Linking actin cytoskeleton to membrane dynamics in mitochondrial fission

我们对肌动蛋白聚合机制有着长期的兴趣，这促使我们研究 在数量上较小的具有重要细胞作用的细丝群体。一个这样的肌动蛋白群体 在线粒体分裂中起作用。线粒体分裂是线粒体正常分布所必需的， 线粒体自噬、氧化应激反应和对不同代谢底物的适应。缺陷 线粒体分裂与主要神经退行性疾病的病理学有关，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。发动蛋白家族GTdR Drp 1是一个核心参与者， 在线粒体分裂中，在分裂位点寡聚化并促进膜收缩。不过 触发线粒体分裂的机制尚不清楚。我们发现肌动蛋白聚合 在哺乳动物的Drp 1募集和线粒体分裂中起着重要作用。这 这一发现来自于我们对内质网结合的INF 2的关注，INF 2组装了这种 丝状体种群通过这些研究，我们开发了用于成像的活细胞系统 线粒体分裂的高空间和时间分辨率，这使我们能够定义顺序 导致线粒体上Drp 1寡聚化的事件。我们还建立了完善的 研究肌动蛋白与Drp 1、INF 2和其他裂变组分相互作用的生化系统 这一过程将使最终的无细胞裂变重建成为可能。这些发现 从根本上改变了我们对线粒体分裂的看法。我们未来五年的目标是 哺乳动物线粒体分裂的一种“类型”（由钙离子载体刺激）， 随后使用这些知识来定义由其他刺激引起的裂变机制。我们有 两个长期目标：使用纯化的组分重建肌动蛋白介导的线粒体分裂 （这将表明完全的机械理解），并定义激活的信号输入 在特定的生理情况下裂变。INF 2的突变与两种人类疾病有因果关系。 疾病：局灶性和节段性肾小球硬化症（一种肾脏疾病）和腓骨肌萎缩症 周围神经病变（peripheral neuropathy）因此，我们的工作影响了基础细胞生物学和疾病- 基于研究。该实验室的第二个重点是由FAMNL 3进行的丝状伪足组装。而 本研究策略中未讨论，我们将在本MIRA中继续我们的丝状伪足研究。类似于我们 INF 2研究，多年来仔细的细胞和生物化学工作正在导致令人惊讶的发现， 包括1）丝状伪足与细胞-细胞和细胞-基质粘附之间的联系，以及2） FMNL 3在核内体动力学中的作用。我们的总体设想是，存在着未被发现的肌动蛋白群体， 丝，短暂的和低丰度，介导关键的细胞功能。合并研究 正在揭示这些肌动蛋白丝的数量。

We have a long-standing interest in actin polymerization mechanisms, which has led us to investigate quantitatively minor populations of filaments with important cellular roles. One such actin population functions in mitochondrial fission. Mitochondrial fission is required 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 focus on an endoplasmic reticulum-bound formin, INF2, which assembles this filament population. 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.