Cytoskeletal effects on mitochondrial dynamics through the ER-bound formin INF2

细胞骨架通过内质网结合的 INF2 对线粒体动力学的影响

• 批准号: 8827186
• 负责人: Thomas A Blanpied
• 金额: $ 39.75万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8827186
• 关键词: Actins; Affect; Alzheimer' s Disease; Area; Attention; Automobile Driving; Binding; Cell physiology; Cells; Charcot-Marie-Tooth Disease; Confocal Microscopy; Cytoskeleton; Defect; Disease; Dynamin; Electron Microscopy; Endocytosis; Endoplasmic Reticulum; Eukaryota; Event; Generations; Genome; Goals; Guanosine Triphosphate Phosphohydrolases; Health; Homeostasis; Human; Huntington Disease; Immunoprecipitation; Lead; Length; Life; Link; Mammals; Mediating; Membrane; Microfilaments; Microscopy; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Morphogenesis; Movement; Mutation; Neurodegenerative Disorders; Organelles; Oxidative Stress; Parkinson Disease; Peripheral Nervous System Diseases; Pharmaceutical Preparations; Play; Preparation; Process; Protein Isoforms; Proteins; Proteomics; Research; Research Personnel; Resolution; Role; Site; Small Interfering RNA; Suggestion; Techniques; Testing; Work; Yeasts; base; constriction; depolymerization; design; human disease; mutant; novel; polymerization; protein protein interaction; receptor; spatial relationship

DESCRIPTION (provided by applicant): In the last several years, appreciation of the importance of mitochondria to cell physiology has risen dramatically. Mitochondria are highly dynamic, undergoing frequent fission and fusion to maintain proper distribution as well as to reduce the effects of oxidative stresses and deleterious mutations in their genome. These attributes are conserved throughout eukaryotes, as demonstrated by evolutionary conservation of the machinery for mitochondrial fission and fusion. The fission machinery is currently thought to consist of a cytosolic dynamin-related GTPase (Drp1 in mammals, Dnm1 in yeast) and mitochondrial protein receptors. However, many aspects of the fission process are unclear. First, the identity of the mitochondrial Drp1 receptor is not clear in mammals, with several proteins (hFis1, Mff, MiD49/51, and GDAP1) being proposed. In addition, it is uncertain whether Drp1-mediated constriction is capable of full mitochondrial fission. Finally, mitochondrial fission appears to be initiated by contact with endoplasmic reticulum (ER), which alone is able to affect a Drp1-independent constriction. We propose a novel mechanism to resolve this issue, with interactions between the ER-mediated actin polymerization driving a primary mitochondrial constriction, which is necessary for a secondary Drp1-based constriction. The ER-bound formin protein INF2 mediates actin polymerization. Our preliminary results provide evidence supporting this mechanism. Our aims utilize cutting-edge techniques (super resolution microscopy, proteomics) to elucidate the macromolecular interactions necessary for ER-mediated mitochondrial fission. Aim 1 uses live-cell confocal and super resolution PALM to track INF2, actin, and Drp1 dynamics during fission at an unprecedented level. Aim 2 uses electron microscopy and super resolution STORM to examine structural features of the fission process and the spatial relationships between the protein components with at least 20 nm resolution. Aim 3 uses proteomics to identify the "INF2 receptor" on mitochondria, which we postulate is one of the currently hypothesized Drp1 receptors. Proteins identified in Aim 3 will be incorporated into Aims 1 and 2. While we test our mechanistic model, we remain open to many other mechanistic possibilities and our aims are designed to distinguish between these possibilities.

描述（由申请人提供）：在过去的几年中，人们对线粒体对细胞生理学重要性的认识急剧提高。线粒体是高度动态的，经历频繁的裂变和融合，以维持适当的分布，并减少氧化应激和基因组中有害突变的影响。这些属性在真核生物中是保守的，正如线粒体裂变和融合机制的进化保守所证明的那样。目前认为裂变机制由胞质动力相关的 GTP 酶（哺乳动物中的 Drp1，酵母中的 Dnm1）和线粒体蛋白受体组成。然而，裂变过程的许多方面尚不清楚。首先，哺乳动物中线粒体 Drp1 受体的身份尚不清楚，有人提出了几种蛋白质（hFis1、Mff、MiD49/51 和 GDAP1）。此外，尚不确定 Drp1 介导的收缩是否能够实现线粒体的完全裂变。最后，线粒体裂变 似乎是通过与内质网 (ER) 接触而启动的，仅内质网就能够影响不依赖于 Drp1 的收缩。我们提出了一种新的机制来解决这个问题，即内质网介导的肌动蛋白聚合之间的相互作用驱动初级线粒体收缩，这对于基于 Drp1 的次级收缩是必要的。内质网结合的福尔明蛋白 INF2 介导肌动蛋白聚合。我们的初步结果提供了支持这一机制的证据。我们的目标是利用尖端技术（超分辨率显微镜、蛋白质组学）来阐明内质网介导的线粒体裂变所需的大分子相互作用。 Aim 1 使用活细胞共聚焦和超分辨率 PALM 以前所未有的水平跟踪裂变过程中的 INF2、肌动蛋白和 Drp1 动态。目标 2 使用电子显微镜和超分辨率 STORM 以至少 20 nm 的分辨率检查裂变过程的结构特征以及蛋白质成分之间的空间关系。目标 3 使用蛋白质组学来识别线粒体上的“INF2 受体”，我们假设它是目前假设的 Drp1 受体之一。目标 3 中鉴定的蛋白质将被纳入目标 1 和 2。在我们测试我们的机械模型时，我们对许多其他机械可能性持开放态度，我们的目标旨在区分这些可能性。