotic exit, as this slk19 sumoylation mutant can partially rescue the spindle disassembly defect of the mitotic exit network mutant cdc15-2. This slk19 mutant also has aberrant spindle elongation dynamics, which might be due to a change in Cdc14 function during anaphase.

In conclusion, my dissertation research has uncovered a number of previously unrecognized interactions among mitotic proteins and has revealed a novel function of sumoylation in the regulation of Cdc14 function during anaphase through the FEAR network protein Slk19.

During mitosis, individual microtubules make attachments to chromosomes via a specialized protein complex called the kinetochore to faithfully segregate the chromosomes to daughter cells. Translocation of kinetochores on the lateral surface of the microtubule has been proposed to contribute to high fidelity chromosome capture and alignment at the mitotic midzone, but has been difficult to observe in vivo because of spatial and temporal constraints. To overcome these barriers, we used total internal reflection fluorescence (TIRF) microscopy to track the interactions between microtubules, kinetochore proteins, and other microtubule-associated proteins in lysates from metaphase-arrestedSaccharomyces cerevisiae. TIRF microscopy and cryo- correlative light microscopy and electron tomography indicated that we successfully reconstituted interactions between intact kinetochores and microtubules. These kinetochores translocate on the lateral microtubule surface toward the microtubule plus end and transition to end-on attachment, whereupon microtubule depolymerization commences.These kinetochore dynamics we were able to reconstitute in vitro resembled the process of chromosome congression seen in cells. Chromosome alignment on the mitotic spindle, also referred to as congression, is facilitated by translocation of side- bound chromosomes along the microtubule surface, which allows the establishment of end-on attachment of kinetochores to microtubule plus ends. This is exactly what we observed in our reconstitution assay. We observed kinetochore translocation on the lateral microtubule surface toward the microtubule plus end, which we found that the directional kinetochore movement is dependent on the highly processive kinesin-8, Kip3. We propose that Kip3 facilitates stable kinetochore attachment to microtubule plus ends through its abilities to move the kinetochore laterally on the surface of the microtubule and to regulate microtubule plus end dynamics. Additionally, we also observed kinetochore motility was dependent on Stu2. However, we showed that these proteins display distinct dynamics on the microtubule. Kip3 is highly processive and moves faster than the kinetochore. Stu2 tracks both growing and shrinking microtubule ends but also colocalizes with moving lattice-bound kinetochores. In cells, we observed that both Kip3 and Stu2 are important for establishing chromosome biorientation, Moreover, when both proteins are absent, biorientation is completely defective. All cells lacking both Kip3 and Stu2 had declustered kinetochores and about half also had at least one unattached kinetochore. Our evidence argues that despite differences in their dynamics, Kip3 and Stu2 share roles in chromosome congression to facilitate proper kinetochore- microtubule attachment.

The timely regulation of spindle disassembly is essential for cells to coordinate mitotic exit with cytokinesis. In the budding yeast Saccharomyces cerevisiae, the microtubule-associated protein She1 functions in one of at least three parallel pathways that promote spindle disassembly. She1 phosphorylation by the Aurora kinase Ipl1 facilitates a role for She1 in late anaphase, when She1 contributes to the depolymerization of microtubules and shrinkage of spindle halves. By examining genetic interactions between known spindle disassembly genes, I identified components of the Hog (High Osmolarity Glycerol response) pathway, and found that three genes in this pathway, SHO1,PBS2, and HOG1, are necessary for proper localization of She1 to the anaphase spindle. Hog pathway mutants exhibited spindle disassembly defects, as well as mislocalization of the NoCut checkpoint proteins Boi1 and Boi2 from the bud neck. Moreover, Boi2, but not Boi1, plays a role in spindle disassembly that places it in a pathway with Sho1, Pbs2, and Hog1. A non-classical nuclear localization signal, the importin Nmd5, and exportin Crm1/Xpo1 - all necessary for Hog1 trafficking - are also necessary for proper She1 localization to the spindle and bud neck. Together, my data suggest a process by which cells monitor events at the spindle and bud neck, and describe a novel role for Hog pathway signaling in mitosis.

I also studied mechanisms by which spindle microtubules attach to kinetochores. I generated two new alleles of NDC80, a component of the essential Ndc80 complex, which tethers the outer kinetochore to spindle microtubules and participates in chromosome segregation. Deletions of an internal loop within the coiled-coil region of Ndc80 render cells temperature sensitive and unable to make stable microtubule-kinetochore attachments, resulting in metaphase arrest and DNA mis-segregation phenotypes. These alleles have been useful in helping us understand the required geometry of proteins in the outer kinetochore, and how protein-protein interactions promote the stable microtubule-kinetochore attachments necessary for high-fidelity chromosome segregation.

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.

The 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.

The 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.

Together, 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.