Regulation of Cytokinesis and Tumor Formation by RhoA

描述（由申请人提供）：细胞质分裂是细胞分裂的最后阶段，一个细胞分裂成两个子细胞。这个过程必须仔细调节，以确保切割沟的位置正确，使遗传物质和细胞器均匀地分布到每个子细胞。更好地理解细胞分裂是基础生物学和癌症研究的一个关键目标。然而，对调节细胞分裂的分子机制的清晰理解仍然是难以捉摸的。在我的实验室里，我计划研究调节细胞分裂的分子机制以及细胞分裂失败如何促进肿瘤的发生。我的长期目标是成为一名独立的研究者，在细胞生物学和肿瘤生物学领域处于领先地位。为了实现这一目标，我建议在K99指导培训阶段，我将专注于发表和展示我的博士后研究，并将我的工作发展成为一个独立的研究项目。我还将接受癌症生物学方面的重要培训，并寻求专业发展活动，以帮助我在就业市场上成为一名强有力的候选人，并建立一个成功的独立研究项目。为了获得精通癌症生物学所需的培训，我将通过以下途径来完成：1)与我的合作伙伴互动，他们是癌症生物学方面的专家：卡罗琳·亚历山大博士、韦德·布什曼博士和贝丝·韦弗博士；2)积极参加癌症生物学文献小组；3)参加肿瘤学703课程；4)参加肿瘤生物学主题的小型会议；5)成为UW Carbone综合癌症中心的准会员，并积极参加他们的培训活动，如Grand Rounds系列研讨会和年度务静会。在我的研究生工作和博士后培训中，我一直在寻找专业发展的机会。具体来说，在K99指导培训阶段，我将参加一个关于撰写R01的研讨会，参加一个为期一个学期的教师指导研究小组，并利用一切机会在当地和全国会议上展示我的工作，与我所在领域的其他研究人员建立牢固的联系，并在我准备进入就业市场时为我的工作带来知名度。K99/R00基金给予我的额外培训时间也将使我能够进一步发展我的独立研究计划。在动物细胞中，细胞分裂是由肌动蛋白丝和肌球蛋白-2的收缩环驱动的。收缩环的形成依赖于小的GTPase Rho，它在细胞赤道的一个精确区域被激活。到目前为止，我的工作已经表明，Rho调节剂MgcRacGAP的GTPase激活蛋白（GAP）活性在整个细胞分裂过程中是必要的，通过GTPase通量形成和维持一个集中的Rho活性区；也就是说，Rho在活性的、gtp结合的状态和非活性的、gdp结合的状态之间快速循环。通过GTPase通量，细胞可以维持一个聚焦的Rho活性区，这是形成聚焦的收缩环和成功的细胞分裂所必需的。我在这里提出的工作是建立在这些发现以及我已经在Bement实验室开发的技能和工具的基础上，同时也通过与威斯康星大学麦迪逊分校的一群优秀合作者的互动，开发了癌症生物学和多光子显微镜方面的新专业知识。Aim 1中描述的实验，我将在该资助的K99指导阶段进行，通过解剖Aurora B和Anillin在非洲爪蟾胚胎细胞质分裂期间调节Rho活性区和GTPase通量的作用，直接建立在GTPase通量发现的基础上。首先，我将通过使用拟磷或不可磷酸化的MgcRacGAP突变体或用Aurora B抑制剂处理细胞来测试GTPase Flux是否需要MgcRacGAP的Aurora B磷酸化。其次，当Rho活性区被MgcRacGAP GAP-DEAD突变体或组成型活性Rho操纵时，我将通过进行Anillin定位的活显微镜来测试Rho活性区的操作是否会影响Anillin定位。第三，我将通过分析Anillin敲除胚胎和内源性Anillin被Anillin突变体取代的胚胎的Rho活性区来测试Anillin是否促进了Rho活性区的正反馈。在Aim 2中描述的实验，我将在该资助的指导K99阶段开始，并在独立的R00阶段继续进行，研究非整倍性，即染色体数量多于或少于正常数量的情况，是否是肿瘤发生的原因或结果这一有争议的问题。这项工作将首次直接解决细胞分裂失败，导致四倍体和非整倍体，是否可以驱动肿瘤发生的问题。首先，我将测试在p53整体被敲除的背景下，MgcRacGAP的靶向敲除是否会诱导爪蟾蝌蚪的肿瘤。其次，我将通过检查肿瘤核、中心体、病理和血管生成来描述肿瘤的特征。第三，我将通过对肿瘤形成区域的高分辨率活体显微镜，测试正在形成肿瘤的爪蟾蝌蚪的细胞分裂是否失败。最后，我将测试其他Rho区调节因子，特别是那些在人类肿瘤中上调或下调或突变的调节因子诱导的细胞分裂失败是否促进肿瘤的形成。

DESCRIPTION (provided by applicant): 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. Public Health Relevance: 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.