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

• 批准号: 10405718
• 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
• 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
• 来源: https://reporter.nih.gov/project-details/10405718
• 关键词: Actins; Acute; Address; Aerobic; Affect; Anaerobic Bacteria; Biochemical; Biology; Calcium; Cell physiology; Cells; Charcot-Marie-Tooth Disease; Complex; Cytoskeleton; Cytosol; Dependence; Disease; Endoplasmic Reticulum; FMNL1 gene; Filament; Filopodia; Focal Segmental Glomerulosclerosis; Glycolysis; Goals; Grant; Health; Human; Hypoxia; Link; Mammalian Cell; Membrane Lipids; Metabolic; Metabolism; Mitochondria; Modeling; Mutation; Myosin Type II; Nature; Neurons; Organelles; Parkin; Pathway interactions; Pharmacology; Polymers; Process; Protein Kinase; Protein Kinase C; Proteins; Proteomics; Research; Respiration; STK11 gene; Stress Fibers; Structure; Work; actin 2; base; fascin; frontier; live cell microscopy; polymerization; reconstitution; recruit

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，而是组装并拆卸的许多不同的结构 不同的目的。在哺乳动物的细胞中，一些丰富且易于识别的结构主导着我们的观点 肌动蛋白的细胞骨架，包括：应力纤维，薄片和丝状肌骨架。但是，越来越多的 已经揭示了丰富和/或高度瞬态肌动蛋白的结构，控制了重要的细胞 过程。此应用的两个这样的肌动蛋白结构是：1）CIA，钙诱导的肌动蛋白；和2） ADA，急性去极化诱导的肌动蛋白。虽然高度瞬态，但两种结构在细胞质中均广泛 并影响重要过程。此外，中央情报局和ADA都会影响线粒体的结构和功能。 中央情报局取决于formin蛋白IFF2的钙激活，这刺激了肌动蛋白聚合在 内质网和整个细胞质。中央情报局的下游效应包括线粒体增加 钙和线粒体裂变的增加。 INF2突变的事实说明了中央情报局的重要性 链接到两种疾病，局灶性节段性肾小球硬化（FSG）和charcot-marie-tooth病（CMTD）。 ADA是由线粒体沉积（药物诱导的或缺氧诱导的）触发的，这是由此触发的） 激活两个平行途径：1）线粒体钙释放激活蛋白激酶C-，依次激活 RAC，Wave复合物和ARP2/3复合物； 2）开发的ATP激活了依赖AMP的蛋白激酶 （AMPK）通过LKB1，依次激活CDC42和FMNL formins。 ADA肌动蛋白网络密切相关 与线粒体。一个令人兴奋的新结果是，ADA的直接后果是快速刺激 糖酵解。此外，ADA暂时抑制线粒体沉积的长期后果 作为线粒体的重组和线粒体蛋白parkin的募集。这个赠款时期的目标 阐明触发CIA和ADA的机制及其下游效应。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（无细胞重构，模型脂质上纯化蛋白的分析 膜）。要问的问题包括以下内容。 1）如何通过增加钙激活INF2？ 2）如何通过肌球蛋白II和Fascin组织到网络中INF2聚合的细丝？ 3）中央情报局如何 与已知的线粒体裂变蛋白（如MFF和DRP1）的接口以刺激裂变？ 4）PKC如何 在ADA期间，AMPK分别激活RAC和CDC42？ 5）ARP2/3复合物和FMNL形式如何 在ADA期间一起工作？ 6）ADA如何刺激糖酵解？这些问题是针对基本的 机械问题对各种哺乳动物细胞很重要，并占据了令人兴奋的边界 细胞骨架生物学，线粒体生物学和代谢。 1