Molecular and cellular mechanisms of the actin cytoskeleton organization and function

肌动蛋白细胞骨架组织和功能的分子和细胞机制

• 批准号: 10419950
• 负责人: Dmitri Kudryashov
• 金额: $ 36.86万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10419950
• 关键词: 3-Dimensional; Actin-Binding Protein; Actins; Adhesions; Age; Allosteric Regulation; Architecture; Attenuated; Autoimmune Diseases; Autoimmune Process; Binding; Biochemical; Biological; Blood; Bundling; Cancer Intervention; Cell Adhesion; Cell division; Cell physiology; Cells; Chronic Lymphocytic Leukemia; Communication; Communities; Complex; Connective Tissue Diseases; Coupling; Crosslinker; Cryo-electron tomography; Cryoelectron Microscopy; Cytokinesis; Cytoskeleton; Data; Deletion Mutation; Diaphragmatic Hernia; Disease; Disseminated Malignant Neoplasm; Endocytosis; Epithelial; Event; F-Actin; Family; Fiber; Filopodia; Fimbrin; Fluorescence; Fluorescence Microscopy; Focal Adhesions; Fostering; Goals; Health; Human; Human Activities; Immune system; Individual; Intestines; Investigation; Kidney; LCP1 gene; Labyrinth; Lead; Libraries; Link; Malignant Neoplasms; Maps; Mediating; Membrane; Metastatic to; Microfilaments; Modeling; Molecular; Molecular Conformation; Mutation; Nutrient; Osteogenesis; Osteoporosis; Pathology; Perception; Personal Satisfaction; Post-Translational Protein Processing; Process; Property; Protein Isoforms; Proteins; Publishing; Recycling; Regulation; Renal dialysis; Research; Resolution; Role; Shapes; Signal Transduction; Site; Structural Models; Structure; Syndrome; Systems Development; Testing; Therapeutic; Therapeutic Intervention; Tissues; Translating; Tropomyosin; Variant; Work; X-Ray Crystallography; bone fragility; calponin; cancer cell; cancer invasiveness; cell motility; cellular microvillus; congenital deafness; congenital hearing loss; coronin protein; crosslink; deafness; flexibility; functional outcomes; hearing impairment; immune activation; immunological synapse formation; improved; interest; member; migration; molecular dynamics; mutant; plastin; protein crosslink; reconstruction; recruit; response; single molecule; stem

PROJECT SUMMARY/ABSTRACT The remarkable functional versatility of the actin cytoskeleton stems from its ability to assemble into a variety of diverse structures – branched networks, meshes, and bundles. This architectural complexity is orchestrated by actin-binding proteins, whose activity is delicately regulated in response to internal and external signals. Our long-term goal is to contribute to human health and well-being by advancing the understanding of the actin cytoskeleton organization by actin-bundling proteins and their contribution to pathologies (e.g., congenital diseases and metastatic cancers). Plastin/fimbrin family of cytoskeleton organizers are conserved proteins that promote assembly of actin filaments into bundles involved in cell migration, adhesion, cytokinesis, and formation of stereocilia and microvilli structures of the inner ear, intestinal and kidney epithelia. Of three human plastin (PLS) isoforms, PLS1 deletion results in deafness, PLS2 contributes to pathologies of the immune system and the development of aggressive metastatic cancers, while mutations in PLS3 lead to severe osteoporosis with bone fragility and other connective tissue disorders. Despite the importance and a long-lasting interest of the research community to these proteins, understanding of their interaction with actin and their regulation is superficial, whereas published structural and biochemical data are incomplete, scattered, and sometimes contradictory. The overall objective of the current proposal is to fill these major gaps by providing a thorough characterization of the molecular and cellular mechanisms governing the function of plastins and to demonstrate how this improved understanding can contribute to explaining the pathology of plastin-related diseases. We propose that the unique domain organization of plastins enables several regulation modes interconnected via a central allosteric mechanism that confers multifaceted contribution to various actin-governed cellular processes. Biochemical characterization of plastin isoforms will reveal mechanisms of their regulation and function at the molecular level (Aim 1a,b); high-resolution cryo-electron microscopy (EM)/cryo-electron tomography (ET) reconstruction will provide structural details of plastin interaction with actin (Aim 1c); structural analysis and atomistic molecular dynamics (MD) simulations will generate a model of the auto-inhibition allowing to predict functional outcomes of congenital mutations (Aim 2); while Aim 3 will focus on understanding functional significance and implications of the allosteric auto-inhibition of plastins and its role in cooperation with other actin- binding proteins. These approaches, supported by single-molecule speckle (SiMS), total internal reflection fluorescence (TIRF), and bulk epi-fluorescence microscopy, will unveil plastin dynamics, cooperation with protein partners, and contribution to actin-dependent processes in living cells. The proposal will result in a breakthrough in the understanding of the actin-dependent cellular events controlled by the plastin/fimbrin family of cytoskeleton organizers, uncover molecular mechanisms behind plastin-linked congenital (deafness, osteoporosis, and diaphragmatic hernia) and acquired (cancer) diseases, opening opportunities for their specific therapeutics.

