Myo3A Motor Activity and Tail Domain Interactions Impact Actin Protrusion Elongation

Class III myosins are actin-based motors that play a role in regulating stereocilia length and morphology. We investigated the impact of a dominant deafness mutation (L697W) in the Myo3A motor, as well as the influence of a tail-binding partner (MORN4) to understand how Myo3A mediates actin protrusion dynamics. We found that the L697W mutation decreases Myo3A's ADP release rate constant by 2-fold and the overall ATPase by more than 5-fold, along with a 2-fold increase in actin affinity Consequently, the mutation also slows actin sliding in the in vitro motility assay 2-fold. Thus, we propose that the L697W mutation increases Myo3A's duty ratio - the fraction of the ATPase spent bound to actin, which increases Myo3A's residence time at stereocilia tips, allowing it to outcompete its WT counterpart. GFP-Myo3A localizes to filopodia tips when expressed in COS7 cells. We found that L697W Myo3A has a 2-fold longer FRAP recovery time at the filopodia tips, as compared to WT. Filopodia extension rates in L697W Myo3A-expressing cells are 1.6-fold slower than WT. We also found that the presence of MORN4, a Myo3A tail binding partner, enhances Myo3A localization to the tips of filopodia and increases the lengths of Myo3A-associated filopodia in COS7 cells. MORN4 alters the dynamics of Myo3A-associated filopodia by increasing the filopodia extension rate 1.3-fold without altering filopodia retraction. The Myo3A:MORN4 complex can sustain actin filament sliding in in vitro motility and also interacts with membranes, as GFP-Myo3A co-expressed with MORN4 localizes with liposomes in vitro and induces membrane tubulation. Overall, our results suggest a model in which Myo3A generates a tip-directed force in actin protrusions and that the motor's duty ratio and tail-membrane interactions are important for mediating protrusion dynamics.