Myosin Va Motor Teams Navigate Vesicle Cargos through 3D Actin Filament Intersections

Abstract

Myosin Va (MyoVa) motor teams collectively move intracellular cargo through a complex, three dimensional (3D), actin meshwork. To address how MyoVa motors navigate this physical challenge, we imaged fluorescently labeled 350nm synthetic lipid vesicles, bound with ∼10 MyoVa HMM motors, moving through 3D actin filament intersections strung between 3µm beads. Intersections were formed by filaments being ∼90 degrees to one another and separated by 30-250nm. As the vesicle surface contacted the intersecting actin filaments, two regions of potential motor engagement were created on the vesicle surface. Geometric constraints limited the engaged motor number at each contact point to no more than 3, as confirmed by laser trap stall force measurements. These points of vesicle-actin filament contact resulted in an effective tug of war between two motor teams that were physically separated on the vesicle surface. The vesicle's directional outcome at an intersection was visualized using high spatial and temporal 3D super resolution imaging. For vesicles that physically contacted both actin filaments at the intersection, 55% of vesicles continued straight through the intersection, 35% switched filaments, while only 10% terminated. These outcomes were successfully simulated by a model in which the directional outcome was dependent on the interfilament gap, the vesicle's azimuthal approach angle to the intersection, the stochastic attachment and detachment of motors within a team (i.e. a team's force generation), and the Brownian motion of the vesicle while attached to the actin filaments. All of these physical factors contributed to which motor ensemble won the tug of war at the intersection and thus the vesicle's directional outcome. This simplified biomimetic system and resultant data provide an experimental framework for understanding how MyoVa motor teams navigate their cargo through evermore complex 3D actin networks that exist within cells.