srGAP and the cadherin complex during morphogenesis in C. elegans

描述（由申请人提供）：了解是什么调节了钙粘蛋白-连环蛋白复合物（CCC）中肌动蛋白细胞骨架的关键连接物-catenin的功能，对于理解和治疗胚胎发育过程中的缺陷，在组织工程应用中调节细胞行为，以及诊断和治疗转移性肿瘤具有广泛的意义。通过功能基因组学和蛋白质组学研究，我们发现srGAP SRGP-1在秀丽隐杆线虫形态发生过程中是一个重要的CCC调控因子。srGAP同源基因的突变导致人类智力迟钝，但对其在上皮细胞-细胞粘附中的作用尚不清楚。我们将使用遗传学、创新的体内成像和生物化学来测试一个模型，在这个模型中，-catenin和SRGP-1共同作用，促进粘附连接的成熟和维持，具体目的如下：

DESCRIPTION (provided by applicant): Understanding what modulates the function of �-catenin, the key linker to the actin cytoskeleton within the cadherin-catenin complex (CCC) has widespread implications for understanding and treating defects during embryonic development, for modulating cell behavior in tissue engineering applications, and for diagnosing and treating metastatic tumors. Using functional genomics and proteomics, we identified the srGAP, SRGP-1, as an important regulator of the CCC during C. elegans morphogenesis. Mutations in srGAP orthologs lead to mental retardation in humans, but nothing is known about their roles in epithelial cell-cell adhesion. We will use genetics, innovative in vivo imaging, and biochemistry to test a model in which �-catenin and SRGP-1 act together to promote maturation and maintenance of adherens junctions in the following specific aims: Aim 1: Role of the SRGP-1 plasma membrane interaction in AJ maturation and maintenance. We will (a) characterize the phospholipid binding and bending properties of SRGP-1 using biochemistry, (b) assess recruitment of SRGP-1 to areas enriched in key phospholipids in vivo using dynamic imaging; and (c) test the importance of SRGP-1's membrane bending activity vs. general membrane recruitment in vivo using point mutations in key residues predicted to mediate convex vs. concave membrane bending and heterologous membrane targeting motifs. Aim 2: Role of the SRGP-1 interaction with actin regulators in AJ maturation and maintenance. We will (a) use a novel tissue-specific inducible expression system to test whether SRGP-1 regulates Rac signaling at nascent and maturing junctions in the epidermis; (b) use epitasis tests and WVE-1/deletion constructs predicted to abrogate SRGP-1 recruitment to the WAVE complex to place SRGP-1 within a genetic hierarchy during epidermal enclosure and embryonic elongation, and (c) we will test whether WVE-1/Wave, components of the C. elegans Sra-1/Nap1/Abi complex, and MIG-10/lamellipodin interact with the homologous region of SRGP-1. Aim 3: Role of SRGP-1 binding to �-catenin in AJ maturation and maintenance. We will (a) assess the temporal order of recruitment of SRGP-1 and the CCC using high-speed in vivo imaging, and test for their mutual dependence; (b) map the interacting domains within each protein, and assess the effects of the HMP-1/SRGP-1 interaction on their dynamics and function at AJs in vivo and on their known binding partners in vitro; and (c) we will validate candidate novel binding partners of the SRGP-1 C terminus, and perform large-scale screens for additional SRGP-1 physical interactors. As a result of these studies, we will gain new insight into how adherens junctions mature during epithelial morphogenesis in a living organism, a process crucial for diverse cellular events during human development and suppression of oncogenesis. We will also gain fundamental new insights in vivo into a class of proteins, the srGAPs, required for normal human development.