Binding partners regulate unfolding of myosin VI to activate the molecular motor

Myosin VI is the only minus-end actin motor and is coupled to various cellular processes ranging from endocytosis to transcription. This multi-potent nature is achieved through alternative isoform splicing and interactions with a network of binding partners. How the regulation mechanism of myosin VI varies between different isoforms and binding partners remains unexplored. Here, we have compared the regulation of two myosin VI splice isoforms by two different binding partners. By combining biochemical and single-molecule approaches, we propose that myosin VI regulation follows a generic mechanism, independently of the spliced isoform and the binding partner involved. We describe how myosin VI adopts an autoinhibited backfolded state which is unfolded by binding partners. This unfolding activates the motor and can subsequently trigger dimerization. We have further expanded our study by using live single molecule imaging to investigate the impact of binding partners upon myosin VI molecular organisation and dynamics. Overall, binding partners determine myosin VI function.