Myo19 tethers mitochondria to endoplasmic reticulum-associated actin to promote mitochondrial fission.

Mitochondrial homeostasis requires a dynamic balance of fission and fusion. The actin cytoskeleton promotes fission; we find that the mitochondrially-localized myosin, Myosin 19 (Myo19), is integral to this process. Myo19 knock-down induces mitochondrial elongation, while Myo19 overexpression induces fragmentation. This mitochondrial fragmentation is blocked by a Myo19 mutation predicted to inhibit ATPase activity and strong actin binding but not by mutations predicted to affect the motor's working stroke that preserve ATPase activity. Super-resolution imaging indicates a dispersed localization of Myo19 on mitochondria, which we find to be dependent on metaxins. These observations suggest that Myo19 acts as a dynamic actin-binding tether that facilitates mitochondrial fragmentation. Myo19-driven fragmentation is blocked by depletion of either the endoplasmic reticulum (ER)-anchored formin INF2-CAAX or the mitochondrially-localized F-actin nucleator Spire1C, which together polymerize actin at sites of mito-ER contact for fission. These observations imply that Myo19 promotes fission by stabilizing mito-ER contacts; we used a split-luciferase system to demonstrate a reduction in these contacts following Myo19 depletion. Our data support a model in which Myo19 tethers mitochondria to ER-associated actin to promote mitochondrial fission.