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: 10797753
• 负责人: Dmitri Kudryashov
• 金额: $ 23.84万
• 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/10797753
• 关键词: 3-Dimensional; Actin-Binding Protein; Actins; Adhesions; Age; Allosteric Regulation; Architecture; Attenuated; Autoimmune Diseases; Autoimmune Process; Binding; Biochemical; Biological; Blood; Bundling; Calcium; 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; Cytoskeletal Modeling; Cytoskeleton; Data; Development; Diaphragmatic Hernia; Disease; Disseminated Malignant Neoplasm; Endocytosis; Epithelium; Event; F-Actin; Family; Fiber; Filopodia; Fimbrin; Fluorescence; 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; Microfilaments; Microscopy; 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; Testing; Therapeutic; Therapeutic Intervention; Tissues; Translating; Tropomyosin; Variant; Work; X-Ray Crystallography; bone fragility; calponin; cancer cell; cancer invasiveness; cell motility; cellular microvillus; 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.

项目摘要/摘要 肌动蛋白细胞骨架的显着功能多功能性源于其组装成各种的能力 各种结构 - 分支网络，网格和捆绑包。这种建筑复杂性是精心策划的 通过肌动蛋白结合蛋白，其活性明确调节了内部和外部信号。我们的 长期目标是通过促进对肌动蛋白的理解来为人类健康和福祉做出贡献 肌动蛋白捆绑蛋白及其对病理的贡献（例如先天性）的细胞骨架组织 疾病和转移性癌症）。质素/纤维蛋白细胞骨架组织者家族是保守的蛋白质， 将肌动蛋白细丝组装成与细胞迁移，粘合剂，细胞因子和形成有关的捆绑包。 内耳，肠道和肾上皮的立体胶质和微绒毛结构。三个人类塑料 （PLS）同工型，PLS1缺失会导致耳聋，PLS2有助于免疫系统的病理和 侵略性转移性癌症的发展，而PLS3中的突变导致严重的骨质疏松症 骨骼脆弱性和其他连接的组织障碍。尽管重要性和持久的兴趣 这些蛋白质的研究界，了解它们与肌动蛋白的相互作用以及其调节的理解是 肤浅，而发表的结构和生化数据是不完整的，分散的，有时是 矛盾。当前建议的总体目标是通过提供详尽 塑料功能的分子和细胞机制的表征，并证明 这种改善的理解如何有助于解释塑料相关疾病的病理。我们 提议，塑料的独特领域组织可以使几种通过A相互联系的法规模式 承认对各种肌动蛋白治疗的细胞过程的多方面贡献的中央变构机制。 塑料同工型的生化表征将揭示其调节和功能的机制 分子水平（AIM 1A，B）；高分辨率的低分辨率显微镜（EM）/冷冻电子断层扫描（ET） 重建将提供与肌动蛋白相互作用的结构细节（AIM 1C）；结构分析和 原子分子动力学（MD）模拟将生成自动抑制模型，以预测 先天性突变的功能结果（AIM 2）；而目标3将专注于理解功能 变构自动抑制塑料及其与其他肌动蛋白合作的作用的意义和含义 结合蛋白。这些方法，由单分子斑点（SIMS）支持，总内部反射 荧光（TIRF）和大量的荧光显微镜将公布塑料动力学，与蛋白质合作 伴侣，以及对活细胞中肌动蛋白依赖性过程的贡献。该提案将导致突破 在理解肌动蛋白依赖性的细胞事件中，由质子素/脂肪骨骼家族控制 组织者，识别塑料与先天性的分子机制（耳聋，骨质疏松症和 diaphragmatic疝）并获得（癌症）疾病，为其特定疗法开辟了机会。