A novel strategy to visualize vesicle‐bound kinesins reveals the diversity of kinesin‐mediated transport

Abstract

In mammals, 15 to 20 kinesins are thought to mediate vesicle transport. Little is known about the identity of vesicles moved by each kinesin or the functional significance of such diversity. To characterize the transport mediated by different kinesins, we developed a novel strategy to visualize vesicle‐bound kinesins in living cells. We applied this method to cultured neurons and systematically determined the localization and transport parameters of vesicles labeled by different members of the Kinesin‐1, ‐2, and ‐3 families. We observed vesicle labeling with nearly all kinesins. Only six kinesins bound vesicles that undergo long‐range transport in neurons. Of these, three had an axonal bias (KIF5B, KIF5C and KIF13B), two were unbiased (KIF1A and KIF1Bβ), and one transported only in dendrites (KIF13A). Overall, the trafficking of vesicle‐bound kinesins to axons or dendrites did not correspond to their motor domain preference, suggesting that on‐vesicle regulation is crucial for kinesin targeting. Surprisingly, several kinesins were associated with populations of somatodendritic vesicles that underwent little long‐range transport. This assay should be broadly applicable for investigating kinesin function in many cell types.

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

“…In an attempt to improve vesicle labeling, we decided to test an approach we previously developed to visualize vesicle‐bound kinesins 23 . Similar to kinesins, most cytoplasmic myosin V molecules are in an autoinhibited conformation in which the globular tail binds to the motor domain 24‐26 .…”

“…When imaging full‐length fluorescently tagged myosin Vs, the bright diffuse signal from cytoplasmic myosin V molecules obscures any labeled vesicles. For kinesins, expressing only the vesicle‐binding domain rather than the full‐length protein dramatically improves vesicle labeling in neurons 23 . We reasoned that because myosin V is structurally similar to kinesin, a comparable approach may enhance myosin V vesicle labeling.…”

“…Vesicle transport parameters, such as run‐length and velocity, differ between different vesicle populations, giving most vesicles a specific transport “signature.” 28‐31 Long‐range transport (excursion lengths greater than ~3‐5 μm) is microtubule‐based and mediated by kinesins and dynein 32,33 . Correspondingly, vesicles labeled by different kinesins also exhibit signature transport parameters 23 . Therefore, even though myosin V itself does not mediate long‐range transport, analysis of transport parameters provides insight into the identity of the kinesin motors that mediate its movement.…”

“…Myosin V‐labeled vesicles undergo long‐range transport, which is mediated by microtubule‐based motors. We utilized the tail‐labeling strategy to identify the kinesins that bind moving myosin Va‐labeled vesicles 23 . We had previously found that dendrite‐selective vesicles are moved by members of the Kinesin‐3 family 37,42 .…”

“…We had previously found that dendrite‐selective vesicles are moved by members of the Kinesin‐3 family 37,42 . KIF13A may be a kinesin that is specialized for dendritic transport, as it binds exclusively to dendrite‐selective vesicles in hippocampal neurons 23 . The closely related KIF13B also interacts with dendrite‐selective TfR vesicles, 37 but also mediates the transport of axonal cargoes 23,43,44 .…”