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.

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Kinesin-driven organelle transport is crucial for neuron development and maintenance, yet the mechanisms by which kinesins specifically bind their organelle cargoes remain undefined. In contrast to other transport kinesins, the neuronal function and specific organelle adaptors of heterodimeric Kinesin-2 family members KIF3AB and KIF3AC remain unknown. We developed a novel microscopy-based assay to define protein–protein interactions in intact neurons. The experiments found that KIF3AB and KIF3AC both bind kinesin-associated protein (KAP). These interactions are mediated by the distal C-terminal tail regions and not the coiled-coil domain. We used live-cell imaging in cultured hippocampal neurons to define the localization and trafficking parameters of KIF3AB and KIF3AC organelle populations. We discovered that KIF3AB/KAP and KIF3AC/KAP bind the same organelle populations and defined their transport parameters in axons and dendrites. The results also show that ∼12% of KIF3 organelles contain the RNA binding protein, adenomatous polyposis coli. These data point towards a model in which KIF3AB and KIF3AC use KAP as their neuronal organelle adaptor and that these kinesins mediate transport of a range of organelles.

Kinesin-driven organelle transport is crucial for neuron development and maintenance, yet the mechanisms by which kinesins specifically bind their organelle cargoes remain undefined. In contrast to other transport kinesins, the neuronal function and specific organelle adaptors of heterodimeric Kinesin-2 family members KIF3AB and KIF3AC remain unknown. We developed a novel microscopy-based assay to define protein-protein interactions in intact neurons. The experiments found that KIF3AB and KIF3AC both bind KAP. These interactions are mediated by the distal C-terminal tail regions and not the coiled-coil domain. We used live-cell imaging in cultured hippocampal neurons to define the localization and trafficking parameters of KIF3AB and KIF3AC organelle populations. We discovered that KIF3AB/KAP and KIF3AC/KAP bind the same organelle populations and defined their transport parameters in axons and dendrites. The results also show that ~12% of KIF3 organelles contain the RNA binding protein, adenomatous polyposis coli. These data point towards a model in which KIF3AB and KIF3AC use KAP as their neuronal organelle adaptor and that these kinesins mediate transport of a range of organelles.

No abstract