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: 9016561
• 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 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9016561
• 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; live cell microscopy; mutant; novel; overexpression; 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，线粒体对细胞生理学的重要性的认识急剧上升。线粒体是高度动态的，经历频繁的分裂和融合以维持适当的分布以及减少其基因组中的氧化应激和有害突变的影响。这些属性在整个真核生物中是保守的，正如线粒体分裂和融合机制的进化保守所证明的那样。裂变机制目前被认为是由细胞溶质动力蛋白相关的GTdR（哺乳动物中的Drp 1，酵母中的Dnm 1）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，线粒体Drp 1受体在哺乳动物中的身份尚不清楚，提出了几种蛋白质（hFis 1，Mff，MiD 49/51和GDAP 1）。此外，目前还不确定Drp 1介导的收缩是否能够完全线粒体分裂。最后，线粒体分裂 似乎是通过与内质网（ER）接触而启动的，内质网单独能够影响Drp 1非依赖性收缩。我们提出了一种新的机制来解决这个问题，与ER介导的肌动蛋白聚合驱动的初级线粒体收缩，这是必要的二级Drp 1为基础的收缩之间的相互作用。ER结合的肌动蛋白INF 2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ER介导的线粒体分裂所必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INF 2，肌动蛋白和Drp 1动态。目标2使用电子显微镜和超分辨率STORM检查裂变过程的结构特征和蛋白质组分之间的空间关系，分辨率至少为20 nm。目的3利用蛋白质组学技术鉴定线粒体上的“INF 2受体”，我们假设它是目前假设的Drp 1受体之一。目标3中鉴定的蛋白质将纳入目标1和2。当我们测试我们的机械模型时，我们仍然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。