Molecular Mechanism of Myosin-7a Translocation and Actin Bundle Assembly Insights from a New Binding Protein

Myosin-7a plays an evolutionarily conserved role in promoting cell protrusions of actin filament bundles. However, little is known about the underlying molecular mechanisms. Here, we report on the identification of a binding protein for Drosophila myosin-7a that we have named M7BP, and describe in detail how M7BP assembles myosin-7a motor complex, and thereby enables robust processive motility as well as actin filament remodeling. Structural and biophysical characterization show that M7BP tightly binds to the C-terminal FERM domain of myosin-7a in a 1:1 stoichiometric ratio, relieving the auto-inhibition and inducing an extended, activated motor. Using single-molecule motility assays, we show that myosin-7a-M7BP complexes move slowly along actin filaments (∼ 8 nm/s) and sustain long distance movements via binding to actin for long durations (∼300 s) without detaching. Moving complexes predominantly contain two motor molecules, and no processive movement can be observed in the absence of M7BP. A modified single-molecule motility assay further elucidates how myosin-7a-M7BP complex drives the alignment of actin filaments during movement. Finally, live cell super-resolution imaging reveals that the myosin-7a-M7BP complex dynamically reshapes the cytoskeleton when the two are co-transfected into S2 cells. Instead of a prominent cortical ring of actin, actin bundles traverse the interior of the cell and filopodial protrusions are formed. The myosin-7a-M7BP complex migrates to the tip of these filopodia and traffics along the bundles formed inside the cells. Together, by discovering the binding protein M7BP, our results describe a new mechanism for myosin-7a complex assembly and its roles in cargo transport and cell protrusion advancement, a strategy that may be used by other monomeric myosins, including myosin-10 and myosin-15.