Final Report Summary - MITOTRAFFICBYMIRO (Differential role of atypical Rho GTPases Miro-1 and Miro-2 for controlling mitochondrial dynamics and transport) Mitochondria are the powerhouse of cells, generating cellular energy in the form of ATP which can in turn drive key cellular functions including ion pumping, cellular trafficking, and communication. Mitochondria are trafficked around the cell to locations where they are needed with the help of motor proteins and adaptors found on the outer mitochondrial membrane. The outer mitochondrial membrane Miro GTPases form complexes with dynein and kinesin microtubule motor proteins and their TRAK adaptors to regulate mitochondrial transport and distribution in metazoans. In lower eukaryotes, Miro exists as a single orthologue important for proper mitochondrial inheritance and viability. Biochemical interactions between Miro proteins and several trafficking partners and mitochondrial fusion/fission machinery are well characterised. However surprisingly little is known about the distribution of Miro proteins on mitochondrial outer membranes and their interactions with other key mitochondrial complexes. We aimed to shed new light on the molecular mechanisms that allow Miro proteins to regulate mitochondrial distribution, morphology and function, in addition to interaction with other key organelles such as the endoplasmic reticulum. Identification of novel Miro interacting partners Using Mass spectrometric and co-immunoprecipitation approaches we identified new Miro protein interacting partners including interactions with components of the Mitochondrial Inner membrane Organizing System (MINOS) complex such as CHCHD3 and SAM50. Blue-Native PAGE and western blotting revealed endogenous Miro protein may exist as dimers and can also be found in higher order complexes with similar molecular weights to those of MINOS components. Nanoscopic localisation of Miro proteins on the outer mitochondrial membraneWe optimized super resolution imaging approaches including stochastic optical reconstrucion microscopy (STORM) and structured illumination microscopy (SIM) to demonstrate the nanoscale organization and protein complex formation of Miro proteins on the outer mitochondrial membrane. Super resolution imaging revealed Miro complexes form nanoclusters on the outer mitochondrial membrane that can contain Miro homodimers and heterodimers. Moreover STORM imaging revealed that subpopulations of Miro clusters can colocalize with MINOS complex components.Miro as a linker between mitochondrial ER-contacts and the mitochondrial inner membraneMitochondria show physical interaction with endoplasmic reticulum (ER) through dedicated protein complexes at mitochondria-ER contact sites. Using deconvolution microscopy and STORM imaging on HeLa cells we found that some Miro nanoclusters can be found aligned with ER tubules. Moreover, Miro loss of function studies revealed that in the absence of Miro proteins, contacts between mitochondria and ER are disrupted leading to altered calcium transfer between the two organelles. Miro proteins were also found to interact with the schizophrenia associated protein DISC1. Super resolution microscopy revealed that DISC1 is localized to endoplasmic reticulum contact sites and that altering DISC1 function decreases endoplasmic reticulum-mitochondria contact area. Thus Miro proteins may link MINOS components to contact sites with endoplasmic reticulum and may act as organizers of molecular complexes spanning from the ER to the mitochondrial inner membrane to facilitate calcium transfer from ER stores to the mitochondrial matrix or other processes such as lipid transfer between the two organelles.
