Regulation of Adherens Junction Trafficking by Polarity Proteins

极性蛋白对粘附连接运输的调节

• 批准号: 8665969
• 负责人: Yang Hong
• 金额: $ 27.45万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8665969
• 关键词: Actins; Address; Adherens Junction; Affinity; Affinity Chromatography; Alleles; Anabolism; Apical; Biological; Biological Assay; Biological Models; Cell Adhesion Molecules; Cell Polarity; Cell-Cell Adhesion; Cells; Complex; Cytoskeleton; Defect; Development Polarity; Diffusion; Drosophila genus; E-Cadherin; Elements; Embryo; Engineering; Epithelial Cells; Epitopes; Freedom; Gene Targeting; Genetic; Genetic Models; Genomics; Homologous Gene; Integral Membrane Protein; Intercellular Junctions; Invertebrates; Knock-in Mouse; Larva; Lateral; Maps; Mass Spectrum Analysis; Mediating; Membrane; Molecular; Molecular Genetics; Normal Cell; Pathway interactions; Phosphorylation; Phosphoserine; Play; Protein Binding Domain; Proteins; Proteomics; Recycling; Regulation; Role; Route; Series; Staging; Testing; Tight Junctions; Vertebrates; Vesicle; atypical protein kinase C; base; cancer cell; cancer therapy; design; human disease; in vivo; insight; larval control; moesin; mutant; novel; novel marker; polarized cell; protein E; rho GTP-Binding Proteins; trafficking; tumor progression

DESCRIPTION (provided by applicant): Regulation of Adherens Junction Dynamics by Polarity Proteins. Key components of cell junctions such as E-Cadherin and claudins are transmembrane proteins that are delivered and regulated primarily through vesicle trafficking. In polarizing epithelial cells, regulating the dynamics of junctional complex through trafficking may be crucial for establishing the apical- basal polarity. Here we propose that control of adherens junction (AJ) dynamics by polarity proteins Stardust (Sdt), Crumbs (Crb) and Lethal giant larvae (Lgl) is an essential cellular mechanism for establishing apical-basal polarity. To test this hypothesis, we have developed a novel genomic engineering approach in Drosophila that enables us to modify a target gene into any desired mutant alleles. By this genomic engineering approach we have generated multiple genetically validated AJ markers such as E-Cadherin::GFP for quantitatively assaying the AJ dynamics in vivo in Drosophila epithelial cells. In this proposal, we will first characterize how AJ dynamics, such as the rates of biosynthesis, degradation, and local (subcellular) turnover, may be differentially regulated during apical- basal polarization, and how polarity proteins Sdt, Crb and Lgl play major regulatory roles in such AJ dynamics. By manipulating the AJ dynamics through both cis- and trans-regulatory mechanisms, we will identify whether regulated AJ dynamics during apical-basal polarity development is essential for establishing the polarity. We will then explore the potential molecular mechanisms by which Sdt/Crb complex controls the AJ dynamics and polarity. We will focus on hypotheses that Sdt/Crb controls the AJ dynamics by regulating the aPKC-mediated DE-Cad phosphorylation and/or by regulating the Moesin- regulated actin-network. Finally, we propose to identify novel mechanisms of DE-Cad, Crb, Lgl, and Sdt function in cell polarization by systematically identifying their interacting partners through proteomics approaches, using genetically validated GFP and high-affinity epitope knock-in alleles generated by genomic engineering. Accomplishing the specific aims in this proposal will establish the regulation of AJ dynamics as a key molecular and cellular mechanism by which Sdt, Crb and Lgl to control the apical-basal polarization in epithelial cells.

描述（由申请人提供）：极性蛋白对粘附连接动力学的调节。细胞连接的关键成分（例如 E-钙粘蛋白和密蛋白）是跨膜蛋白，主要通过囊泡运输进行递送和调节。在极化上皮细胞中，通过运输调节连接复合物的动力学对于建立顶端-基底极性可能至关重要。在这里，我们提出极性蛋白 Stardust (Sdt)、Crumbs (Crb) 和致死巨幼虫 (Lgl) 对粘附连接 (AJ) 动力学的控制是建立顶端-基底极性的重要细胞机制。为了检验这一假设，我们在果蝇中开发了一种新颖的基因组工程方法，使我们能够将目标基因修改为任何所需的突变等位基因。通过这种基因组工程方法，我们生成了多个经过基因验证的 AJ 标记，例如 E-Cadherin::GFP，用于定量分析果蝇上皮细胞体内的 AJ 动态。在本提案中，我们将首先描述 AJ 动力学，例如生物合成、降解和局部（亚细胞）周转率，在顶端-基底极化过程中如何受到差异性调节，以及极性蛋白 Sdt、Crb 和 Lgl 如何在这种 AJ 动力学中发挥主要调节作用。通过顺式和反式调节机制操纵 AJ 动力学，我们将确定顶端-基底极性发育过程中受调节的 AJ 动力学是否对于建立极性至关重要。然后我们将探索 Sdt/Crb 复合物控制 AJ 动力学和极性的潜在分子机制。我们将重点关注 Sdt/Crb 通过调节 aPKC 介导的 DE-Cad 磷酸化和/或通过调节 Moesin 调节的肌动蛋白网络来控制 AJ 动力学的假设。最后，我们建议通过蛋白质组学方法，使用基因验证的 GFP 和基因组工程产生的高亲和力表位敲入等位基因，系统地识别它们的相互作用伙伴，从而确定 DE-Cad、Crb、Lgl 和 Sdt 在细胞极化中功能的新机制。实现该提案中的具体目标将建立 AJ 动力学的调节作为 Sdt、Crb 和 Lgl 控制上皮细胞顶底极化的关键分子和细胞机制。