Probing Lipid Vesicle Transport in 3D by Teams of Myosin Va Motors at Suspended Actin Intersections in vitro

Abstract

In cells, myosin Va motor teams are forced to transport vesicles through a complex three-dimensional (3D) actin meshwork. To characterize how these motors navigate this physically challenging meshwork, we created actin filament intersections adhered to a coverslip surface and confronted a team of ∼10 myosin Va HMM motors carrying a 350nm synthetic lipid vesicle. When approaching the intersection on the lower filament, the motor-vesicle complex (MVC) switched to the upper intersecting filament with a 51% probability while crossing over the intersection with only a 33% probability. If approaching the intersection on the upper filament, the MVC preferred staying on the upper filament through the intersection and only switched to the lower filament with a 31% probability. These data suggest that the extent of MVC surface contact with the target binding zone of the intersecting actin filaments dictates the directional outcome. To build complexity, actin filaments were strung between 3μm beads, creating suspended filament intersections. The 3D spatial relations between the MVC and the individual filaments were determined by super-resolution STORM imaging. As the MVC approached the intersection, the complex spiraled around the actin filament with a pitch of ∼1.3µm. If the MVC was forced to navigate through the intersection and the separation between actin filaments (<100nm) was less than the vesicle diameter, the MVC initially paused and then switched to the intersecting filament. For this to occur, motors must be free to diffuse on the fluid lipid (DOPC) vesicle surface and engage an actin filament anywhere that the vesicle surface contacts an actin filament. This 3D model system with added complexity will provide an experimental platform to understand how myosin Va motor ensembles maneuver their cargo through the complex actin cytoskeletal network.