Motoneuron mortality in neurodegenerative diseases induced by homeostatic dysregulation of excitability

Here we seek to understand the mechanism by which mitochondria adapt the machinery of cellular motors in order to move within the cell - thereby enabling, for example, neuronal mitochondria to transport along axons to locations requiring increased energy density, or, during cell division, mitochondria segregation to each daughter cell. Our specific focus is the mitochondrial outer membrane protein, Miro, which appears to serve as a structural nexus to adapt molecular motors for mitochondria-as-cargo, and as a regulatory nexus for small molecule (Ca2+, GTP) modulation of mitochondrial motility. Miro comprises a pair of distinct GTPase domains which sandwich a pair of EF-hand-like domains. While cell-biological approaches have shown that each of the four domains of Miro plays some role in Miro function, the extrapolation of 'role' to mechanism remains fraught in the absence of a detailed understanding of the structural basis for their function. At the center of the puzzle are the roles of calcium and GTP. We have previously determined the crystal structures of each of the functional domains of Miro. However, critically, these structures of the isolated Miro have not revealed significant structural changes in response to Ca2+ or GTP binding. That is, the small-molecule regulatory mechanisms are almost certainly dependent on the structural context(s) of the interactions between Miro and its auxiliary adapter proteins and the motors themselves. We hypothesize that the GTPase domains function not as canonical 'switches', but rather provide an 'assembly-activation' mechanism to promote formation of mitochondrial transport complexes, and we hypothesize that the quiescent Ca2+ sites only become functionally responsive to [Ca2+] after formation of a transport complex. To test these ideas we seek to develop a detailed structural, biochemical and biophysical understanding of the interactions between Miro and its known binding partners, focusing initially on Milton (TRAK1/2), kinesin, and Cenp-F. We will determine the structural basis for Miro's macromolecular interactions using a variety of biophysical and biochemical approaches, including fluorescence spectroscopy, luminescence spectroscopy, ITC, and X-ray crystallography. Critical to understanding the biological role of Ca2+ or GTP as regulators of mitochondrial motility will be to understand how their regulatory interactions change within the context of functional macromolecular assemblies. To that end, we will characterize how binding of these small molecule regulators modulate and are modulated by Miro complex assembly, using kinetics studies and luminescence spectroscopy in addition to structural biological methods. As work under this project matures, we anticipate developing collaboratory mutagenesis and in vivo studies testing new hypotheses that should illuminate the biological mechanisms of Miro as an adapter for mitochondrial motility.