The Structural Basis of Shroom-Mediated Cell Contractility

描述(申请人提供)：细胞结构和形态的调节在脊椎动物的发育、器官发生和组织动态平衡过程中是必不可少的。肌动蛋白相关的蘑菇蛋白是上皮细胞形状的关键决定因素，控制着顶端收缩和细胞收缩的过程。蘑菇蛋白通过将Rock-myosin II途径定位于细胞的特定区域来发挥作用，在那里它会导致收缩肌球蛋白网络的形成。蘑菇参与Rock-肌球蛋白途径的能力依赖于一个直接与Rock结合的保守结构域。根据我们的工作，我们预测这一途径代表了一个进化上保守的信号模块，在脊椎动物的许多发育事件中都需要它，包括中枢神经系统、眼睛和肠道的形成。该提案将在结构、生化和细胞水平上研究该途径的组装和功能。我们将通过以下目标实现这一目标。目的I.SHRM SD2结构域的结构分析和鉴定。我们将使用X射线结晶学来确定SD2结构域的结构，作为产生关于SD2如何与Rock相互作用控制收缩的假设的起点。我们将使用生化和细胞生物学的方法，结合我们对结构的分析得出的突变来检验这些假设。目的II.岩石蘑菇结合结构域(SBD)的特征。我们将确定SBD的结构以深入了解其构象，并设计信息丰富的突变体来确定SBD的哪些部分与SHRM物理相互作用。我们还将检查这些突变在体内的影响。结合SD2的结构，我们将能够生成简单的可测试模型，描述SD2与SBD的相互作用以及它如何调节岩石活动。目的III.SD2-SBD复合体的结构及其对信号的影响。SHRM和Rock之间的相互作用足以调节下游细胞骨架的变化和改变细胞的形态。我们将通过求解SD2-SBD复合体的结构来解决相互作用的性质。我们将扩展我们的分析，以检查每个结构域在调节细胞形态中所起的作用，并分析SHRM-Rock途径和Rho-Rock途径之间的相互作用。这些研究意义重大，因为只有通过阐明信号复合体是如何组装的，我们才能了解它们的功能和调节方式。由于Rock-myosin II途径是大量细胞过程的中心，了解该途径如何发挥作用可能为了解正常和疾病条件下细胞调控的基本机制提供宝贵的见解。此外，靶向干扰特定的岩石依赖事件可能被证明是治疗某些人类疾病和障碍的有效治疗方法。

DESCRIPTION (provided by applicant): The regulation of cell architecture and morphology is essential during the course of vertebrate development, organogenesis, and tissue homeostasis. The actin-associated Shroom proteins are critical determinants of epithelial cell shape, controlling the process of apical constriction and cellular contractility. Shroom proteins work by targeting the Rock-Myosin II pathway to specific regions of the cells where it causes the formation of a contractile actomyosin network. The ability of Shroom to engage the Rock-myosin pathway is dependent on a conserved domain that binds directly to Rock. Based on our work, we predict that this pathway represents an evolutionarily conserved signaling module that is required for a number of developmental events in vertebrates, including formation of the central nervous system, eye, and intestines. This proposal will investigate the assembly and function of this pathway at the structural, biochemical, and cellular levels. We will accomplish this through the following aims. Aim I. Structural Analysis and Characterization of a Shrm SD2 domain. We will use X-ray crystallography to determine the structure for an SD2 domain as a starting point for generating hypotheses about how SD2 interacts with Rock to control contractility. We will use biochemical and cell biological approaches, combined with mutants derived from our analysis of the structure, to test these hypotheses. Aim II. Characterization of the Shroom-Binding Domain (SBD) of Rock. We will determine the structure of SBD to give insight into its conformation and design informative mutants to determine which portions of SBD are physically interacting with Shrm. We will also examine the effects of these mutations in vivo. Coupled with the structure of SD2, we will be able to generate simple testable models that describe the SD2-SBD interaction and how it regulates Rock activity. Aim III. Structure of the SD2-SBD complex and implications for signaling. The interaction between Shrm and Rock is sufficient to regulate downstream cytoskeletal changes and alter cell morphology. We will address the nature of the interaction by solving the structure of the SD2- SBD complex. We will expand our analysis to examine the role each domain plays in regulating cell morphology and analyze the interplay between the Shrm-Rock pathway and the Rho-Rock pathway. These studies are significant because only by elucidating how signaling complexes are assembled can we understand their manner of function and regulation. Because the Rock- myosin II pathway is central to a vast number of cellular processes, understanding how this pathway functions may provide invaluable insight into the basic mechanisms of cellular regulation under both normal and disease conditions. In addition, targeted disruption of specific Rock-dependent events may prove to be a powerful therapeutic approach to treating certain human diseases and disorders.