The Structural Basis of Shroom-Mediated Cell Contractility

蘑菇介导的细胞收缩性的结构基础

• 批准号: 8499374
• 负责人: ANDREW Paul VANDEMARK
• 金额: $ 26.66万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8499374
• 关键词: Actins; Actomyosin; Address; Adopted; Animals; Apical; Architecture; Area; Binding; Biochemical; Biological; Biological Assay; C-terminal; Cell Differentiation process; Cell Shape; Cell Survival; Cell physiology; Cells; Cellular Morphology; Cellular biology; Complex; Core Protein; Coupled; Data; Defect; Development; Dimerization; Disease; Embryonic Development; Ensure; Epithelial Cells; Event; Eye; Homeostasis; Homologous Gene; Human; Intestines; Learning; Malignant - descriptor; Malignant Neoplasms; Mediating; Modeling; Molecular; Molecular Conformation; Molecular Models; Morphogenesis; Morphology; Mus; Mutation; Myosin Phosphatase Pathway; Myosin Type II; Nature; Neuraxis; Neuro-Ocular System; Organ; Organogenesis; Outcome; Pathway interactions; Phosphotransferases; Play; Process; Property; Proteins; Publications; Reagent; Regulation; Research; Rho-associated kinase; Role; Shapes; Signal Transduction; Stem cells; Structure; Testing; Therapeutic; Tissue Engineering; Tissues; Vertebrates; Work; X-Ray Crystallography; base; cell behavior; cell growth regulation; cell type; constriction; design; human disease; in vivo; injury and repair; insight; molecular modeling; mutant; rho

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.

描述（由申请人提供）：在脊椎动物发育，器官发生和组织稳态过程中，细胞结构和形态的调节至关重要。与肌动蛋白相关的扫描室蛋白是上皮细胞形状的关键决定因素，可控制顶端收缩和细胞收缩的过程。塑料蛋白通过将岩石丝素II途径靶向到引起收缩肌动蛋白网络的形成的细胞的特定区域来起作用。汉室参与岩石丝素途径的能力取决于直接与岩石结合的保守域。基于我们的工作，我们预测该途径代表了脊椎动物中许多发育事件所需的进化保守的信号模块，包括形成中枢神经系统，眼睛和肠道。该建议将研究该途径在结构，生化和细胞水平上的组装和功能。我们将通过以下目标实现这一目标。 AIM I. SHRM SD2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SD2结构域的结构，作为生成有关SD2如何与岩石相互作用以控制收缩力的假设的起点。我们将使用生化和细胞生物学方法，并结合从我们对结构分析的突变体结合来检验这些假设。目标II。岩石的剪室结合域（SBD）的特征。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。我们还将检查体内这些突变的效果。再加上SD2的结构，我们将能够生成简单的可测试模型来描述SD2-SBD相互作用以及它如何调节岩石活动。目标三。 SD2-SBD复合物的结构及其对信号的影响。 SHRM与岩石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SD2-SBD复合物的结构来解决相互作用的性质。我们将扩大分析，以检查每个领域在调节细胞形态中的作用，并分析SHRM-ROCK途径与Rho-Rock途径之间的相互作用。这些研究很重要，因为只有通过阐明信号复合物的组装方式，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途径对于大量细胞过程至关重要，因此了解该途径的功能如何提供对正常和疾病条件下细胞调节的基本机制的宝贵见解。此外，针对特定岩石依赖性事件的有针对性的破坏可能被证明是治疗某些人类疾病和疾病的强大治疗方法。