PROJECT SUMMARY/ABSTRACT The goal of the proposed research is to define the molecular mechanisms that govern intracellular transport by teams of myosin-Va and kinesin-1 molecular motors in a 3-dimensional (3D) network of actin filaments and microtubules (MTs). In Aim 1, in vitro biophysical assays will be used to measure the transport of fluid-like liposome cargoes coated with teams of myosin-Va and kinesin-1 motors in a reconstituted 3D network of actin filaments and MTs. Liposome trajectories in the 3D filament network will be correlated with the underlying filament position and orientation to determine how the liposome’s modes of motion (directed, diffusive, or stationary) are related to the local filament network architecture. To achieve this goal, training will be acquired in motor protein expression (Trybus Lab), 3D super-resolution microscopy, complex system reconstitution, and actomyosin biology (Warshaw Lab). In Aim 2, mechanistic mathematical modeling will be utilized to understand the biophysical parameters that govern liposome motility observed in vitro in Aim 1. How properties of the motors, the cargo, and the tracks influence liposome trajectories in in silico cytoskeletal networks identical to those mapped in vitro will highlight this modeling effort. New expertise in modeling will be gained by a mini “sabbatical” in the Walcott Lab (WPI). Aim 3 will bridge the gap between in vitro cargo transport studies in Aim 1 and that which occurs within a cell. Therefore, liposome transport by teams of kinesin-1 and myosin-Va motors will be characterized in exposed cytoskeletal networks of permeabilized cultured, COS7 cells. By growing cells on patterned substrates, these “designer” cells adopt to the desired geometry (e.g. circular or square) and thus organize their actin filament and MT cytoskeleton accordingly. The proposed experiments represent new training in novel tissue culture techniques (Howe Lab). The knowledgebase generated in Aims 1 and 2 will test the hypothesis that principles governing liposome transport in vitro are operative for transport along cellular cytoskeletal networks ex vivo. This proposal represents a synergistic, multidisciplinary training experience in which the trainee will gain and utilize new expertise in protein expression, new biophysical techniques, mathematical modeling, and cell biological techniques.