Spatio-temporal assembly of respiratory chain complexes RCCs in mitochondrial fusion and fission dynamics
Biochemistry
Final Report Abstract
Mitochondria are highly dynamic organelles that fuse, divide and move through cells. At the protein level, proteins assemble dynamically into super complexes, most likely in the course of short-term adaptive processes. This dynamic is essential to maintain mitochondrial morphology, functionality and plasticity. A result of mitochondrial fusion and fission dynamics is the formation of hybrid mitochondria. The mixing of components during these events was discussed in the context of mitochondrial quality control, with the hypothesis that mixing prevents accumulation of damaged compounds. In order to better understand the significance of mitochondrial dynamics, we have followed the dissemination of fluorescence-labelled complexes of the respiratory chain (RCC) from different mitochondria. The mixing dynamics were documented by fluorescence microscopy, two-color high-resolution microscopy and immuno-EM. We found out that the limited diffusion of single respiratory chain complexes in dynamic mitochondria delays the exchange between the cristae compartments and creates a mosaiclike distribution. Our data thus suggest that the composition of cristae mainly remains preserved during the fusion of mitochondria and that cristae with mixed RCC are formed only slowly and by repetitive fusion and division events. Apparently RCC proteins are trapped in cristae due to their specific ultrastructures. We were able to demonstrate this in detail by quantifying the spatiotemporal behaviour of F1FO-ATP synthase. In addition, the dimer to monomer ratio determines the mobility and distribution of ATP synthase. This in turn is adjusted by the relative proportion of certain subunits of the F1FO-ATP synthase, Su e and IF1. The extent to which metabolic conditions influence these values will have to be investigated in future studies. The composition of RCC into supercomplexes (SCs) was investigated in the context of the functional plasticity of mitochondria. For the first time, we were able to monitor SC formation in living cells using FRET/FLIM approaches. To this end, subunits of the RCC were fused with fluorescent donor and acceptor proteins, taking into account a spatially close localization within the supercomplex. We were able to detect FRET between the labelled subunits of CIII and CIV, which can be explained by CIII2/CIV or CI/CIII2/CIV supercomplex formation. In addition, SC formation was induced using the FRB-FKBP- rapamyin system. Finally, we were able to demonstrate SC formation based on fluorescence lifetime image microscopy (FLIM) with a single fluorescence sensor. For this purpose, the sensor was positioned at the interface between CIII and CIV. This was made possible by the fact that the fluorescence lifetime of fluorescent proteins depends on the nano-environment, which is characterized by a high density of molecules in supercomplexes. The many possible dipole-dipole interactions reduce the fluorescence lifetime of the sensor protein. These techniques allowed us to show changes in SC formation depending on the presence and absence of SC assembly factors and are currently used to monitor SC formation depending on metabolic conditions.
Publications
- (2013). Restricted diffusion of OXPHOS complexes in dynamic mitochondria delays their exchange between cristae and engenders a transitory mosaic distribution. Journal of Cell Science 126, 103-116
Wilkens V, Kohl W, and Busch K
(See online at https://doi.org/10.1242/jcs.108852) - (2014) Lateral pH-gradient between OXPHOS proton-pump and FOF1-ATP-synthase in folded cristae membranes of active mammalian mitochondria. Nature communications 5:3103
Rieger B, Junge W, Busch KB
(See online at https://doi.org/10.1038/ncomms4103) - (2016). Probing of protein localization and shuttling in mitochondrial microcompartments by FLIM with sub-diffraction resolution. Biochim Biophys Acta 1857, 1290-1299
Sohnel AC, Kohl W, Gregor I, Enderlein J, Rieger B, and Busch, KB
(See online at https://doi.org/10.1016/j.bbabio.2016.03.021) - (2017) Lifetime imaging of GFP at CoxVIIIa reports respiratory supercomplex assembly in live cells. Sci Rep 7, 46055
Rieger B, Shalaeva DN, Söhnel A-CC, Kohl W, Duwe P, Mulkidjanian AY & Busch KB
(See online at https://doi.org/10.1038/srep46055)
