Mitochondrial positioning regulates redox-signaling during cell migration

项目总结 线粒体通过适配蛋白、微管马达的作用而战略性地在细胞内运输 和肌动蛋白细胞骨架。线粒体在细胞内的定位支持亚细胞水平的ATP、Ca2+ 和活性氧物种(ROS，即过氧化氢、过氧化氢)。我们已经提供了直接证据 线粒体主动进入迁移细胞的前沿以支持与细胞相关的表型 迁移。线粒体适配蛋白Miro1缺失导致线粒体核周受限 小鼠胚胎成纤维细胞，细胞外围没有线粒体。重要的是，缺少Miro1的细胞 保留正常的线粒体生物能量。我们的实验室已经证明，Miro1的缺失会扰乱亚细胞 能量梯度、焦点粘连(FA)动力学，并显著减少细胞迁移。细胞迁移是 当Miro1被重新引入到缺少Miro1的细胞中时被拯救。然而，支持的特定信令事件 由当地线粒体群体控制的细胞迁移仍然不清楚。我们的初步数据提供了 线粒体分布决定亚细胞内H_2O_2梯度从而决定核周的有力证据 限制缺少Miro1的细胞中的线粒体会影响最前沿的过氧化氢水平。双氧水起信号的作用 分子，氧化靶蛋白中特定的半胱氨酸残基，影响蛋白质结构和功能 这一过程称为氧化还原依赖信号传递。过氧化氢在污染源附近迅速被消耗。 (线粒体)，因此必须在离目标很近的地方产生。我们发现KEY的氧化 在缺乏细胞的情况下，驱动FA动态和细胞迁移的蛋白质中的半胱氨酸残基被氧化得明显较少 Miro1.在细胞外环境中加入过氧化氢部分挽救缺乏细胞的细胞迁移表型 Miro1.因此，我们假设miro1介导的线粒体亚细胞定位导致局部 依赖氧化还原的信号事件，以支持细胞骨架和FA动力学。我们将通过以下方式验证这一假设 生成依赖于Miro1介导的线粒体的亚细胞过氧化氢水平的空间和时间地图 在细胞附着和迁移过程中定位与FA和细胞骨架动力学的关系。我们会 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、FA和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 迁徙基因。在拟议的研究结束时，我们将建立一个详细的机制关系 线粒体亚细胞运输与氧化还原依赖的信号事件之间的关系 表达、蛋白质翻译后修饰以及细胞附着过程中FA和细胞骨架的动态变化 和迁徙。

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.