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

描述(由申请人提供)：在过去的几年里，人们对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，经历着频繁的分裂和融合，以保持适当的分布，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，线粒体分裂和融合机制的进化保守证明了这一点。分裂机制目前被认为是由胞浆动力蛋白相关的GTPase(哺乳动物中的Drp1，酵母中的DNM1)和线粒体蛋白受体组成。然而，裂变过程的许多方面都不清楚。首先，线粒体Drp1受体在哺乳动物中的身份尚不清楚，有几种蛋白质(hFis1、Mff、MiD49/51和GDAP1)被提出。此外，目前还不确定Drp1介导的收缩是否能够完全分裂线粒体。最后，线粒体分裂 似乎是通过与内质网(ER)接触而启动的，内质网本身就能够影响DRp1非依赖的收缩。我们提出了一种新的机制来解决这个问题，内质网介导的肌动蛋白聚合之间的相互作用驱动了初级线粒体收缩，这是基于Drp1的二级收缩所必需的。内质网结合的福尔曼蛋白INF2介导肌动蛋白聚合。我们的初步结果为这一机制提供了证据。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在前所未有的水平上跟踪INF2、肌动蛋白和Drp1在分裂过程中的动态。目的2使用电子显微镜和超分辨率STORM来研究分裂过程的结构特征和至少20 nm分辨率的蛋白质组分之间的空间关系。目的3利用蛋白质组学鉴定线粒体上的“INF2受体”，我们推测它是目前假想的Drp1受体之一。目标3中确定的蛋白质将被合并到目标1和2中。当我们测试我们的机械论模型时，我们仍然对许多其他机械论可能性持开放态度，我们的目标旨在区分这些可能性。

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.