Mitochondria respiratory complexes and supercomplexes co-exist simultaneously in mammalian cells

Abstract The mitochondrial electron transport chain was understood as four respiratory complexes (RCs) independently moving in the mitochondrial inner membrane and electrons transferred between them by CoQ and cytochrome c1,2. The disruption of the mitochondrial membranes with mild detergents followed by blue native electrophoresis (BNGE), allowed visualization of RCs as independent entities, but also associated forming a variety of supercomplexes (SCs)1,3. This led to the proposal that RCs are not moving free entities but instead they super-assembly into structures, called supercomplexes (SCs) or respirasomes3. The use of detergents raised concerns on the existence of either free RCs or SCs in the cellular context4, and does not allow determining the relevance of additional SCs. To address this controversy, it was proposed the plasticity model that considered that both RCs and SCs co-existed in a dynamic organization1,5,6. Over the last 20 years the reality of respiratory supercomplexes was confirmed by functional3,6–8, genetic6,9 and structural10–15 studies. However, the co-existence of free RCs and SCs is still unanswered. We applied multi-color Stimulated Emission Depletion (STED) microscopy in whole cells in a panel of cell lines: mtDNA depleted cells (p0), complex III (CYTbM) or complex IV (COX10KO) depleted cell lines and their isogenic controls. We demonstrate the co-existence of RCs and SCs in large cell areas. Our observations support the plasticity organization model of the mitochondrial respiratory chain were RCs and SCs co-exist and reveal an unexpected heterogeneous distribution of RCs and SCs within mitochondria in-situ.