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: 8488671
• 负责人: Thomas A Blanpied
• 金额: $ 42.85万
• 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 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8488671
• 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; public health relevance; 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，对线粒体对细胞生理的重要性的欣赏显着增长。线粒体是高度动态的，经常进行裂变和融合，以保持适当的分布，并减少其基因组中氧化应激和有害突变的影响。这些属性在整个真核生物中都是保守的，如将线粒体裂变和融合机械的进化保护所证明的那样。目前，裂变机器由胞质动力蛋白相关的GTPase（哺乳动物中的DRP1，酵母中的DNM1）和线粒体蛋白受体组成。但是，裂变过程的许多方面尚不清楚。首先，在哺乳动物中，线粒体DRP1受体的身份尚不清楚，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完全线粒体裂变。最后，线粒体裂变 似乎是通过与内质网（ER）接触而引发的，仅凭内质网（ER）就能影响独立于DRP1的收缩。我们提出了一种解决此问题的新型机制，与ER介导的肌动蛋白聚合驱动主要线粒体收缩之间的相互作用，这对于基于次级DRP1的狭窄是必不可少的。 ER结合的formin蛋白INF2介导肌动蛋白聚合。我们的初步结果提供了支持这种机制的证据。我们的目标利用尖端技术（超级分辨率显微镜，蛋白质组学）来阐明ER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级分辨率手掌在前所未有的水平下跟踪INF2，肌动蛋白和DRP1动力学。 AIM 2使用电子显微镜和超级分辨率风暴来检查裂变过程的结构特征以及至少20 nm分辨率的蛋白质成分之间的空间关系。 AIM 3使用蛋白质组学来鉴定线粒体上的“ INF2受体”，我们假设这是当前假设的DRP1受体之一。 AIM 3中鉴定出的蛋白质将纳入目标1和2。尽管我们测试机械模型，但我们仍然对许多其他机械可能性开放，我们的目标旨在区分这些可能性。