项目概要/摘要 肌动蛋白细胞骨架卓越的功能多样性源于其组装成多种细胞的能力 不同的结构——分支网络、网格和束。这种架构的复杂性是精心策划的 由肌动蛋白结合蛋白产生，其活性根据内部和外部信号进行精细调节。我们的 长期目标是通过增进对肌动蛋白的理解来为人类健康和福祉做出贡献 肌动蛋白捆绑蛋白的细胞骨架组织及其对病理学的贡献（例如先天性 疾病和转移性癌症）。细胞骨架组织者的塑性蛋白/纤毛蛋白家族是保守的蛋白质， 促进肌动蛋白丝组装成参与细胞迁移、粘附、胞质分裂和形成的束 内耳、肠和肾上皮细胞的静纤毛和微绒毛结构。三种人类塑性蛋白 (PLS) 同种型，PLS1 缺失会导致耳聋，PLS2 会导致免疫系统病变， 侵袭性转移性癌症的发展，而 PLS3 突变会导致严重的骨质疏松症 骨质脆弱和其他结缔组织疾病。尽管重要性和长期的兴趣 研究界对这些蛋白质的了解，了解它们与肌动蛋白的相互作用及其调节 肤浅，而已发表的结构和生化数据不完整、分散，有时 矛盾的。当前提案的总体目标是通过提供彻底的解决方案来填补这些主要空白 表征控制塑蛋白功能的分子和细胞机制并证明 这种加深的理解如何有助于解释与塑性蛋白相关的疾病的病理学。我们 提出塑料蛋白独特的结构域组织使得多种调节模式通过 中央变构机制，为各种肌动蛋白控制的细胞过程提供多方面的贡献。 塑性蛋白亚型的生化表征将揭示其调节和功能的机制 分子水平（目标 1a、b）；高分辨率冷冻电子显微镜 (EM)/冷冻电子断层扫描 (ET) 重建将提供塑性蛋白与肌动蛋白相互作用的结构细节（目标 1c）；结构分析和 原子分子动力学（MD）模拟将生成一个自动抑制模型，可以预测 先天性突变的功能结果（目标 2）；而目标 3 将侧重于理解功能性 塑蛋白变构自动抑制的意义和含义及其与其他肌动蛋白合作的作用 结合蛋白。这些方法由单分子散斑 (SiMS)、全内反射支持 荧光 (TIRF) 和散装落射荧光显微镜将揭示塑性蛋白动力学以及与蛋白质的合作 合作伙伴，以及对活细胞中肌动蛋白依赖性过程的贡献。该提案将带来突破 理解由细胞骨架的塑性蛋白/纤毛蛋白家族控制的肌动蛋白依赖性细胞事件 组织者，揭示与塑性蛋白相关的先天性疾病（耳聋、骨质疏松症和 膈疝）和获得性（癌症）疾病，为其特定疗法提供了机会。