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”不是一种结构，而是许多不同结构的组装和拆卸在哺乳动物细胞中，一些丰富且易于识别的结构主导了我们的视野。肌动蛋白细胞骨架包括：应力纤维、板状伪足和丝状伪足，但数量越来越少。丰富和/或高度瞬时的肌动蛋白结构已被揭示，控制着重要的细胞两种这样的肌动蛋白结构是本申请的主题：1) CIA，钙诱导的肌动蛋白；2) ADA，急性去极化诱导肌动蛋白，虽然高度短暂，但两种结构都广泛存在于细胞质中。此外，CIA 和 ADA 都会影响线粒体的结构和功能。 CIA 依赖于福尔明蛋白 INF2 的钙激活，从而刺激肌动蛋白聚合 CIA 的下游影响包括增加线粒体。 INF2 突变这一事实说明了 CIA 的重要性。与局灶节段性肾小球硬化症（FSGS）和腓骨肌萎缩症（CMTD）这两种疾病有关。 ADA 由线粒体去极化（药理诱导或缺氧诱导）触发，激活两条平行途径：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；2) ATP 减少激活 AMP 依赖性蛋白激酶 (AMPK) 通过 LKB1，依次激活 Cdc42 和 FMNL 形成蛋白 ADA 肌动蛋白网络紧密相关。一个令人兴奋的新结果是 ADA 的一个直接后果是快速刺激此外，ADA 会暂时抑制线粒体去极化的长期后果，例如作为线粒体重组和线粒体自噬蛋白 Parkin 的招募，是本资助期的目标。旨在阐明 CIA 和 ADA 的触发机制及其下游影响。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（无细胞重建、模型脂质上纯化蛋白质的分析）需要提出的问题包括： 1) INF2 是如何被增加的钙激活的？ 2) INF2 聚合丝如何通过肌球蛋白 II 和肌成束蛋白组织成网络？与已知的线粒体裂变蛋白（例如 Mff 和 Drp1）结合以刺激裂变？ 4) PKC 如何作用？ ADA 期间，AMPK 和 AMPK 分别激活 Rac 和 Cdc42 5) Arp2/3 复合体和 FMNL 如何形成？ ADA 期间如何协同工作？ 6) ADA 如何刺激糖酵解？对于广泛的哺乳动物细胞来说很重要的机制问题，并且占据了一个令人兴奋的前沿领域细胞骨架生物学、线粒体生物学和新陈代谢。 1