Regulation of localized RhoA activity in dividing epithelial cells

 描述(申请人提供)：令人惊讶的是，人们对细胞质分裂如何在完整的上皮环境中发挥作用知之甚少，在这种环境中，分裂的细胞通过细胞与细胞的连接与相邻细胞相连。值得注意的是，失败的胞质分裂可以促进肿瘤的形成；超过85%的 癌症起源于上皮组织，细胞-细胞连接缺陷可能导致癌症转移。因此，这项研究的目的是描述上皮细胞在细胞分裂过程中维持和重塑其细胞-细胞连接的分子机制。我们的中心假设是，支架蛋白Anlin调节活性RhoA的适当时空模式(RhoA-GTP)，对于调节分裂和非分裂上皮细胞的连接结构、重塑和张力是必不可少的。我们关注RhoA，一种促进肌动球蛋白收缩的小GTP酶，源于有证据表明，RhoA的精确定位区域对于胞质分裂以及细胞-细胞连接的形成和维持都是必需的。我们的团队处于独特的地位，能够实现所述目标 因为我们在非洲爪哇胚胎的完整脊椎动物上皮中使用了活性RhoA动态的高分辨率实时成像。我们将通过追求三个具体目标来检验我们的中心假设。在目标1中，我们将确定阿尼林在维持细胞间连接方面的功能。或令人兴奋的初步数据表明，已知在调节胞质分裂中发挥重要作用的支架蛋白Anlin，也在调节细胞-细胞连接完整性方面发挥了新的作用。我们将验证阿尼林通过控制连接的RhoA-GTP的分布和稳定顶端肌动球蛋白带来调节细胞-细胞连接的假设。在目标2中，我们将描述动态的细胞-细胞连接重塑在细胞分裂中是如何被调节的。我们将测试 这一假说认为，由于张力的变化，连接蛋白在分裂细胞中的动力学发生了变化，Anlin对定位的RhoA-GTP进行了适当的调节，这是连接重建所必需的。在目标3中，我们将确定分裂细胞调节和响应张力变化的机制。在这里，我们将检验这样的假设，即RhoA在机械力的作用下被激活，而阿尼林稳定RhoA-GTP并促进连接张力的增加。使用创新的方法，包括使用专门突出细胞中活性RhoA的荧光探针进行实时成像，首次表征细胞-细胞连接蛋白质动态在脊椎动物细胞分裂过程中是如何调节的，以及研究RhoA介导的细胞力学-所有这些都在完整的脊椎动物上皮组织中-将使我们能够对HW细胞在上皮细胞中的分裂工作有新的见解。这项拟议研究的完成有望确定调控局部RhoA活性和肌动球蛋白介导的张力的新机制，这些张力是在完整的脊椎动物上皮细胞胞质分裂期间维持细胞-细胞连接和重塑所需的。

 DESCRIPTION (provided by applicant): Surprisingly little is known about how cytokinesis works in an intact epithelial environment where the dividing cell is connected to its neighboring cells via cell-cell junctions. Notably, failed cytokinesis can promote tumor formation; over 85% of cancers arise from epithelial tissues, and cell-cell junction defects can contribute to cancer metastasis. Therefore, the objective of the proposed research is to characterize the molecular mechanisms by which epithelial cells maintain and remodel their cell-cell junctions during cell division. Our central hypothesis is that the scaffolding protein Anillin regulates proper spatiotemporal patterning of active RhoA (RhoA-GTP) and is essential for regulating junction structure, remodeling, and tension in dividing and non-dividing epithelial cells. Our focus on RhoA, a small GTPase that promotes actomyosin contractility, stems from evidence that precisely localized zones of active RhoA are required both for cytokinesis and for formation and maintenance of cell-cell junctions. Our group is uniquely positioned to tackle the stated objective because we use high-resolution live imaging of active RhoA dynamics in the intact vertebrate epithelium of the Xenopus laevis embryo. We will test our central hypothesis by pursuing three specific aims. In Aim 1, we will determine Anillin's function in maintaining cell-cell junctions. Or exciting preliminary data indicate that the scaffolding protein Anillin, which is known to play an important role in regulating cytokinesis, also plays a novel role in regulating cell-cell junction integrity. We will test the hypothesis that Anillin regulates cell-cell junctions by controlling th distribution of junctional RhoA-GTP and stabilizing the apical actomyosin belt. In Aim 2, we will characterize how dynamic cell-cell junction remodeling is regulated in dividing cells. We will test the hypothesis that junction protein dynamics are altered in dividing cells due to changes in tension, and proper regulation of localized RhoA-GTP by Anillin is required for junction remodeling. In Aim 3, we will identify mechanisms by which dividing cells regulate and respond to tension changes. Here, we will test the hypothesis that RhoA is activated in response to mechanical force, and Anillin stabilizes RhoA-GTP and promotes increased junctional tension. Using innovative approaches that include live imaging with a fluorescent probe that specifically highlights active RhoA in the cell, characterizing for the first time how cell-cell junction protei dynamics are regulated during vertebrate cell division, and examining RhoA-mediated cellular mechanics - all in intact vertebrate epithelial tissue - will allow us to gain new insights about hw cell division works in epithelial cells. Completion of the proposed research is expected to identif novel mechanisms that regulate localized RhoA activity and the actomyosin-mediated tension required for cell-cell junction maintenance and remodeling during cytokinesis in the intact vertebrate epithelium.