Cytokinesis is the final stage of cell division where one cell is separated into two daughter cells. This process must be carefully regulated to ensure that the cleavage furrow is positioned correctly so that the genetic material and cellular organelles are distributed equally to each daughter cell. Gaining a better understanding of cytokinesis represents a key goal for both basic biology and cancer research. However, a clear understanding of the molecular mechanisms that regulate cytokinesis remains elusive. In my lab, I plan to study the molecular mechanisms that regulate cytokinesis and how cytokinesis failure can promote tumorigenesis. My long-term goal is to become an independent investigator who is a leader in the fields of cell biology and tumor biology. To meet this goal, I propose that during the K99 mentored training phase, I will focus on publishing and presenting my postdoctoral research and developing my work into an independent research program. I will also obtain crucial training in cancer biology and seek out professional development activities to help position me to be a strong candidate on the job market and establish a successful independent research program. Obtaining the training I need to be well-versed in cancer biology will be accomplished by: 1) interactions with my collaborators, who are experts in cancer biology: Dr. Caroline Alexander, Dr. Wade Bushman, and Dr. Beth Weaver, 2) actively participating in a cancer biology literature group, 3) taking the course Oncology 703: Carcinogenesis and Tumor Cell Biology, 4) attending small meetings on topics of tumor biology, and 5) becoming an associate member of the UW Carbone Comprehensive Cancer Center and actively participating in their training activities such as the Grand Rounds seminar series and the Annual Retreat. I have sought out professional development opportunities throughout my graduate work and postdoctoral training. Specifically, during the K99 mentored training phase, I will participate in a workshop on writing an R01, take part in a semester-long Faculty Mentoring Research Group, and take every opportunity I can to present my work both locally and at national meetings to develop strong connections with other researchers in my fields and bring visibility to my work as I prepare to go on the job market. The additional training time afforded to me by the K99/R00 grant would also allow me to further develop my independent research program. In animal cells, cytokinesis is powered by a contractile ring of actin filaments and myosin-2. Formation of the contractile ring is dependent on the small GTPase Rho, which is activated in a precise zone at the cell equator. My work thus far has shown that the GTPase activating protein (GAP) activity of the Rho regulator MgcRacGAP is necessary throughout cytokinesis for the formation and maintenance of a focused Rho activity zone via GTPase Flux;that is, Rho cycles rapidly between the active, GTP-bound state and the inactive, GDP-bound state. Through GTPase Flux, cells can maintain a focused Rho activity zone, which is necessary for forming a focused contractile ring and for successful cytokinesis. The work I propose here builds on these findings along with the skills and tools I have already developed in the Bement lab, while also developing new expertise in cancer biology and multiphoton microscopy through interactions with a group of excellent collaborators here at UW-Madison. The experiments described in Aim 1, which I will carry out during the mentored K99 phase of this grant, build directly on the GTPase Flux finding by dissecting the roles of Aurora B and Anillin in regulating the Rho activity zone and GTPase Flux during cytokinesis in Xenopus embryos. First, I will test whether Aurora B phosphorylation of MgcRacGAP is required for GTPase Flux by using phosphomimetic or non-phosphorylatable MgcRacGAP mutants or treating cells with Aurora B inhibitors. Second, I will test whether manipulation of the Rho activity zone affects Anillin localization by conducting live microscopy of Anillin localization when the Rho activity zone is manipulated by expression of MgcRacGAP GAP-DEAD mutants or constitutively active Rho. Third, I will test whether Anillin promotes positive feedback in the Rho activity zone by analyzing Rho activity zones in Anillin knockdown embryos and embryos where endogenous Anillin is replaced by Anillin mutants. The experiments described in Aim 2, which I will initiate during the mentored K99 phase of this grant and continue in the independent R00 phase, examine the controversial question of whether aneuploidy, the condition of having more than or less than the normal number of chromosomes, is a cause or consequence of tumorigenesis. This work will directly address for the first time the question of whether cytokinesis failure, which leads to tetraploidy then aneuploidy, can drive tumorigenesis. First, I will test whether targeted knockdown of MgcRacGAP will induce tumors in Xenopus tadpoles in a background where p53 is globally knocked down. Second, I will characterize the tumors by examining tumor nuclei, centrosomes, pathology, and angiogenesis. Third, I will test whether cytokinesis fails in live Xenopus tadpoles that are forming tumors by live, high-resolution microscopy of regions where tumors are forming. Finally, I will test whether cytokinesis failure induced by other Rho zone regulators, especially those that are up- or down-regulated or mutated in human tumors, promotes tumor formation.
The work proposed here is exciting because, it will help us gain a better understanding of how the process of cytokinesis is regulated and will for the first time allow us to image at high resolution the process of tumor formation as it is happening. This work may provide critical insights about whether cytokinesis failure is a mechanism that can drive tumor formation. Learning more about the molecular mechanisms by which Rho activity regulates cytokinesis and tumorigenesis will advance our understanding of basic cell biology and could potentially identify new targets for cancer therapeutics.
