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.

项目总结/摘要 肌动蛋白细胞骨架的显著功能多样性源于其组装成各种 不同的结构-分支网络，网格和束。这种架构复杂性是精心安排的 肌动蛋白结合蛋白，其活性根据内部和外部信号进行微妙调节。我们 长期目标是通过促进对人类行为的理解， 通过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Plastin/fimplastin家族是保守的蛋白质， 促进肌动蛋白丝组装成纤维束，参与细胞迁移、粘附、胞质分裂和形成 内耳、肠和肾上皮的静纤毛和微绒毛结构。三种人的纤维蛋白 (PLS)在同种型中，PLS 1缺失导致耳聋，PLS 2导致免疫系统的病理学， 侵袭性转移性癌症的发展，而PLS 3的突变导致严重的骨质疏松症， 骨脆性和其他结缔组织疾病。尽管重要性和长期的利益， 研究社区对这些蛋白质，了解他们的相互作用与肌动蛋白和他们的调控是 表面的，而出版的结构和生化数据是不完整的，分散的，有时， 自相矛盾本提案的总体目标是通过提供一个全面的 表征控制质体蛋白功能的分子和细胞机制，并证明 这种改进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plastins独特的域组织使多种调节模式能够通过一个相互连接 中心变构机制，赋予多方面的贡献，各种肌动蛋白支配的细胞过程。 对质体异构体的生化表征将揭示它们在细胞内的调节和功能机制。 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） 重建将提供与肌动蛋白（目的1c）的结构细节的质体相互作用;结构分析和 原子分子动力学（MD）模拟将产生一个模型的自动抑制允许预测 先天性突变的功能结局（目标2）;而目标3将侧重于了解功能性 的意义和影响的别构自动抑制的plastins和它的作用，在合作与其他肌动蛋白， 结合蛋白这些方法，支持单分子散斑（西姆斯），全内反射 TIRF和大体积落射荧光显微镜，将揭示质体动力学，与蛋白质的合作， 合作伙伴，并在活细胞中的肌动蛋白依赖过程的贡献。该提案将带来突破性进展 在理解肌动蛋白依赖的细胞事件控制的纤维蛋白酶/fimplants家庭的细胞骨架 组织者，揭示背后的分子机制plastin连锁先天性（耳聋，骨质疏松症， 疝气）和获得性（癌症）疾病，为他们的特定治疗开辟了机会。