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: 7770196
• 负责人: Ann Louise Miller
• 金额: $ 9万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 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 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7770196
• 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; TP53 gene; 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; mgcRacGAP; mutant; oncology; post-doctoral training; programs; public health relevance; research study; rho; skills; tool; tumor; tumorigenesis

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.

描述（由申请人提供）：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。必须仔细调节此过程，以确保将裂解沟正确放置，以使遗传物质和细胞细胞器平均分布到每个子细胞上。更好地了解细胞因子是基本生物学和癌症研究的关键目标。但是，对调节细胞因子的分子机制的清晰了解仍然难以捉摸。在我的实验室中，我计划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研究者，他是细胞生物学和肿瘤生物学领域的领导者。为了实现这一目标，我建议在K99的指导培训阶段，我将重点介绍发布和介绍我的博士后研究，并将工作发展为独立的研究计划。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，并建立成功的独立研究计划。获得我需要精通癌症生物学的培训将通过：1）与我的合作者进行互动，这些合作者是癌症生物学专家：Caroline Alexander博士，Wade Bushman博士和Beth Weaver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）参加有关肿瘤生物学主题的小型会议，5）参加他们的培训活动，例如大回合研讨会系列和年度务虚会。在整个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的指导培训阶段，我将参加一个关于编写R01的研讨会，参加了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与我的领域其他研究人员的牢固联系，并在准备进入就业市场时为我的工作带来知名度。 K99/R00赠款给我提供的额外培训时间也将使我能够进一步制定我的独立研究计划。在动物细胞中，细胞因子由肌动蛋白丝和肌球蛋白2的收缩环提供动力。收缩环的形成取决于小的GTPase RHO，该RHO在细胞赤道的精确区域中被激活。到目前为止，我的工作表明，RHO调节剂MGCRACGAP的GTPase激活蛋白（GAP）活性在整个细胞因子中都是通过GTPase Flux形成和维持RHO活性区的整个细胞因子。也就是说，RHO在活跃的，GTP结合的状态和不活跃的GDP结合状态之间迅速循环。通过GTPase通量，细胞可以维持聚焦的RHO活性区，这对于形成聚焦的收缩环和成功的细胞因子是必不可少的。我在这里提出的工作以及我在Bement Lab中已经开发的技能和工具的基础上，同时还通过与UW-Madison的一组优秀的合作者进行互动来开发癌症生物学和多光子显微镜的新专业知识。 AIM 1中描述的实验，我将在该赠款的指导K99阶段进行，直接基于GTPa​​se通量发现，通过剖析Aurora B和Anillin在调节Rho活性区和GTPase中的Anillin在Xenopus中的细胞动物期间的作用。胚胎。首先，我将通过使用磷酸化或非磷酸化的mgcracgap突变体或用Aurora b抑制剂治疗细胞来测试GTPase通量需要MGCRACGAP的Aurora B磷酸化。其次，我将测试对Rho活性区的操纵是否通过通过MGCRACGAP差距死亡突变体的表达或强度活跃的Rho来操纵RHO活性区时通过进行Anillin定位的实时显微镜来影响Anillin的定位。第三，我将通过分析Anillin敲低胚胎和胚胎中的Rho活性区域来测试Anillin是否促进RHO活性区中的正反馈，其中内源性anillin被anillin突变体取代。在AIM 2中描述的实验，我将在该赠款的指导K99阶段启动并在独立R00阶段继续进行，研究了有争议的问题，即非整倍性，具有超过正常染色体数量的状况，是肿瘤发生的原因或结果。这项工作将首次直接解决细胞因子衰竭是否会导致四倍体然后是非倍倍倍的问题，这是否会驱动肿瘤发生。首先，我将测试MGCRACGAP的靶向敲低是否会在全球撞击p53的背景下诱导爪蟾t骨的肿瘤。其次，我将通过检查肿瘤核，中心体，病理和血管生成来表征肿瘤。第三，我将测试在活的爪蟾t骨中的细胞因子是否失败，这些曲to骨通过现场形成肿瘤的区域的高分辨率显微镜形成肿瘤。最后，我将测试其他RHO区调节剂诱导的细胞因子衰竭，尤其是在人类肿瘤中被调节或下调或突变的细胞因子衰竭会促进肿瘤的形成。 公共卫生相关性：这里提出的工作令人兴奋，因为它将帮助我们更好地了解细胞因子的过程如何受到调节，并首次允许我们在高分辨率上进行肿瘤形成过程，以实现它的发生。 。这项工作可能会提供有关细胞因子衰竭是否可以驱动肿瘤形成的机制的关键见解。了解更多有关调节细胞因子和肿瘤发生的分子机制的分子机制，将提高我们对碱性细胞生物学的理解，并有可能识别癌症治疗剂的新靶标。