Regulation of Cytokinesis and Tumor Formation by RhoA

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。这一过程 必须仔细调节，以确保乳沟的位置正确，以便遗传 物质和细胞器均匀地分布在每个子细胞上。更好地了解 细胞质分裂是基础生物学和癌症研究的一个关键目标。然而，一个明确的 对调节胞质分裂的分子机制的了解仍然是难以捉摸的。在我的实验室里，我计划 研究调节胞质分裂的分子机制以及胞质分裂失败如何促进 肿瘤发生学。我的长期目标是成为一名在细胞领域处于领先地位的独立研究员 生物学和肿瘤生物学。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一个独立的 研究计划。我还将接受癌症生物学方面的重要培训，并寻求职业发展。 帮助我在就业市场上成为一个强有力的候选人并建立一个成功的 独立研究计划。获得精通癌症生物学所需的培训将是 完成方式：1)与我的合作者互动，他们都是癌症生物学专家：卡罗琳博士 亚历山大、韦德·布什曼博士和贝丝·韦弗博士积极参与癌症生物学文献的研究 组，3)选修《肿瘤学703：癌变与肿瘤细胞生物学》课程，4)选修Small 肿瘤生物学专题会议，成为威斯康星大学Carbone准会员 综合癌症中心，并积极参加他们的培训活动，如大轮 系列研讨会和年度务虚会。我一直在寻找职业发展的机会 我的研究生工作和博士后培训。具体地说，在K99指导培训阶段，我将 参加R01写作工作坊，参加为期一学期的教师指导研究组， 并抓住一切可能的机会，在当地和全国会议上展示我的工作，以发展 与我所在领域的其他研究人员建立联系，并在我准备上岗时为我的工作带来可见性 市场。 K99/R00赠款为我提供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，胞质分裂是由肌动蛋白细丝的收缩环驱动的。 和肌球蛋白-2。收缩环的形成依赖于小的GTP酶Rho，该酶在 在单元格赤道的精确区域。到目前为止，我的工作表明GTP酶激活蛋白(GAP) 在整个胞质分裂过程中，Rho调节因子MgcracGAP的活性对于形成和 通过GTP酶通量维持集中的Rho活动区；即Rho在活跃的、 GTP绑定状态和非活动GDP绑定状态。通过GTP酶通量，细胞可以保持聚焦的Rho 活动区，这是形成集中的收缩环和成功的胞质分裂所必需的。这项工作 我建议在这里建立在这些发现的基础上，以及我已经在Bement中开发的技能和工具 实验室，同时通过相互作用开发癌症生物学和多光子显微镜的新专业知识 在威斯康星大学麦迪逊分校有一群优秀的合作者。目标1中描述的实验，我将 在这项资助的指导K99阶段进行，通过解剖直接建立在GTPase通量发现的基础上 极光B和阿尼林在调节Rho活动区和GTP酶通量中的作用 非洲爪哇的胚胎。首先，我将测试GTP酶是否需要Aurora B的MGRACGAP磷酸化 使用拟磷化或非磷酸化的MgcracGAP突变体或用Aurora B处理细胞所产生的通量 抑制剂。其次，我将测试Rho活动区的操纵是否会通过以下方式影响Anlin的本地化 当Rho活动区被表达时进行苯胺素定位的活显微镜观察 MgcracGAP缺口-死亡突变体或结构性活性Rho。第三，我会测试阿尼林是否会提升 通过分析阿尼林基因敲除胚胎中的Rho活动区和 内源苯胺素被苯胺素突变体取代的胚胎。目标2中描述的实验，即 我将在这项资助的指导K99阶段启动，并在独立的R00阶段继续，检查 一个有争议的问题，即非整倍体是否高于或低于正常水平 染色体的数目，是肿瘤发生的原因或结果。这项工作将直接针对 第一次提出了细胞质分裂失败是否会导致四倍体和非整倍体的问题 肿瘤发生学。首先，我将测试靶向敲除MgcracGAP是否会在非洲爪哇诱导肿瘤 在全球范围内P53被击倒的背景下的蝌蚪。其次，我将通过以下方式描述肿瘤的特征 检查肿瘤细胞核、中心体、病理学和血管生成。第三，我将测试胞质分裂是否失败 在活的非洲爪哇蝌蚪中，通过活的高分辨率显微镜观察肿瘤所在区域 正在形成。最后，我将测试其他Rho区调节剂是否导致胞质分裂失败，特别是 那些在人类肿瘤中上调或下调或突变的基因，会促进肿瘤的形成。

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.