srGAP and the cadherin complex during morphogenesis in C. elegans

描述(由申请人提供)：了解是什么调节钙粘素-连环蛋白复合体(CCC)内肌动蛋白细胞骨架的关键连接物？-连环蛋白的功能，对于理解和治疗胚胎发育过程中的缺陷，调节组织工程应用中的细胞行为，以及诊断和治疗转移肿瘤具有广泛的意义。利用功能基因组学和蛋白质组学方法，我们确定srGAP，SRGP-1，是线虫形态发生过程中CCC的重要调控因子。SrGAP同源基因的突变会导致人类的智力低下，但关于它们在上皮细胞-细胞黏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，在该模型中，？-catenin和SRGP-1共同作用，促进黏附连接的成熟和维持，具体目标如下： 目的1：SRGP-1质膜相互作用在AJ成熟和维持中的作用。 我们将(A)用生化方法表征SRGP-1的磷脂结合和弯曲特性；(B)使用动态成像技术评估SRGP-1在体内富含关键磷脂的区域的募集情况；以及(C)通过预测介导膜凹凸弯曲和异源膜靶向基序的关键残基的点突变，测试SRGP-1‘S膜弯曲活性与体内普通膜募集的重要性。 目的2：SRGP-1与肌动蛋白调节蛋白的相互作用在AJ成熟和维持中的作用。我们将(A)使用一种新的组织特异性诱导表达系统来测试SRGP-1是否在表皮的新生和成熟连接处调节RAC信号；(B)使用外移测试和WVE-1/缺失构建体来消除SRGP-1在表皮包围和胚胎伸长过程中招募到WAVE复合体中的位置，将SRGP-1置于遗传层次中；以及(C)我们将测试WVE-1/Wave、线虫SRA-1/NAP1/Abi复合体的成分以及MIG-10/lamelLipodin是否与SRGP-1的同源区域相互作用。 目的3：SRGP-1与β-连环蛋白结合在AJ成熟和维持中的作用。我们将(A)利用高速体内成像评估SRGP-1和CCC的招募时间顺序，并测试它们的相互依赖性；(B)绘制每个蛋白质内的相互作用结构域，并评估HMP-1/SRGP-1相互作用对它们在体内AJ及其已知结合伙伴的动力学和功能的影响；(C)我们将验证SRGP-1 C末端的候选新结合伙伴，并对其他SRGP-1物理相互作用因子进行大规模筛选。作为这些研究的结果，我们将对 黏附连接如何在活体的上皮形态发生过程中成熟，这一过程对人类发育和抑制肿瘤发生的各种细胞事件至关重要。我们还将在体内获得对一类蛋白质的基本新见解，srGAP是人类正常发育所必需的。

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.