Investigations of human myosin VI targeting using optogenetically-controlled cargo loading

Abstract

Myosins play countless critical roles in the cell, each requiring it to be activated at a specific location and time. To control myosin VI with this specificity, we created a novel optogenetic tool for activating myosin VI by fusing the light-sensitive Avena sativa phototropin1 LOV2 domain to a peptide from Dab2 (LOVDab), a myosin VI cargo protein. Our approach harnesses the native targeting and activation mechanism of myosin VI, allowing direct inferences on myosin VI function. LOVDab robustly recruits human full length myosin VI to various organelles in vivo and hinders peroxisome motion in a light-controllable manner. LOVDab also activates myosin VI in an in vitro gliding filament assay. Our data suggest that protein and lipid cargoes cooperate to activate myosin VI, allowing myosin VI to integrate Ca2+, lipid, and protein cargo signals in the cell to deploy in a site-specific manner.\n\nSignificance StatementMyosins are a broad class of motor proteins that generate force on actin filaments and fulfill contractile, transport, and anchoring roles. Myosin VI, the only myosin to walk toward the pointed end of actin filaments, is implicated in cancer metastasis and deafness. Intriguingly, myosin VI may play both transport and anchoring roles, depending upon where it is activated in the cell. Here we develop an optogenetic tool for studying myosin VI activation with high spatial and temporal resolution. Our approach photoactivates unmodified myosin VI through its native cargo pathway, enabling investigation of motor function and activation partners with minimal perturbation. This approach allows us to detect how and where myosin VI integrates multiple protein and second messenger signals to activate.