Regulation of Planar Cell Polarity in Migrating Cells

描述（由申请人提供）：平面细胞极性（PCP）描述了组织平面内细胞的极化，是胚胎形态发生的基本特征。脊椎动物PCP的破坏与多种疾病相关，包括神经管闭合缺陷、纤毛病、耳聋和潜在的癌症。PCP研究的所有领域都面临两个主要问题。首先，将核心PCP蛋白的信号转化为细胞极性变化的下游效应物是什么？第二，在平面组织中，细胞之间如何传递信息以建立和维持极性？苍蝇体内五氯苯酚的广泛特征 翅上皮显示了货车高和卷曲（Fz）/凌乱（Dsh）信号传导功能之间的那些拮抗性相互作用，以阻止富含肌动蛋白的毛发的形成。相比之下，尽管十年的推测，这些核心PCP蛋白的不对称表达极化膜突起的形成在原肠胚细胞运动，实际的分子机制仍然不清楚。该建议的目标是提供一个新的实验范式，解释PCP信号如何协调极化原肠胚细胞群体的定向迁移。我们的中心假设是Vang-like 2（Vangl 2）和Fz/Dsh信号传导分别不同地影响细胞-基质相互作用和细胞-细胞粘附，以控制极化的膜粘附活性。具体目的：首先，我们将建立Vangl 2调节Mmp 14活性、ECM蛋白水解和PCP的机制。我们将使用体内分子和遗传操作，细胞移植方法和共聚焦/延时成像的组合来鉴定Vangl 2表达的结构域并分析膜突起动力学。我们将使用体外生物化学测定来鉴定连接Vangl 2和整合素α 5 β 1功能的信号传导机制。在第二个目标中，我们将确定钙粘蛋白介导的粘附Fz/Dsh依赖性调节ECM组装和PCP的贡献。我们将使用分子和遗传学的方法，移植和显微成像，以确定FZ，DSH和钙粘蛋白的表达域，操纵细胞粘附，并分析膜突起的动力学。我们将使用生物化学方法来确定机制，Fz/Dsh信号调节细胞表面N-钙粘蛋白的表达在原肠胚形成。在第三个目标中，我们将剖析Vangl 2和Fz/Dsh信号转导协调PCP穿过迁移原肠胚细胞的能力。在这里，我们将使用马赛克分析和共聚焦成像的强大组合来量化膜突起方向，细胞极性和PCP蛋白定位的非自主效应。使用这些方法，我们将识别短距离和长距离场效应，并首次确定 在原肠胚细胞的集体运动过程中，ECM结构和细胞凝聚力的变化如何影响PCP。这项资助的完成将大大促进我们对脊椎动物的了解， 五氯酚 由于PCP发生在ECM微环境和细胞-细胞粘附的背景下，我们的数据应该对其他发育和疾病过程具有广泛的影响。

DESCRIPTION (provided by applicant): Planar cell polarity (PCP) describes the polarization of cells within the plane of a tissue and is an essential feature of embryonic morphogenesis. Disruption of vertebrate PCP is associated with multiple disorders including neural tube closure defects, ciliopathies, deafness, and potentially cancer. Two major questions face all fields of PCP research. First, what are the downstream effectors that transform signals from core PCP proteins into changes in cell polarity? Second, how is information communicated between cells to establish and maintain polarity in a planar tissue? Extensive characterization of PCP in the fly wing epithelium demonstrates those antagonistic interactions between Van Gogh and Frizzled (Fz)/Disheveled (Dsh) signaling function to polarize the formation of actin-rich hairs. By contrast despite a decade of speculation that asymmetric expression of these core PCP proteins polarizes the formation of membrane protrusions during gastrulation cell movements, the actual molecular mechanisms remain unclear. The goal of this proposal is to provide a new experimental paradigm explaining how PCP signaling coordinates the directed migration of polarized gastrula cell populations. Our central hypothesis is that Vang-like 2 (Vangl2) and Fz/Dsh signaling differentially impact cell-matrix interactions and cell-cell adhesion, respectivel, to control polarized membrane protrusive activity. Specific Aims: First, we will establish the mechanism whereby Vangl2 regulates Mmp14 activity, ECM proteolysis, and PCP. We will use a combination of in vivo molecular and genetic manipulations, cell transplantation methods, and confocal/time-lapse imaging to identify domains of Vangl2 expression and analyze membrane protrusion dynamics. We will use in vitro biochemical assays to identify signaling mechanisms connecting Vangl2 and integrin alpha5beta1 function. In the second aim, we will determine the contribution of cadherin-mediated adhesion to Fz/Dsh-dependent regulation of ECM assembly and PCP. We will use molecular and genetic methods, transplantations, and microscopic imaging to identify Fz, Dsh, and cadherin expression domains, manipulate cell adhesion, and analyze membrane protrusion dynamics. We will use biochemical methods to identify mechanisms whereby Fz/Dsh signaling regulates cell surface N-cadherin expression during gastrulation. In the third aim, we will dissect the abilities of Vangl2 and Fz/Dsh signaling to coordinate PCP across a field of migrating gastrula cells. Here, we will use a powerful combination of mosaic analyses and confocal imaging to quantify non-autonomous effects on membrane protrusion orientation, cell polarity, and PCP protein localization. Using these methods, we will identify both short- and long-range field effects and determine for the first time how changes in ECM structure and cell cohesion impact PCP during the collective movement of gastrula cells. Completion of this grant will significantly advance our understanding of vertebrate PCP. Since PCP occurs in the context of an ECM microenvironment and cell-cell adhesion, our data should have broad implications for other developmental and disease processes.