Specific Protein Isoforms of Miro and Milton Have Divergent Roles Controlling Mitochondrial Transport, Fusion & Fission, and Health

Abstract

Neurons depend on mitochondria to produce ATP, regulate calcium signaling/homeostasis, and control apoptosis. Motor proteins of the kinesin and dynein families actively transport mitochondria through axons. Miro and Milton are two proteins responsible for linking kinesin to mitochondria, and Miro regulates dynein-based transport. Drosophila Miro (dMiro) has three isoforms (dMiro-M, -L, and -S). Milton has four: Milton-A, -B, -C, and -D. To study the role of the dMiro isoforms, we expressed a transgene of each in a dmiro null background, and examined their phenotypic effects on mitochondrial distribution, transport, and morphology in motor neuron axons by using live time-lapse imaging of GFP-tagged mitochondria. The potential role of the Milton-A isoform was examined by chronically or acutely overexpressing (OE) the transgene in motor neurons. We found that Milton-A OE causes a massive loss of mitochondria within 20 hours. In dMiro-L and -S rescues, compared to wildtype, there was decrease the total mitochondrial density, but not the percent of motile mitochondria. To determine components of the signaling pathway controlled by Miro-L and -S, we performed a genetic screen to isolate dominant suppressor mutations of the lethal overexpression phenotypes of dMiro-L and -S. Many suppressor mutations were isolated and mapped to specific chromosomes.