Restricted diffusion of OXPHOS complexes in dynamic mitochondria delays their exchange between cristae and engenders a transitory mosaic distribution

Abstract

SummaryMitochondria are involved in cellular energy supply, signaling and apoptosis. Their ability to fuse and divide provides functional and morphological flexibility and is a key feature in mitochondrial quality maintenance. To study the impact of mitochondrial fusion/fission on the reorganization of inner membrane proteins, oxidative phosphorylation (OXPHOS) complexes in mitochondria of different HeLa cells were tagged with fluorescent proteins (GFP and DsRed-HA), and cells were fused by polyethylene glycol treatment. Redistribution of the tagged OXPHOS complexes was then followed by means of immunoelectron microscopy, two color super-resolution fluorescence microscopy and single molecule tracking. In contrast to outer membrane and matrix proteins, which mix quickly and homogeneously upon mitochondrial fusion, the mixing of inner membrane proteins was decelerated. Our data suggest that the composition of cristae is preserved during fusion of mitochondria and that cristae with mixed OXPHOS complexes are only slowly and successively formed by restricted diffusion of inner membrane proteins into existing cristae. The resulting transitory mosaic composition of the inner mitochondrial membrane illuminates mitochondrial heterogeneity and potentially is linked to local differences in function and membrane potential.

Superresolution microscopy reveals spatial separation of UCP4 and F 0 F 1 -ATP synthase in neuronal mitochondria

“…As a result of both the membrane proteins' activity and proton surface to bulk release, the local surface proton concentration varies along the membrane (32,33). It implies that proteins colocalized with the proton pumps on the CM (e.g., ATP synthase) (34,35) will experience the highest pmf, whereas the pmf is much lower for proteins localized at the IBM (e.g., UCP4). Fig.…”

Quality matters: how does mitochondrial network dynamics and quality control impact on mtDNA integrity?

“…The experimental results presented here that demonstrate the restriction of nucleoids and respiratory chain complexes to nearby matrix space or individual mitochondrial cristae [39] renders some of the original assumptions of the organelle control theory unnecessary. The experiments indicate that no direct connection among the OXPHOS complexes and mtDNA molecules is necessary to create a genotype-phenotype link.…”

“…OXPHOS complexes showed a patchy distribution in cell fusion assays with OXPHOS-GFP and OXPHOS-RFP from different sources [62]. A more detailed study revealed that the reason for the retarded mixing is most likely based on the fact that cristae are preserved during fusion and fission and proteins in the cristae membranes are limited in their diffusion [39,63]. It was similarly shown that cristae architecture limits matrix diffusion [63].…”

“…It has been suggested that nucleoids contain multiple copies of mtDNA [36], but recent work using superresolution microscopy indicates that each nucleoid contains on average only a single mtDNA copy in various cell types [37]. Furthermore, the confinement of OXPHOS complexes to single cristae owing to their restricted diffusion adds to the focal perspective [38,39]. Together, this would suggest that nucleoids containing a deleterious mutant mtDNA might, indeed, express a focal OXPHOS phenotype allowing it, in principal, to be selectively degraded via fission/ mitophagy.…”

Lateral pH gradient between OXPHOS complex IV and F0F1 ATP-synthase in folded mitochondrial membranes

“…The proton pumps and the proton-driven ATP synthase can be segregated. Complexes I-IV are mainly found in the flat sheet membrane 6,7 , whereas immuno-EM and EM-tomography have revealed ribbons of F 0 F 1 dimers lining the highly curved rim [8][9][10][11][12] . These ribbons seem to be involved in folding the crista membrane.…”