Melanophilin, a Myosin Va Adapter Protein, Biases Track Selection of Myosin Va-and Kinesin-1-Transported Liposomes at Actin-Microtubule Intersections In Vitro

Abstract

Secretory vesicle transport from the Golgi to the cell membrane involves kinesin and myosin Va motors on the vesicle surface cooperatively navigating their shared cargo through numerous actin-microtubule (MT) intersections. How the track on which the cargo exits the intersection is selected so that vesicles are delivered to their destination with spatial and temporal fidelity remains unclear. Here we hypothesized that melanophilin – the adapter that links myosin Va to pigmented melanosomes and can bind to both actin and MTs – acts as a phosphorylation-dependent switch to bias track preference at actin-MT intersections. To test this, we modeled melanosome transport in vitro using 350-nm liposomes with ∼5 surface-bound molecules each of constitutively active myosin Va, kinesin-1, and full-length melanophilin with varying phosphorylation levels. Liposomes were then challenged with actin-MT intersections. Regardless of the track the liposomes entered the intersection on, liposomes with phosphorylated melanophilin were biased towards exiting the intersection on actin filaments while those with dephosphorylated melanophilin were biased to exit on MTs. Consistent with this, phosphorylated melanophilin showed a 2-fold preference to bind actin over MTs, and slowed liposome transport by myosin Va along actin filaments by ∼40% by effectively acting as an anchor. Conversely, dephosphorylated melanophilin preferentially bound (2-fold) MTs over actin and, by acting as a tether, increased the kinesin-1 liposome transport distance on MTs. Therefore, melanophilin, based on its phosphorylation state, can bias track selection of cargo transported by kinesin-1 and myosin Va through the cell’s complex cytoskeletal network with its numerous actin-MT intersections. Summary Statement Intracellular vesicular cargo transport (e.g., insulin granule trafficking) requires cargo transitions between microtubule (MT)- and actin-based transport as cargos navigate numerous actin-MT intersections in the dense cytoskeleton. How such transitions occur with temporal and spatial control is unclear. Using liposomes transported by actin-based myosin Va and MT-based kinesin-1 motors as a model system, we hypothesized that melanophilin, a myosin Va cargo adapter that preferentially binds actin or MTs depending on its phosphorylation state, steers liposomes through actin-MT intersections. Liposomes with melanophilin, regardless of the track they entered the intersection on, prefer to exit on the track dictated by melanophilin’s track binding preference. This observation highlights an under-appreciated role for adapter proteins (e.g., melanophilin) in ensuring proper cargo delivery in cells.