Regulation of localized RhoA activity in dividing epithelial cells

 描述（由申请人提供）：令人惊讶的是，人们对胞质分裂在完整的上皮环境中如何工作知之甚少，其中分裂细胞通过细胞-细胞连接与其邻近细胞相连。值得注意的是，失败的胞质分裂可以促进肿瘤形成;超过85%的肿瘤患者， 癌症起源于上皮组织，细胞-细胞连接缺陷可导致癌症转移。因此，拟议的研究的目的是表征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，支架蛋白苯胺调节适当的时空模式的活性RhoA（RhoA-GTP），是必不可少的调节交界处的结构，重塑，并在分裂和非分裂上皮细胞的张力。我们专注于RhoA，一个小的GTdR，促进肌动球蛋白收缩性，源于证据表明，精确定位区域的活性RhoA细胞质分裂和细胞连接的形成和维持。我们的集团在实现既定目标方面处于独特的地位 因为我们在非洲爪蟾胚胎的完整脊椎动物上皮中使用了活性RhoA动力学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，我们将确定苯胺在维持细胞-细胞连接中的功能。或者令人兴奋的初步数据表明，支架蛋白Anilin，已知在调节胞质分裂中起重要作用，也在调节细胞-细胞连接完整性中起着新的作用。我们将检验这一假设，即苯胺通过控制连接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。在目标2中，我们将描述动态细胞-细胞连接重塑在分裂细胞中是如何调节的。我们将测试 假设连接蛋白动力学在分裂细胞中由于张力的变化而改变，并且连接重塑需要苯胺对局部RhoA-GTP的适当调节。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，我们将测试的假设，RhoA被激活响应机械力，和苯胺稳定RhoA-GTP和促进连接张力增加。使用创新的方法，包括用荧光探针实时成像，特别突出细胞中的活性RhoA，首次表征脊椎动物细胞分裂期间细胞-细胞连接蛋白动力学如何调节，并检查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-将使我们能够获得关于上皮细胞中细胞分裂工作的新见解。完成拟议的研究，预计将确定新的机制，调节局部RhoA活性和肌动球蛋白介导的张力所需的细胞间连接的维持和重塑在完整的脊椎动物上皮细胞胞质分裂。

 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.