Supplement - Linking actin cytoskeleton to membrane dynamics in mitochondrial fission

家长奖摘要 线粒体的分裂对于线粒体的正常分布、有丝分裂、氧化应激是必不可少的。 对不同代谢底物的反应和适应。线粒体分裂缺陷与 主要神经退行性疾病的病理学，包括阿尔茨海默氏症，亨廷顿氏症，帕金森氏症， 和肌萎缩侧索硬化症动力蛋白家族GTP酶Drp1是线粒体分裂的中心参与者，寡聚在 裂解部位和促进膜收缩。尽管如此，触发线粒体的机制 裂变是不透明的。我们已经发现，肌动蛋白在分裂部位的聚合在 哺乳动物中的drp1募集和线粒体分裂。这一发现来自于我们对 肌动蛋白通过Forin蛋白聚合，特别关注内质网结合 福尔明，INF2。通过这些研究，我们开发了用于线粒体成像的活细胞系统 高空间和时间分辨率的裂变，使我们能够定义事件的顺序 线粒体上的Drp1寡聚。我们还建立了精细的生化系统来研究 肌动蛋白与Drp1、INF2和裂变过程的其他成分的相互作用，这将使 最终进行无细胞的裂变重组。这些发现从根本上改变了我们对 线粒体分裂。我们在未来五年的目标是定义一种哺乳动物线粒体的“类型” 详细的裂变(由钙离子载体刺激)，并随后利用这一知识来定义 其他刺激引起的裂变机制。我们有两个更长期的目标：重组肌动蛋白- 使用纯化的成分(这将表明完全机械性的)介导的线粒体分裂 理解)，并定义在特定生理情况下激活裂变的信号输入。 INF2基因突变与人类两种疾病有关：局灶性和节段性肾小球硬化 (肾脏疾病)和Charcot-Marie-Tooth病(一种外周神经病变)。因此，我们的工作影响到 包括基础细胞生物学和基于疾病的研究。该实验室的第二个重点是丝状伪足。 以FMNL3为模板组装。虽然没有在本研究战略中讨论，但我们将继续我们的 丝状伪足在这个Mira中起作用。与我们的INF2研究类似，多年的仔细细胞和生化工作 导致了令人惊讶的发现，包括1)丝状伪足与细胞-细胞和细胞-之间的联系 底物黏附，以及2)FMNL3在内体动力学中的作用。我们的总体愿景是 是未被发现的肌动蛋白细丝种群，它们是瞬时的和低丰度的，它们介导了关键 细胞功能。我实验室的联合研究揭示了这些肌动蛋白细丝群体。

Abstract of Parent Award Mitochondrial fission is essential for proper mitochondrial distribution, mitophagy, oxidative stress response, and adaptation to varying metabolic substrates. Defects in mitochondrial fission are linked to the pathology of major neurodegenerative diseases, including Alzheimer’s, Huntington’s, Parkinson’s, and ALS. The dynamin family GTPase Drp1 is a central player in mitochondrial fission, oligomerizing at fission sites and promoting membrane constriction. Still, the mechanisms that trigger mitochondrial fission are murky. We have discovered that actin polymerization at fission sites plays a major role in Drp1 recruitment and mitochondrial fission in mammals. This finding came from our long-term interest in actin polymerization through formin proteins, with particular focus on an endoplasmic reticulum-bound formin, INF2. Through these studies, we have developed live-cell systems for imaging mitochondrial fission at high spatial and temporal resolution, which have allowed us to define the order of events leading to Drp1 oligomerization on mitochondria. We have also established refined biochemical systems to study interaction of actin with Drp1, INF2 and other components of the fission process, which will enable eventual cell-free reconstitution of fission. These discoveries have fundamentally changed our view of mitochondrial fission. Our goal in the next five years is to define one “type” of mammalian mitochondrial fission in detail (stimulated by calcium ionophore), and subsequently to use this knowledge to define fission mechanisms induced by other stimuli. We have two longer-term goals: to reconstitute actin- mediated mitochondrial fission using purified components (which would indicate full mechanistic understanding), and to define the signaling in-puts that activate fission in specific physiological situations. Mutations in INF2 are causally linked to two human diseases: focal and segmental glomerulosclerosis (a kidney disease) and Charcot-Marie-Tooth disease (a peripheral neuropathy). Thus, our work impacts both fundamental cell biology and disease-based research. A second focus of the laboratory is filopodia assembly by the formin FMNL3. While not discussed in this Research Strategy, we will continue our filopodia work in this MIRA. Similar to our INF2 studies, years of careful cellular and biochemical work are leading to surprising discoveries, including 1) links between filopodia and both cell-cell and cell- substratum adhesion, and 2) a role for FMNL3 in endosomal dynamics. Our overall vision is that there are undiscovered populations of actin filaments, transient and of low abundance, which mediate key cellular functions. The combined studies in my laboratory are revealing these actin filament populations.