An Optogenetic Method for Controlling Full-Length Myosin VI through its Cargo Binding Domain

Abstract

Motor proteins are force-producing proteins that play countless critical roles in the cell, each requiring them to be activated at a specific location and time. Therefore, if researchers could control motor proteins with the same specificity they could much better understand motor protein function and mechanical processes in the cell. A promising technique for doing this is optogenetics, which involves the engineering of optically gated, genetically encoded protein tools that activate biomolecules upon absorption of light. One available method for putting proteins under optogenetic control is through coupling with the light-sensitive LOV2 domain. LOV2 is a 150 AA region in Avena sativa phototropin1 that undergoes a conformational change involving the unfolding of its terminal helices upon absorbing blue light. This conformational change can be engineered to control the binding of a peptide fused to the LOV2 C-terminus in a light-dependent manner.Here we present our results of controling full-length myosin VI activity using a LOV2 with a C-terminal fusion to a peptide derived from Dab2 (LOV2-Dab2pep), an adaptor protein that recruits and activates myosin VI in vivo. Because LOV2-Dab2pep activates myosin VI through a single event involving the dimerization of the cargo binding domain, our approach can be applied to activating endogenous myosin VI. Thus, this strategy is appealing because it harnesses the native activation mechanism of myosin VI and it avoids the use of a constitutively active forced-dimer that can interfere with normal cell function. We are able to demonstrate light-dependent activation of processive motion of myosin VI on actin in an in vitro gliding filament assay, as well as reversible recruitment of myosin VI in vivo.