Load dependent properties of a single molecular motor determines ensemble behavior

Project Summary Many complex cellular processes from muscle contraction to vesicular transport are driven, not by a single myosin motor, but by the coordinated action of teams of motors. A key unanswered question is how do single motors couple and coordinate their activity in ensembles to give rise to these complex cellular processes? This knowledge gap is preventing a detailed molecular understanding of a host of intracellular processes, as well as the development of therapies for diseases that result from the dysfunction of these processes. A widely held view is that teams of myosin molecules coordinate their activity through their ability to sense the magnitude and direction of an applied load. However, this idea has not been fully, nor directly, tested in part because myosin’s biochemical and mechanical transitions occur faster than most existing technologies can resolve. Thus, it is still not clear which step or steps in the cross-bridge cycle are load sensitive, and what the specific nature of these load-sensitivities are. To gain this knowledge I have developed novel laser trap assay methods and analyses with the necessary spatial and time resolution to directly characterize the load-sensitive steps in myosin’s mechanochemical cycle. These data will be used to develop molecular models of single molecule behavior under a variety of conditions. I then use the mini-ensemble laser trap assay to directly characterize how ensembles of motors work together to generate force and motion, providing the first direct test of our models and the information needed to refine and improve the models. To date I have focused primarily on muscle myosin II, but recently I have begun taking similar approach to characterize the behavior of the processive motor, myosin Va. And I now plan to expand this approach to investigate the two other classes of myosin V motors (Vb and Vc) and myosin III to better understand the complex processes that they drive within the cell. If this work is successful the information will transform our understanding of molecular motors by providing the first complete molecular explanation of how these prototypical molecular motors coordinate their activity. This will then provide crucial insight into how these motors drive many complex cellular processes, and by doing so will reveal novel molecular targets for more effective therapies for a host of myosin associated diseases.