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.