Regulation of Cytokinesis and Tumor Formation by RhoA

描述(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。这一过程必须仔细管理，以确保卵裂沟的位置正确，以便遗传物质和细胞器均匀分布到每个子细胞。更好地了解胞质分裂是基础生物学和癌症研究的关键目标。然而，对调控胞质分裂的分子机制的清楚理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的发生。我的长期目标是成为一名独立的研究员，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导师培训阶段，我将专注于发表和展示我的博士后研究成果，并将我的工作发展成一个独立的研究项目。我还将获得癌症生物学方面的重要培训，并寻找专业发展活动，帮助我成为就业市场上的有力候选人，并建立一个成功的独立研究项目。获得精通癌症生物学所需的培训将通过以下方式完成：1)与我的同事们进行互动，他们都是癌症生物学方面的专家：Caroline Alexander博士、Wade Bushman博士和Beth Weaver博士；2)积极参加癌症生物学文献小组，3)学习Oncology 703：癌症发生与肿瘤细胞生物学，4)参加关于肿瘤生物学主题的小型会议，5)成为华盛顿大学Carbone综合癌症中心的准成员，并积极参加他们的培训活动，如Grand Runds系列研讨会和年度务虚会。在我的研究生工作和博士后培训期间，我一直在寻找职业发展机会。具体地说，在K99指导培训阶段，我将参加一个关于撰写R01的研讨会，参加一个为期一学期的教师指导研究小组，并尽可能利用一切机会在当地和全国会议上展示我的工作，以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提高我的工作的知名度。K99/R00拨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，胞质分裂由肌动蛋白细丝和肌球蛋白-2组成的收缩环提供动力。收缩环的形成依赖于小的GTP酶Rho，它在细胞赤道的精确区域被激活。到目前为止，我的工作表明，Rho调节因子MgcracGAP的GTP酶激活蛋白(GAP)活性在细胞质分裂过程中是通过GTP酶通量形成和维持集中的Rho活动区所必需的；即Rho在活跃的GTP结合状态和非活跃的GDP结合状态之间快速循环。通过GTP酶通量，细胞可以保持一个集中的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。我在这里提出的工作建立在这些发现的基础上，以及我已经在Bement实验室开发的技能和工具，同时还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。目标1中描述的实验，我将在这项资助的指导K99阶段进行，直接建立在GTP酶通量发现的基础上，通过解剖Aurora B和Anlin在非洲爪哇胚胎胞质分裂过程中调节Rho活动区和GTP酶通量的作用。首先，我将通过使用拟磷酸化或非磷酸化的镁cracGAP突变体或用Aurora B抑制剂处理细胞来测试GTP酶通量是否需要Aurora B磷酸化的MgcracGAP。其次，我将通过对Rho活动区进行活显微镜观察，来测试Rho活动区的操纵是否会影响ANO的定位。第三，我将通过分析阿尼林基因敲除胚胎和内源性阿尼林被阿尼林突变体取代的胚胎中的Rho活跃区，来测试阿尼林是否促进了Rho活跃区的正反馈。目标2中描述的实验，我将在这笔赠款的指导K99阶段启动，并在独立的R00阶段继续，研究有争议的问题，即非整倍体，即具有多于或少于正常数目的染色体的条件，是肿瘤发生的原因或结果。这项工作将首次直接解决细胞质分裂失败--导致四倍体然后非整倍体--是否可以驱动肿瘤形成的问题。首先，我将测试在P53被全球击倒的背景下，靶向敲除MgcracGAP是否会在非洲爪哇蝌蚪中诱导肿瘤。其次，我将通过检查肿瘤细胞核、中心体、病理学和血管生成来确定肿瘤的特征。第三，我将通过对正在形成肿瘤的区域进行高分辨率的活显微镜观察，来测试正在形成肿瘤的活的非洲爪哇蝌蚪的胞质分裂是否失败。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引起的细胞质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.