Mitochondrial positioning regulates redox-signaling during cell migration

线粒体定位调节细胞迁移过程中的氧化还原信号

• 批准号: 10520211
• 负责人: Brian Cunniff
• 金额: $ 33.08万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-15 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10520211
• 关键词: Actins; Active Biological Transport; Adaptor Signaling Protein; Adhesions; Architecture; Automobile Driving; BCAR1 gene; Bioenergetics; Bypass; Cell physiology; Cell-Matrix Junction; Cells; Complex; Consumption; Cysteine; Cytoplasm; Cytoskeleton; Data; Embryo; Event; Exhibits; Fibroblasts; Focal Adhesions; Frustration; Gene Expression; Gene Expression Profile; Genes; Hydrogen Peroxide; Label; Laboratories; Maps; Mediating; Membrane; Microtubules; Mitochondria; Mitochondrial Membrane Protein; Molecular; Motor; Movement; Mus; N-Cadherin; Nuclear; Outer Mitochondrial Membrane; Oxidation-Reduction; PTPN12 gene; Peripheral; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Population; Positioning Attribute; Post-Translational Protein Processing; Process; Proteins; Publishing; Reactive Oxygen Species; Reporting; Role; Signal Transduction; Signaling Molecule; Site; Source; Structure; Supporting Cell; Testing; Time; Vinculin; cell motility; cofilin; density; experimental study; extracellular; insight; link protein; live cell imaging; migration; mimetics; mutant; oxidation; protein function; protein structure; trafficking; transcriptome sequencing

PROJECT SUMMARY Mitochondria are strategically trafficked throughout the cell by the action of adapter proteins, microtubule motors and the actin cytoskeleton. The intracellular positioning of mitochondria supports subcellular levels of ATP, CA2+ and reactive oxygen species (ROS, i.e. hydrogen peroxide, H2O2). We have provided direct evidence that mitochondria actively traffic into the leading edge of migrating cells to support phenotypes associated with cell migration. Deletion of the mitochondrial adapter protein Miro1 leads to perinuclear restriction of mitochondria in mouse embryonic fibroblasts, leaving the cell periphery devoid of mitochondria. Importantly, cells lacking Miro1 retain normal mitochondrial bioenergetics. Our laboratory has shown that deletion of Miro1 disrupts subcellular energy gradients, focal adhesion (FA) dynamics and significantly reduces cell migration. Cell migration is rescued when Miro1 is reintroduced into cells lacking Miro1. However, the specific signaling events supporting cell migration that are governed by local mitochondrial populations are still unclear. Our preliminary data provides strong evidence that mitochondrial distribution dictates subcellular H2O2 gradients and therefore perinuclear restriction of mitochondria in cells lacking Miro1 compromises leading edge H2O2 levels. H2O2 acts as a signaling molecule, oxidizing specific cysteine residues in target proteins, influencing protein structure and function, a process termed redox dependent signaling. H2O2 is rapidly consumed at sites proximal to the source (mitochondria) and therefore must be produced in close proximity to the target. We find the oxidation of key cysteine residues in proteins driving FA dynamics and cell migration are significantly less oxidized in cells lacking Miro1. Addition of H2O2 to the extracellular milieu partially rescues cell migration phenotypes in cells lacking Miro1. Therefore, we hypothesize that Miro1-mediated subcellular positioning of mitochondria induces localized redox-dependent signaling events to support cytoskeleton and FA dynamics. We will test this hypothesis by generating a spatial and temporal map of subcellular H2O2 levels dependent on Miro1-mediated mitochondrial positioning in relationship to FA and cytoskeleton dynamics during cell attachment and migration. We will investigate the importance of specific cysteine oxidation events dependent on Miro1-mediated mitochondrial trafficking in supporting protein phosphorylation and FA and cytoskeleton dynamics. Lastly, we will investigate how the subcellular architecture of mitochondria influences gene-expression patterns, with an emphasis on cell migration genes. At the end of the proposed studies, we will have established a detailed mechanistic relationship between the subcellular trafficking of mitochondria and redox-dependent signaling events governing gene expression, protein post-translational modifications and FA and cytoskeleton dynamics during cell attachment and migration.

项目概要 线粒体通过衔接蛋白、微管马达的作用在整个细胞中进行战略性运输 和肌动蛋白细胞骨架。线粒体的细胞内定位支持 ATP、CA2+ 的亚细胞水​​平 和活性氧（ROS，即过氧化氢，H2O2）。我们已经提供了直接证据表明 线粒体主动进入迁移细胞的前缘以支持与细胞相关的表型 迁移。线粒体衔接蛋白 Miro1 的缺失会导致线粒体的核周限制 小鼠胚胎成纤维细胞，使细胞外周没有线粒体。重要的是，缺乏 Miro1 的细胞 保留正常的线粒体生物能。我们的实验室表明，Miro1 的缺失会破坏亚细胞 能量梯度、粘着斑 (FA) 动力学并显着减少细胞迁移。细胞迁移是 当 Miro1 被重新引入缺乏 Miro1 的细胞时，该细胞被拯救。然而，具体的信令事件支持 由局部线粒体群控制的细胞迁移仍不清楚。我们的初步数据提供 强有力的证据表明线粒体分布决定了亚细胞 H2O2 梯度，因此决定了核周 缺乏 Miro1 的细胞中线粒体的限制会损害前沿的 H2O2 水平。 H2O2 充当信号 分子，氧化靶蛋白中的特定半胱氨酸残基，影响蛋白质结构和功能， 过程称为氧化还原依赖性信号传导。 H2O2 在靠近源头的地方被迅速消耗 （线粒体）因此必须在靠近目标的地方产生。我们发现key被氧化了 驱动 FA 动力学和细胞迁移的蛋白质中的半胱氨酸残基在缺乏 米罗1.在细胞外环境中添加 H2O2 可部分挽救缺乏细胞迁移表型的细胞 米罗1.因此，我们假设 Miro1 介导的线粒体亚细胞定位诱导局部化 氧化还原依赖性信号事件支持细胞骨架和 FA 动力学。我们将通过以下方式检验这个假设 生成依赖于 Miro1 介导的线粒体的亚细胞 H2O2 水平的空间和时间图 细胞附着和迁移过程中与 FA 和细胞骨架动力学相关的定位。我们将 研究依赖于 Miro1 介导的线粒体的特定半胱氨酸氧化事件的重要性 支持蛋白质磷酸化和 FA 以及细胞骨架动力学的运输。最后，我们将调查 线粒体的亚细胞结构如何影响基因表达模式，重点是细胞 迁移基因。在拟议的研究结束时，我们将建立详细的机制关系 线粒体的亚细胞运输与氧化还原依赖性信号事件控制基因之间 细胞附着过程中的表达、蛋白质翻译后修饰以及 FA 和细胞骨架动力学 和迁移。