Role of Milton Domains in the Calcium-Dependent Regulation of Mitochondrial Motility

Abstract

The mammalian GRIF1 and OIP106, and the drosophila Milton are kinesin-binding proteins that form a complex with the Miro GTPase, an outer mitochondrial membrane EF-hand protein, to support the movement of mitochondria along the microtubules. Our study demonstrates that in H9c2 cells overexpressing OIP106 or GRIF1, the basal motility of mitochondria is increased, whereas the sensitivity to calcium-induced movement inhibition is decreased. To dissect the interaction between Milton, kinesin and Miro, three different Milton constructs were tested: Milton (1-450), the soluble domain of Milton; Milton (750-1116), lacking the kinesin heavy chain binding site and Milton (847-1116) that lacks additional ∼100 amino acid presumably containing part of the Miro binding site. Immunohistochemistry revealed that the overexpressed Milton (1-450) was cytoplasmic, whereas the other two Milton constructs showed mitochondrial localization. The basal mitochondrial motility was increased by Milton (750-1116) but was not altered by Milton (847-1116) or Milton (1-450). A plot of mitochondrial motility against slowly rising cytoplasmic [Ca2+] induced by thapsigargin (2μM), shows that overexpression of Milton (750-1116) significantly reduced the calcium sensitivity of mitochondrial motility reminiscent of OIP106 and GRIF1. By contrast, Milton (847-1116) or Milton (1-450) did not have any effect. The thapsigargin-induced cytoplasmic calcium signal was not affected by any of the Milton constructs. These data indicate that the C terminus of Milton is an important regulator of the mitochondria associated motors and is involved in conferring the calcium sensitivity from Miro to the motors. Milton 847-1116 is sufficient for the mitochondrial binding, whereas the 750-847 amino acids are critical for the control of calcium sensitivity of mitochondrial motility.