Regulation of Planar Cell Polarity in Migrating Cells

迁移细胞中平面细胞极性的调节

• 批准号: 8738685
• 负责人: Jason Robert Jessen
• 金额: $ 24.7万
• 依托单位: MIDDLE TENNESSEE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-20 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8738685
• 关键词: Actins; Address; Adhesions; Affect; Biochemical; Biological Assay; Cadherins; Cell Adhesion; Cell Polarity; Cell Transplantation; Cell surface; Cell-Cell Adhesion; Cell-Matrix Junction; Cells; Cellular Structures; Communication; Complex; Data; Defect; Development; Disease; Embryo; Endocytosis; Epithelium; Extracellular Matrix; Face; Focal Adhesion Kinase 1; Gastrula; Goals; Grant; Hair; Image; In Vitro; Integrin alpha5beta1; Integrins; Malignant Neoplasms; Mediating; Membrane; Methods; Microscopic; Molecular; Molecular Genetics; Morphogenesis; Movement; N-Cadherin; Neural Tube Closure; Neural Tube Defects; Population; Process; Proteins; Proteolysis; Regulation; Research; Signal Transduction; Site; Time; Tissues; Transplantation; Wing; Zebrafish; cancer cell; cell motility; ciliopathy; cohesion; deafness; fibrillogenesis; fly; gastrulation; genetic manipulation; human FZD2 protein; human MMP14 protein; in vivo; migration; novel; polarized cell; prevent; public health relevance; receptor

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.

描述(申请人提供)：平面细胞极化(PCP)描述细胞在组织平面内的极化，是胚胎形态发生的基本特征。脊椎动物PCP的中断与多种疾病有关，包括神经管闭合缺陷、纤毛疾病、耳聋和潜在的癌症。PCP研究的所有领域都面临着两个主要问题。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器是什么？第二，在平面组织中，如何在细胞之间传递信息以建立和维持极性？五氯酚在苍蝇体内的广泛特性 翼上皮细胞显示了梵高和FrizzledFz/Disheveled(Dsh)之间的拮抗相互作用，以极化富含肌动蛋白的毛发的形成。相比之下，尽管十年来一直推测这些核心PCP蛋白的不对称表达会极化原肠形成细胞运动过程中膜突起的形成，但实际的分子机制仍不清楚。这项建议的目的是提供一个新的实验范式，解释PCP信号如何协调极化的原肠胚细胞群体的定向迁移。我们的中心假设是Vang-like 2(Vangl2)和Fz/Dsh信号分别不同地影响细胞-基质相互作用和细胞-细胞黏附，从而控制极化的膜突起活动。具体目标：首先，我们将建立Vangl2调节Mmp14活性、ECM蛋白分解和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/时间推移成像相结合的方法来确定Vangl2的表达区域，并分析膜突出的动力学。我们将使用体外生化分析来确定连接Vangl2和整合素α5beta1功能的信号机制。在第二个目标中，我们将确定钙粘附素介导的黏附在Fz/Dsh依赖的ECM组装和PCP调节中的作用。我们将使用分子和遗传学方法、移植和显微成像来鉴定FZ、Dsh和钙粘附素的表达结构域，操纵细胞黏附，并分析膜突出的动力学。我们将使用生化方法来确定FZ/Dsh信号在原肠形成过程中调节细胞表面N-钙粘附素表达的机制。在第三个目标中，我们将剖析Vangl2和Fz/Dsh信号在一片迁移的原肠胚细胞中协调PCP的能力。在这里，我们将使用镶嵌分析和共聚焦成像的强大组合来量化对膜突起方向、细胞极性和PCP蛋白定位的非自主影响。使用这些方法，我们将识别短程和长程场效应，并首次确定 在原肠胚细胞的集体运动过程中，细胞外基质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 五氯苯酚。由于PCP发生在细胞外基质微环境和细胞-细胞黏附的背景下，我们的数据应该对其他发育和疾病过程具有广泛的影响。