Abstract
Intracellular cargo transport relies on myosin Va molecular motor ensembles to travel along the cell’s three-dimensional (3D) highway of actin filaments. At actin filament intersections, the intersecting filament is a structural barrier to and an alternate track for directed cargo transport. Here we use 3D super-resolution fluorescence imaging to determine the directional outcome (that is, continues straight, turns or terminates) for an ∼10 motor ensemble transporting a 350 nm lipid-bound cargo that encounters a suspended 3D actin filament intersection in vitro. Motor–cargo complexes that interact with the intersecting filament go straight through the intersection 62% of the time, nearly twice that for turning. To explain this, we develop an in silico model, supported by optical trapping data, suggesting that the motors’ diffusive movements on the vesicle surface and the extent of their engagement with the two intersecting actin tracks biases the motor–cargo complex on average to go straight through the intersection.
Highlights
Intracellular cargo transport relies on myosin Va molecular motor ensembles to travel along the cell’s three-dimensional (3D) highway of actin filaments
Motor–cargo complexes travelling along an actin filament that encounter and physically interact with a suspended intersecting filament continue straight through the intersection on the original filament it is travelling on 62% of the time
We develop an in silico, mechanistic model that describes the diffusive movement of motors on the vesicle surface, their engagement with the two intersecting actin tracks (Fig. 1b) and the ensuing ‘tug-of-war’ between the two myosin Va (myoVa) ensembles that eventually dictates the directional outcome at an intersection
Summary
Intracellular cargo transport relies on myosin Va molecular motor ensembles to travel along the cell’s three-dimensional (3D) highway of actin filaments. The actin cortex is a dense, three-dimensional (3D) cytoskeletal highway in which the plus-ends of individual actin filaments are biased towards the cell membrane, which is the direction in which myoVa travels[3] This seemingly random highway, with its numerous actin filament intersections, makes efficient straight-line cargo delivery from point A to B directionally challenging (Fig. 1a). As the step to understanding how myoVa motor ensembles meet the mechanical and directional challenges of the cell’s complex 3D actin cytoskeleton, we create an in vitro 3D network of suspended actin filaments with numerous intersections (Fig. 1c) This network is designed to directionally challenge constitutively active myoVa motor ensembles transporting more physiologically relevant, lipid-bound vesicle cargos[9,13]. We develop an in silico, mechanistic model that describes the diffusive movement of motors on the vesicle surface, their engagement with the two intersecting actin tracks (Fig. 1b) and the ensuing ‘tug-of-war’ between the two myoVa ensembles that eventually dictates the directional outcome at an intersection
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