The Phosphorylation State of Melanophilin, a Myosin Va Adapter Protein, Biases the Tug-of-War Between Myosin Va and Kinesin-1 on Liposomes at Actin-Microtubule Intersections In Vitro

In cells, myosin Va and kinesin-1 motors navigate their shared vesicular cargos through numerous actin-microtubule (MT) intersections. How cargos exit these intersections to ensure precise destination delivery remains unclear. We hypothesized that the myosin Va adapter melanophilin -- a phosphorylated protein that links myosin Va to melanosomes but can also bind actin and MTs -- acts as a phosphorylation-dependent switch to bias the tug-of-war between cargo-bound myosin Va and kinesin-1 at actin-MT intersections. We modeled melanosome transport in vitro using 350-nm liposomes with ~5 each of surface-bound constitutively active myosin Va and kinesin-1, and ~5 full-length melanophilin with varying phosphorylation levels. Regardless of the track the liposome entered the actin-MT intersection on, phosphorylated melanophilin biased liposomes to exit on actin, whereas dephosphorylated melanophilin biased liposomes to exit on MTs. Consistent with this, phosphorylated melanophilin preferentially bound actin 2-fold over MTs, and slowed myosin Va liposome transport on actin by ~40%, effectively acting as an anchor. Conversely, dephosphorylated melanophilin preferentially bound (2-fold) MTs over actin, and increased kinesin-1 liposome transport distance on MTs by acting as a tether. Therefore, melanophilin, depending on its phosphorylation state, biases track selection by kinesin-1/myosin Va transported cargos in actin-MT intersections that exist in complex cellular cytoskeletal networks.