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）内肌动蛋白细胞骨架的关键接头-连环蛋白的功能，对于了解和治疗胚胎发育期间的缺陷、调节组织工程应用中的细胞行为以及诊断和治疗转移性肿瘤具有广泛的意义。利用功能基因组学和蛋白质组学的方法，我们鉴定了srGAP，SRGP-1，作为C.秀丽线虫形态发生srGAP同源基因的突变导致人类智力迟钝，但对它们在上皮细胞-细胞粘附中的作用一无所知。我们将使用遗传学，创新的体内成像和生物化学来测试一种模型，其中连环蛋白和SRGP-1共同作用，以促进粘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）使用生物化学表征SRGP-1的磷脂结合和弯曲性质，（B）使用动态成像评估SRGP-1向体内富含关键磷脂的区域的募集;（c）测试SRGP的重要性-1的膜弯曲活性与体内一般膜募集的关系，使用预测介导凸面与凹面膜弯曲的关键残基中的点突变和异源膜目标图案 目的2：SRGP-1与肌动蛋白调节因子的相互作用在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是否调节表皮中新生和成熟连接处的Rac信号传导;（B）使用预测消除SRGP-1向WAVE复合物募集的表位测试和WVE-1/缺失构建体，以在表皮封闭和胚胎伸长期间将SRGP-1置于遗传等级中，和（c）我们将测试WVE-1/Wave，C.线虫Sra-1/Nap 1/Abi复合物，和Sra-10/lamellipodin与SRGP-1的同源区域相互作用。 目的3：SRGP-1与<$-catenin结合在AJ成熟和维持中的作用。我们将（a）使用高速体内成像评估SRGP-1和CCC募集的时间顺序，并测试它们的相互依赖性;（B）绘制每种蛋白质内的相互作用结构域，并评估HMP-1/SRGP-1相互作用对其体内AJs的动力学和功能以及对其体外已知结合配偶体的影响;以及（c）我们将验证SRGP-1 C末端的候选新结合配偶体，并对额外的SRGP-1物理相互作用物进行大规模筛选。通过这些研究，我们将获得新的见解， 粘附连接如何在活生物体的上皮形态发生过程中成熟，这是人类发育和抑制肿瘤发生过程中各种细胞事件的关键过程。我们还将在体内获得对一类蛋白质srGAP的基本新见解，这是正常人类发育所需的。

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.