A novel labeling strategy reveals that myosin Va and myosin Vb bind the same dendritically polarized vesicle population

Neurons are specialized cells with a polarized geometry and several distinct subdomains that require specific complements of proteins. Delivery of transmembrane proteins requires vesicle transport, which is mediated by molecular motor proteins. The myosin V family of motor proteins mediates transport to the barbed end of actin filaments, and little is known about the vesicles bound by myosin V in neurons. We developed a novel strategy to visualize myosin V-labeled vesicles in cultured hippocampal neurons and systematically characterized the vesicle populations labeled by myosin Va and Vb. We find that both myosins bind vesicles that are polarized to the somatodendritic domain where they undergo bidirectional long-range transport. A series of two-color imaging experiments showed that myosin V specifically colocalized with two different vesicle populations: vesicles labeled with the transferrin receptor and vesicles labeled by low-density lipoprotein receptor. Finally, coexpression with Kinesin-3 family members found that myosin V binds vesicles concurrently with KIF13A or KIF13B, supporting the hypothesis that coregulation of kinesins and myosin V on vesicles is likely to play an important role in neuronal vesicle transport. We anticipate that this new assay will be applicable in a broad range of cell types to determine the function of myosin V motor proteins.