The Structural Basis of Shroom-Mediated Cell Contractility

描述（由申请人提供）：细胞结构和形态的调节在脊椎动物发育、器官发生和组织稳态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，控制顶端收缩和细胞收缩的过程。蘑菇蛋白通过将Rock-肌球蛋白II途径靶向细胞的特定区域而起作用，在那里它引起收缩性肌动球蛋白网络的形成。蘑菇参与Rock-肌球蛋白途径的能力取决于直接与Rock结合的保守结构域。基于我们的工作，我们预测这条通路代表了一个进化上保守的信号传导模块，它是脊椎动物中许多发育事件所必需的，包括中枢神经系统、眼睛和肠道的形成。本提案将在结构、生物化学和细胞水平上研究该通路的组装和功能。我们将通过以下目标实现这一目标。艾姆岛Shrm SD 2结构域的结构分析和表征。我们将使用X射线晶体学来确定SD 2结构域的结构，作为产生关于SD 2如何与Rock相互作用以控制收缩性的假设的起点。我们将使用生物化学和细胞生物学的方法，结合突变体来自我们的结构分析，以测试这些假设。Aim II.岩石的蘑菇结合结构域（SBD）的表征。我们将确定SBD的结构，以深入了解其构象和设计信息突变体，以确定SBD的哪些部分与Shrm物理相互作用。我们还将研究这些突变在体内的影响。结合SD 2的结构，我们将能够生成简单的可测试模型，描述SD 2-SBD相互作用及其如何调节Rock活性。Aim III. SD 2-SBD复合物的结构及其对信号传导的影响。Shrm和Rock之间的相互作用足以调节下游细胞骨架变化并改变细胞形态。我们将通过解决SD 2- SBD复合物的结构来解决相互作用的性质。我们将扩大我们的分析，以检查每个域在调节细胞形态中发挥的作用，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，因为只有阐明信号复合物是如何组装的，我们才能理解它们的功能和调节方式。由于岩石-肌球蛋白II途径是中央的大量的细胞过程，了解这一途径的功能可能会提供宝贵的洞察细胞调节的基本机制，在正常和疾病的条件下。此外，靶向破坏特定的Rock依赖性事件可能被证明是治疗某些人类疾病和病症的强大治疗方法。 公共卫生相关性：细胞必须能够调节其形状，以形成正确的身体计划和功能组织和器官。这项研究调查了一种蛋白质网络，该网络控制细胞如何处理这一复杂的任务。通过从原子到细胞水平了解这些过程，我们希望了解这些途径中的缺陷如何导致人类疾病。

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. PUBLIC HEALTH RELEVANCE: Cells must be able to regulate their shape in order to form the correct body plan and functional tissues and organs. This research investigates a network of proteins that controls how cells manage this complicated task. By understanding these processes from the atomic to the cellular level we hope to learn how defects in these pathways may cause human disease.