Ann L. Miller, Ph.D. - US grants

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.

? DESCRIPTION (provided by applicant): Surprisingly little is known about how cytokinesis works in an intact epithelial environment where the dividing cell is connected to its neighboring cells via cell-cell junctions. Notably, failed cytokinesis can promote tumor formation; over 85% of cancers arise from epithelial tissues, and cell-cell junction defects can contribute to cancer metastasis. Therefore, the objective of the proposed research is to characterize the molecular mechanisms by which epithelial cells maintain and remodel their cell-cell junctions during cell division. Our central hypothesis is that the scaffolding protein Anillin regulates proper spatiotemporal patterning of active RhoA (RhoA-GTP) and is essential for regulating junction structure, remodeling, and tension in dividing and non-dividing epithelial cells. Our focus on RhoA, a small GTPase that promotes actomyosin contractility, stems from evidence that precisely localized zones of active RhoA are required both for cytokinesis and for formation and maintenance of cell-cell junctions. Our group is uniquely positioned to tackle the stated objective because we use high-resolution live imaging of active RhoA dynamics in the intact vertebrate epithelium of the Xenopus laevis embryo. We will test our central hypothesis by pursuing three specific aims. In Aim 1, we will determine Anillin's function in maintaining cell-cell junctions. Or exciting preliminary data indicate that the scaffolding protein Anillin, which is known to play an important role in regulating cytokinesis, also plays a novel role in regulating cell-cell junction integrity. We will test the hypothesis that Anillin regulates cell-cell junctions by controlling th distribution of junctional RhoA-GTP and stabilizing the apical actomyosin belt. In Aim 2, we will characterize how dynamic cell-cell junction remodeling is regulated in dividing cells. We will test the hypothesis that junction protein dynamics are altered in dividing cells due to changes in tension, and proper regulation of localized RhoA-GTP by Anillin is required for junction remodeling. In Aim 3, we will identify mechanisms by which dividing cells regulate and respond to tension changes. Here, we will test the hypothesis that RhoA is activated in response to mechanical force, and Anillin stabilizes RhoA-GTP and promotes increased junctional tension. Using innovative approaches that include live imaging with a fluorescent probe that specifically highlights active RhoA in the cell, characterizing for the first time how cell-cell junction protei dynamics are regulated during vertebrate cell division, and examining RhoA-mediated cellular mechanics - all in intact vertebrate epithelial tissue - will allow us to gain new insights about hw cell division works in epithelial cells. Completion of the proposed research is expected to identif novel mechanisms that regulate localized RhoA activity and the actomyosin-mediated tension required for cell-cell junction maintenance and remodeling during cytokinesis in the intact vertebrate epithelium.

PROJECT SUMMARY / ABSTRACT Cell-cell junctions adhere epithelial cells to one another, transmit forces from cell to cell, and generate biological barriers that selectively regulate what can pass between cells in an epithelial tissue. Fundamental questions about how epithelial cell-cell junctions dynamically remodel in response to physiological forces that that challenge cell adhesion and barrier function remain unanswered. In addition to being absolutely essential for development and maintenance of organ homeostasis, disruption of adhesion and barrier function contributes to diseases including cancer cell metastasis and Inflammatory Bowel Disease. Therefore, it is critical to determine the mechanisms that control cell-cell junction remodeling as epithelial cells change shape. This proposal builds on recent discoveries from the lab showing that Rho flares locally reinforce tight junctions following leaks in barrier function, and that adherens junctions are reinforced by recruitment of Vinculin to the cleavage furrow of dividing epithelial cells. The overall objective of this application is to identify mechanisms that promote maintenance of adhesion and barrier function at sites of epithelial cell division and junction elongation. Our central hypothesis is that locally applied mechanical forces challenge adherens junctions and tight junctions and elicit actomyosin-mediated reinforcement required for maintenance of adhesion and barrier function at these sites. The central hypothesis will be tested by pursuing three specific aims: 1) Identify how mechanically-induced tight junction leaks trigger Rho flares; 2) Determine how Rho flare-mediated junction contractility repairs tight junctions; 3) Define mechanisms that mediate tension transmission and barrier maintenance at sites of locally increased tension. The proposed research is innovative because it applies powerful experimental tools including: a developing vertebrate model system (Xenopus laevis embryos), a live imaging barrier assay recently developed in the lab, proven approaches to locally or globally manipulate tension in the intact epithelium, probes for live imaging of active Rho dynamics as well as a host of cytoskeletal proteins, junction proteins, Rho regulators, and cytoplasmic calcium, and specialized analysis tools to quantitatively analyze live imaging data. The proposed research is significant because it will advance our knowledge about a fundamentally important problem in epithelial cell biology: how epithelial cells undergo dramatic cell shape changes like cytokinesis yet maintain tissue integrity and barrier function.