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Macromolecules of various sizes induce crowding of the cellular environment. This crowding impacts on biochemical reactions by increasing solvent viscosity, decreasing the water-accessible volume and altering protein shape, function, and interactions. Although mitochondria represent highly protein-rich organelles, most of these proteins are somehow immobilized. Therefore, whether the mitochondrial matrix solvent exhibits macromolecular crowding is still unclear. Here, we demonstrate that fluorescent protein fusion peptides (AcGFP1 concatemers) in the mitochondrial matrix of HeLa cells display an elongated molecular structure and that their diffusion constant decreases with increasing molecular weight in a manner typical of macromolecular crowding. Chloramphenicol (CAP) treatment impaired mitochondrial function and reduced the number of cristae without triggering mitochondrial orthodox-to-condensed transition or a mitochondrial unfolded protein response. CAP-treated cells displayed progressive concatemer immobilization with increasing molecular weight and an eightfold matrix viscosity increase, compatible with increased macromolecular crowding. These results establish that the matrix solvent exhibits macromolecular crowding in functional and dysfunctional mitochondria. Therefore, changes in matrix crowding likely affect matrix biochemical reactions in a manner depending on the molecular weight of the involved crowders and reactants.

OBJECTIVE: In patients with mitochondrial DNA (mtDNA) maintenance disorders and with aging, mtDNA deletions sporadically form and clonally expand within individual muscle fibers, causing respiratory chain deficiency. This study aimed to identify the sub-cellular origin and potential mechanisms underlying this process. METHODS: Serial skeletal muscle cryosections from patients with multiple mtDNA deletions were subjected to subcellular immunofluorescent, histochemical, and genetic analysis. RESULTS: We report respiratory chain-deficient perinuclear foci containing mtDNA deletions, which show local elevations of both mitochondrial mass and mtDNA copy number. These subcellular foci of respiratory chain deficiency are associated with a local increase in mitochondrial biogenesis and unfolded protein response signaling pathways. We also find that the commonly reported segmental pattern of mitochondrial deficiency is consistent with the three-dimensional organization of the human skeletal muscle mitochondrial network. INTERPRETATION: We propose that mtDNA deletions first exceed the biochemical threshold causing biochemical deficiency in focal regions adjacent to the myonuclei, and induce mitochondrial biogenesis before spreading across the muscle fiber. These subcellular resolution data provide new insights into the possible origin of mitochondrial respiratory chain deficiency in mitochondrial myopathy.