Engineering LOV2 for the Control and Study of Motor Proteins

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 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. We show for the first time that protein and lipid cargoes cooperate to activate myosin VI, supporting a new model of myosin VI regulation whereby myosin VI integrates Ca2+, lipid, and protein cargo signals in the cell to deploy in a site-specific manner.