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The impact of dynamic actin polymerization on mitochondrial dynamics and function

动态肌动蛋白聚合对线粒体动力学和功能的影响

基本信息

批准号：
10670903
负责人：
HENRY N HIGGS
金额：
$ 79.44万
依托单位：
DARTMOUTH COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2027-08-31
项目状态：
未结题

项目摘要

The ‘actin cytoskeleton’ is not one structure but a number of distinct structures assembled and disassembled for different purposes. In mammalian cells, a few abundant and easily recognizable structures dominate our view of the actin cytoskeleton, including: stress fibers, lamellipodia and filopodia. However, a growing number of less abundant and/or highly transient actin-based structures have been revealed, controlling important cellular processes. Two such actin structures are the subject of this application: 1) CIA, calcium-induced actin; and 2) ADA, acute depolarization-induced actin. Though highly transient, both structures are extensive in the cytosol and affect important processes. In addition, both CIA and ADA impact the structure and function of mitochondria. CIA depends on calcium activation of the formin protein INF2, which stimulates actin polymerization on the endoplasmic reticulum and throughout the cytosol. Downstream effects of CIA include increased mitochondrial calcium and increased mitochondrial fission. The importance of CIA is illustrated by the fact that INF2 mutations link to two diseases, focal segmental glomerulosclerosis (FSGS) and Charcot-Marie-Tooth disease (CMTD). ADA is triggered by mitochondrial depolarization (either pharmacologically-induced or hypoxia-induced), which activates two parallel pathways: 1) mitochondrial calcium release activates protein kinase C-, activating in turn Rac, WAVE complex, and Arp2/3 complex; and 2) decreased ATP activates AMP-dependent protein kinase (AMPK) through LKB1, activating in turn Cdc42 and FMNL formins. The ADA actin network is tightly associated with mitochondria. An exciting new result is that one immediate consequence of ADA is rapid stimulation of glycolysis. Additionally, ADA temporarily inhibits longer-term consequences of mitochondrial depolarization such as mitochondrial reorganization and recruitment of the mitophagy protein Parkin. The goals in this grant period are to elucidate both the mechanisms triggering CIA and ADA, as well as their downstream effects. These goals will be accomplished using a combination of cellular approaches (live-cell microscopy, proteomics, metabolic analysis) and biochemical approaches (cell-free reconstitution, analysis of purified proteins on model lipid membranes). The questions to be asked include the following. 1) How is INF2 activated by increased calcium? 2) How are INF2-polymerized filaments organized into a network by myosin II and fascin? 3) How does CIA interface with known mitochondrial fission proteins such as Mff and Drp1 to stimulate fission? 4) How do PKC and AMPK activate Rac and Cdc42, respectively, during ADA? 5) How do Arp2/3 complex and FMNL formins work together during ADA? 6) How does ADA stimulate glycolysis? These questions address fundamental mechanistic questions important to a wide range of mammalian cells, and occupy an exciting frontier between cytoskeletal biology, mitochondrial biology, and metabolism. 1

肌动蛋白细胞骨架不是一个结构，而是许多不同的结构，为了不同的目的。在哺乳动物细胞中，一些丰富且容易辨认的结构主导着我们的视野肌动蛋白的细胞骨架，包括：应力纤维、片脂和丝足。然而，越来越多的人大量和/或高度瞬时的肌动蛋白结构已被揭示，控制着重要的细胞流程。两种这样的肌动蛋白结构是本应用的主题：1)CIA，钙诱导的肌动蛋白；2) 急性去极化诱导的肌动蛋白。虽然高度瞬变，但这两种结构在胞质中分布广泛。并影响重要的进程。此外，CIA和ADA都影响线粒体的结构和功能。 CIA依赖于Forin蛋白INF2的钙激活，INF2刺激肌动蛋白聚合在内质网和整个胞浆。CIA的下游效应包括增加线粒体钙和线粒体分裂增加。INF2突变的事实说明了CIA的重要性与两种疾病有关，局灶性节段性肾小球硬化(FSGS)和夏科-玛丽-图斯病(CMTD)。 ADA是由线粒体去极化(药物诱导或缺氧诱导)触发的，这激活两条平行的途径：1)线粒体钙释放激活蛋白激酶C-，依次激活 RAC、WAVE复合体和Arp2/3复合体；以及2)降低ATP激活AMP依赖的蛋白激酶 (AMPK)通过LKB1，依次激活CDC42和FMNL Forins。ADA肌动蛋白网络密切相关有线粒体。一个令人兴奋的新结果是，ADA的一个直接后果是迅速刺激糖酵解。此外，ADA暂时抑制线粒体去极化的长期后果，如作为线粒体重组和招募的有丝分裂蛋白Parkin。这一批款期的目标旨在阐明CIA和ADA的触发机制及其下游影响。这些目标将使用细胞方法的组合(活细胞显微镜、蛋白质组学、代谢分析)和生化方法(无细胞重组、纯化蛋白质对模型脂的分析膜)。将提出的问题包括以下几个方面。1)增加的钙是如何激活INF2的？ 2)肌球蛋白II和筋膜蛋白是如何将INF2聚合的细丝组织成网络的？3)CIA是如何与已知的线粒体分裂蛋白如Mff和Drp1相互作用以刺激分裂？4)PKC是如何和AMPK在ADA过程中分别激活RAC和CDC42？5)Arp2/3复合体和FMNL Forins是如何在ADA期间一起工作？6)ADA如何刺激糖酵解？这些问题涉及基本问题机制问题对广泛的哺乳动物细胞很重要，并占据了令人兴奋的前沿细胞骨架生物学、线粒体生物学和新陈代谢。 1