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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.

Propofol is a widely used general anesthetic, yet the understanding of its cellular effects is fragmentary. General anesthetics are not as innocuous as once believed and have a wide range of molecular targets that include kinesin motors. Propofol, ketamine, and etomidate reduce the distances that Kinesin-1 KIF5 and Kinesin-2 KIF3 travel along microtubules in vitro. These transport kinesins are highly expressed in the CNS, and their dysfunction leads to a range of human pathologies including neurodevelopmental and neurodegenerative diseases. While in vitro data suggest that general anesthetics may disrupt kinesin transport in neurons, this hypothesis remains untested. Here we find that propofol treatment of hippocampal neurons decreased vesicle transport mediated by Kinesin-1 KIF5 and Kinesin-3 KIF1A ∼25-60%. Propofol treatment delayed delivery of the KIF5 cargo NgCAM to the distal axon. Because KIF1A participates in axonal transport of presynaptic vesicles, we tested if prolonged propofol treatment affects synaptic vesicle fusion mediated by VAMP2. The data show that propofol-induced transport delay causes a significant decrease in vesicle fusion in distal axons. These results are the first to link a propofol-induced delay in neuronal trafficking to a decrease in axonal vesicle fusion, which may alter physiological function during and post-anesthesia.

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