Regulation of Cytokinesis and Tumor Formation by RhoA

RhoA 对细胞分裂和肿瘤形成的调节

• 批准号: 8328727
• 负责人: Ann Louise Miller
• 金额: $ 24.48万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8328727
• 关键词: Address; Affect; Aneuploidy; Animals; Binding; Biology; Cancer Biology; Cell Nucleus; Cell division; Cells; Cellular biology; Centrosome; Chromosomes; Comprehensive Cancer Center; Cytokinesis; Development; Educational workshop; Embryo; Ensure; Faculty; Failure; Feedback; GTP Binding; GTPase-Activating Proteins; Genetic Materials; Goals; Grant; Guanosine Triphosphate Phosphohydrolases; Human; Image; Lead; Learning; Life; Literature; Maintenance; Malignant Neoplasms; Marketing; Mentors; Microfilaments; Microscopy; Molecular; Monomeric GTP-Binding Proteins; Mutate; Myosin ATPase; Occupations; Organelles; Pathology; Phase; Phosphorylation; Positioning Attribute; Process; Proteins; Publishing; Regulation; Research; Research Personnel; Resolution; Role; Series; Staging; Tadpoles; Testing; Tetraploidy; Therapeutic; Time; Training; Training Activity; Tumor Biology; Tumor Cell Biology; Tumor-Associated Process; Work; Writing; Xenopus; angiogenesis; anillin; anticancer research; carcinogenesis; daughter cell; inhibitor/antagonist; insight; knock-down; meetings; member; mutant; oncology; post-doctoral training; programs; research study; rho; skills; tool; tumor; tumorigenesis

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.

细胞因子是细胞分裂的最后阶段，其中一个细胞分为两个子细胞。这个过程 必须仔细调节以确保裂解沟的位置正确，以便遗传 材料和细胞细胞器平均分布到每个子细胞。获得更好的理解 细胞因子的作用是基本生物学和癌症研究的关键目标。但是，很清楚 了解调节细胞因子的分子机制仍然难以捉摸。在我的实验室里，我打算 研究调节细胞因子的分子机制以及细胞因子衰竭如何促进 肿瘤发生。我的长期目标是成为一名独立调查员，他是细胞领域的领导者 生物学和肿瘤生物学。为了实现这一目标，我建议在K99的指导培训阶段，我将 专注于发布和介绍我的博士后研究，并将我的工作发展成独立 研究计划。我还将获得癌症生物学的关键培训，并寻求专业发展 活动以帮助我成为就业市场上有力的候选人并建立成功的活动 独立研究计划。获得我需要精通癌症生物学的培训将是 完成：1）与癌症生物学专家的合作者互动：Caroline博士 亚历山大，韦德·布什曼博士和贝丝·韦弗博士，2）积极参与癌症生物学文献 组，3）参加课程肿瘤学703：癌变和肿瘤细胞生物学，4）参加小 关于肿瘤生物学主题的会议，以及5）成为UW Carbone的副成员 全面的癌症中心，积极参加他们的培训活动，例如大轮 研讨会系列和年度务虚会。我一直在寻找专业发展的机会 我的研究生工作和博士后培训。具体来说，在K99指导培训阶段，我将 参加关于编写R01的研讨会，参加一个学期的教师指导研究小组， 并借此机会在当地和国家会议上展示我的工作以发展强大 与我领域的其他研究人员的联系，并在准备工作时为我的工作带来可见性 市场。 K99/R00赠款给我提供的额外培训时间也将使我进一步发展我的 独立研究计划。在动物细胞中，细胞因子由肌动蛋白丝的收缩环提供动力 和肌球蛋白2。收缩环的形成取决于小的GTPase Rho，该rho被激活 细胞赤道的精确区域。到目前为止，我的工作表明GTPase激活蛋白（GAP） RHO调节剂MGCRACGAP的活性在整个细胞因子中都是必要的，以形成和 通过GTPase通量维持聚焦的RHO活动区；也就是说，Rho循环迅速在活动中， GTP结合的状态和不活跃的GDP结合状态。通过GTPase通量，细胞可以保持聚焦的Rho 活动区，这对于形成集中的收缩环和成功的细胞因子是必需的。工作 我在这里提出了这些发现以及我已经在Bement中已经开发的技能和工具的基础 实验室，同时还通过相互作用开发了癌症生物学和多光子显微镜的新专业知识 与UW-Madison的一群优秀的合作者一起。 AIM 1中描述的实验，我将 在这笔赠款的指导K99阶段进行，直接建立在GTPase Flux发现的基础上 Aurora B和Anillin在调节细胞因子过程中调节Rho活性区和GTPase通量的作用 爪蟾胚胎。首先，我将测试GTPase是否需要MGCRACGAP的Aurora B磷酸化 通过使用磷酸化或不可磷酸化的mgcracgap突变体或用极光B处理细胞的通量 抑制剂。其次，我将测试操纵RHO活动区是否会影响Anillin的定位 当Rho活性区通过表达来操纵RHO活性区时，进行Anillin定位的实时显微镜检查 mgcracgap差距已故突变体或组成型活性Rho。第三，我将测试阿尼林是否促进 通过分析Anillin敲低胚胎中的Rho活性区和RHO活动区域的积极反馈 内源性阿尼林的胚胎被阿尼林突变体取代。 AIM 2中描述的实验，该实验 我将在这笔赠款的指导K99阶段发起，并在独立R00阶段继续进行检查，检查 非整倍性的有争议的问题，具有超过或小于正常状况的状况 染色体的数量是肿瘤发生的原因或结果。这项工作将直接解决 第一次关于导致四倍体的细胞因子衰竭然后是非整倍性的问题，可以驱动 肿瘤发生。首先，我将测试MGCRACGAP的靶向敲低是否会在Xenopus中诱导肿瘤 在全球撞击p53的背景下t。其次，我将通过 检查肿瘤核，中心体，病理学和血管生成。第三，我将测试细胞因子是否失败 在活的爪蟾t中，通过现场的高分辨率显微镜形成肿瘤的区域，其中肿瘤 正在形成。最后，我将测试其他RHO区调节剂引起的细胞因子衰竭，尤其是 在人类肿瘤中被上调或下调或突变的人会促进肿瘤的形成。