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: 8534180
• 负责人: Ann Louise Miller
• 金额: $ 23.35万
• 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 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8534180
• 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.

细胞分裂是细胞分裂的最后阶段，其中一个细胞分成两个子细胞。这个过程 必须仔细调节以确保卵裂沟位置正确，以便遗传 物质和细胞器平均分配给每个子细胞。获得更好的理解 胞质分裂代表了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理解仍然难以捉摸。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。我的长期目标是成为一名独立研究者，在细胞领域处于领先地位 生物学和肿瘤生物学。为了实现这个目标，我建议在K99辅导培训阶段，我将 专注于出版和展示我的博士后研究，并将我的工作发展成为独立的 研究计划。我还将获得癌症生物学方面的重要培训并寻求专业发展 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是 通过以下方式完成：1）与我的合作者（癌症生物学专家：Caroline 博士）互动 Alexander、Wade Bushman 博士和 Beth Weaver 博士，2) 积极参与癌症生物学文献研究 组，3) 参加肿瘤学 703 课程：癌发生和肿瘤细胞生物学，4) 参加小型课程 肿瘤生物学主题的会议，以及 5) 成为威斯康星大学卡本分校的准会员 综合癌症中心积极参与大查房等培训活动 研讨会系列和年度静修会。我自始至终都在寻找职业发展机会 我的研究生工作和博士后培训。具体来说，在K99辅导培训阶段，我会 参加编写 R01 的研讨会，参加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，以发展强大的 与我所在领域的其他研究人员建立联系，并在我准备开始工作时让我的工作更加可见 市场。 K99/R00 补助金为我提供的额外培训时间也将使我能够进一步发展我的能力 独立研究计划。在动物细胞中，胞质分裂由肌动蛋白丝的收缩环提供动力 和肌球蛋白-2。收缩环的形成依赖于小 GTP 酶 Rho，它在 细胞赤道处的精确区域。迄今为止，我的工作表明 GTP 酶激活蛋白 (GAP) Rho 调节剂 MgcRacGAP 的活性在整个胞质分裂过程中是必需的，以形成和 通过 GTPase Flux 维持集中的 Rho 活性区；也就是说，Rho 在活性、 GTP 约束状态和不活跃、GDP 约束状态。通过 GTPase Flux，细胞可以维持集中的 Rho 活动区，这是形成集中收缩环和成功胞质分裂所必需的。工作 我在此建议以这些发现以及我在 Bement 中开发的技能和工具为基础 实验室，同时还通过相互作用开发癌症生物学和多光子显微镜方面的新专业知识 与威斯康星大学麦迪逊分校的一群优秀合作者一起。目标 1 中描述的实验，我将 在本次资助的 K99 指导阶段进行，通过剖析直接建立在 GTPase Flux 发现的基础上 Aurora B 和 Anillin 在胞质分裂过程中调节 Rho 活性区和 GTP 酶通量的作用 爪蟾胚胎。首先，我将测试 GTPase 是否需要 Aurora B 磷酸化 MgcRacGAP 通过使用磷酸化或不可磷酸化的 MgcRacGAP 突变体或用 Aurora B 处理细胞来通量 抑制剂。其次，我将测试 Rho 活动区的操纵是否会影响 Anillin 定位 当 Rho 活性区通过表达进行操纵时，进行 Anillin 定位的实时显微镜检查 MgcRacGAP GAP-DEAD 突变体或组成型活性 Rho。三、我来测试一下Anillin是否有推广 通过分析 Anillin 敲低胚胎中的 Rho 活性区，发现 Rho 活性区的正反馈 内源性 Anillin 被 Anillin 突变体取代的胚胎。目标 2 中描述的实验 我将在本次资助的指导 K99 阶段启动，并继续在独立 R00 阶段，检查 有争议的问题是非整倍体，即多于或少于正常值的情况 染色体数目是肿瘤发生的原因或结果。这项工作将直接针对 第一次提出细胞分裂失败是否会导致四倍体然后非整倍体的问题 肿瘤发生。首先，我将测试 MgcRacGAP 的靶向敲低是否会在非洲爪蟾中诱导肿瘤 p53 被全面敲除的背景中的蝌蚪。其次，我将通过以下方式来描述肿瘤的特征： 检查肿瘤细胞核、中心体、病理学和血管生成。第三，我会测试胞质分裂是否失败 在活的非洲爪蟾蝌蚪中，通过对肿瘤所在区域进行活体高分辨率显微镜观察，这些蝌蚪正在形成肿瘤 正在形成。最后，我将测试其他 Rho 区调节因子是否会导致胞质分裂失败，尤其是 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。

项目成果

Actin organization and dynamics: novel regulatory mechanisms from the biophysical to the tissue level.

肌动蛋白组织和动力学：从生物物理到组织水平的新型调节机制。

• DOI: 10.1091/mbc.e12-12-0879
• 发表时间: 2013
• 期刊: Molecular biology of the cell
• 影响因子: 3.3
• 作者: Miller,AnnL;DeLaCruz,EnriqueM
• 通讯作者: DeLaCruz,EnriqueM

The contractile ring.

• DOI: 10.1016/j.cub.2011.10.044
• 发表时间: 2011-12-20
• 期刊: CURRENT BIOLOGY
• 影响因子: 9.2
• 作者: Miller, Ann L.

MgcRacGAP restricts active RhoA at the cytokinetic furrow and both RhoA and Rac1 at cell-cell junctions in epithelial cells.

• DOI: 10.1091/mbc.e14-11-1553
• 发表时间: 2015-07-01
• 期刊: Molecular biology of the cell
• 影响因子: 3.3
• 作者: Breznau EB;Semack AC;Higashi T;Miller AL
• 通讯作者: Miller AL

Anillin regulates cell-cell junction integrity by organizing junctional accumulation of Rho-GTP and actomyosin.

• DOI: 10.1016/j.cub.2014.04.021
• 发表时间: 2014-06-02
• 期刊: CURRENT BIOLOGY
• 影响因子: 9.2
• 作者: Reyes, Ciara C.;Jin, Meiyan;Breznau, Elaina B.;Espino, Rhogelyn;Delgado-Gonzalo, Ricard;Goryachev, Andrew B.;Miller, Ann L.
• 通讯作者: Miller, Ann L.