Mitochondrial positioning regulates redox-signaling during cell migration

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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.