In mammals, there are two Miro family members, Miro1 and Miro2, that share ~60% sequence identity, comprising two GTPase domains flanking two EF-hand Ca 2+-binding domains and a C-terminal transmembrane domain that targets them to the OMM 10. In yeast, Miro exists as a single orthologue, Gem1, important for correct mitochondrial inheritance and cellular viability 8, 9. Recently, an actin-dependent transport of the mitochondria has also been linked to Miro regulation through the recruitment and stabilization of the mitochondrial myosin 19 (Myo19) to the outer mitochondrial membrane (OMM) 6, 7. Miro proteins form complexes with the TRAK adaptors and dynein/kinesin motors to regulate the microtubule-dependent transport of the mitochondria 4, 5. The mitochondrial population are trafficked to where they are needed to meet local energy and Ca 2+ buffering demands 3.
Mitochondria generate ATP to drive key cellular functions, including ion pumping, intracellular trafficking and cellular signaling cascades 1, 2. The Miro nanoscale organization, association with MICOS complex and regulation of ERMCS reveal new levels of control of the Miro GTPases on mitochondrial functionality. Further, we show that Miro couples MICOS to TRAK motor protein adaptors to ensure the concerted transport of the two mitochondrial membranes and the correct distribution of cristae on the mitochondrial membrane. Using knockout mouse embryonic fibroblasts we show that Miro1 and Miro2 are required for normal mitochondrial cristae architecture and Endoplasmic Reticulum-Mitochondria Contacts Sites (ERMCS). Here, using super-resolution fluorescence microscopy, we report that Miro proteins form nanometer-sized clusters along the mitochondrial outer membrane in association with the Mitochondrial Contact Site and Cristae Organizing System (MICOS). However, their sub-mitochondrial localization and relationship with other critical mitochondrial complexes remains poorly understood. Mitochondrial Rho (Miro) GTPases localize to the outer mitochondrial membrane and are essential machinery for the regulated trafficking of mitochondria to defined subcellular locations.