Myosin Va Vesicular Transport Is Modulated By Actin Filament Density, Orientation, And Polarity In An In Vitro 3d Actin Network

The cell's dense three dimensional (3D) actin filament network presents an obstacle course that physically and directionally challenges vesicular transport via teams of Myosin Va (MyoVa) motors. Complicating matters is that actin filament density, orientation and polarity within such networks can vary within a cell. To define how a MyoVa team maneuvers its cargo through such complex networks, we created actin filament networks in vitro that were suspended from silica structures and then characterized the 3D trajectories of fluorescent, 350nm fluid-like, lipid vesicles transported by a MyoVa team (∼10 motors). Every filament's 3D spatial orientation was defined using STORM microscopy as was its polarity (plus ends), as reported by the movement of single MyoVa. Model networks of randomly oriented actin filaments were assembled as well as branched actin networks using the Arp2/3 complex to create an inherent plus end polarity bias. For every vesicle, its modes of motion (i.e., stationary, diffusive-like, directed) and the spatial orientation/polarity for each filament the vesicle engaged during its trajectory were used to constrain an in silico model (Lombardo et al., 2017). The model predicts that the modes of vesicular motion result from tug-of-wars between motors on the vesicle surface that are engaged with different filaments. For example, directed vesicular transport is enhanced when filament polarity is biased, as in Arp2/3-based networks, where force vectors are predicted to be aligned and cooperative for engaged motors on separate filaments. As filament density in the network increases, a bias in filament polarity is even more critical for directed transport. This model transport system provides a broader platform to understand how cellular regulation of the cytoskeleton's architecture can be employed to fine-tune intracellular cargo transport.