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.
BackgroundMitochondria, the main suppliers of cellular energy, are dynamic organelles that fuse and divide frequently. Constraining these processes impairs mitochondrial is closely linked to certain neurodegenerative diseases. It is proposed that functional mitochondrial dynamics allows the exchange of compounds thereby providing a rescue mechanism.Methodology/Principal FindingsThe question discussed in this paper is whether fusion and fission of mitochondria in different cell lines result in re-localization of respiratory chain (RC) complexes and of the ATP synthase. This was addressed by fusing cells containing mitochondria with respiratory complexes labelled with different fluorescent proteins and resolving their time dependent re-localization in living cells. We found a complete reshuffling of RC complexes throughout the entire chondriome in single HeLa cells within 2–3 h by organelle fusion and fission. Polykaryons of fused cells completely re-mixed their RC complexes in 10–24 h in a progressive way. In contrast to the recently described homogeneous mixing of matrix-targeted proteins or outer membrane proteins, the distribution of RC complexes and ATP synthase in fused hybrid mitochondria, however, was not homogeneous but patterned. Thus, complete equilibration of respiratory chain complexes as integral inner mitochondrial membrane complexes is a slow process compared with matrix proteins probably limited by complete fusion. In co-expressing cells, complex II is more homogenously distributed than complex I and V, resp. Indeed, this result argues for higher mobility and less integration in supercomplexes.Conclusion/SignificanceOur results clearly demonstrate that mitochondrial fusion and fission dynamics favours the re-mixing of all RC complexes within the chondriome. This permanent mixing avoids a static situation with a fixed composition of RC complexes per mitochondrion.
The assembly of respiratory complexes into macromolecular supercomplexes is currently a hot topic, especially in the context of newly available structural details. However, most work to date has been done with purified detergent-solubilized material and in situ confirmation is absent. We here set out to enable the recording of respiratory supercomplex formation in living cells. Fluorescent sensor proteins were placed at specific positions at cytochrome c oxidase suspected to either be at the surface of a CI1CIII2CIV1 supercomplex or buried within this supercomplex. In contrast to other loci, sensors at subunits CoxVIIIa and CoxVIIc reported a dense protein environment, as detected by significantly shortened fluorescence lifetimes. According to 3D modelling CoxVIIIa and CoxVIIc are buried in the CI1CIII2CIV1 supercomplex. Suppression of supercomplex scaffold proteins HIGD2A and CoxVIIa2l was accompanied by an increase in the lifetime of the CoxVIIIa-sensor in line with release of CIV from supercomplexes. Strikingly, our data provide strong evidence for defined stable supercomplex configuration in situ.
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