scratch-migration-assay-dorsal-skinfold-chamber-for-vitro-vivo

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

Saarland University

Oxidation of cysteine residues to disulfides drives import of many proteins into the intermembrane space of mitochondria. Recent studies in yeast unraveled the basic principles of mitochondrial protein oxidation, but the kinetics under physiological conditions is unknown. We developed assays to follow protein oxidation in living mammalian cells, which reveal that import and oxidative folding of proteins are kinetically and functionally coupled and depend on the oxidoreductase Mia40, the sulfhydryl oxidase augmenter of liver regeneration (ALR), and the intracellular glutathione pool. Kinetics of substrate oxidation depends on the amount of Mia40 and requires tightly balanced amounts of ALR. Mia40-dependent import of Cox19 in human cells depends on the inner membrane potential. Our observations reveal considerable differences in the velocities of mitochondrial import pathways: whereas preproteins with bipartite targeting sequences are imported within seconds, substrates of Mia40 remain in the cytosol for several minutes and apparently escape premature degradation and oxidation.

Anouar Belkacemi

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JoVE Journal Publications

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