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. 2003 Sep 15;162(6):1045–1055. doi: 10.1083/jcb.200302175

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Copyright © 2003, The Rockefeller University Press

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Figure 3. — Tailless motors were destined for different regions of neurons. Data were obtained by CLSM. (a) Full-length KIF5B::GFP was transfected into hippocampal neurons. It was distributed throughout the cell body, dendrites, and the axon (arrows). The cell body and dendrites were brightly stained, just because of their thickness compared with the axon. (b) Tailless KIF5B::YFP were transfected into hippocampal neurons. Tailless KIF5B::YFP accumulated at the tips of axonal branches (arrows) but not dendrite tips. Cell body and proximal axon are indicated by arrowheads. (c) Tailless KIF17::YFP accumulated at the tips of dendrites (arrowheads) and axons (arrow). (d and e) Experiments on exchange of head domains revealed that head domains determine the destination of the tailless motors. MAP2 antibody is used as dendrite marker. (d) The KIF5B head and the KIF17 neck and rod constructs were destined for axons (green, arrows). The arrowhead indicates the cell body of transfected neuron. (e) The KIF17 head and the KIF5B neck and rod constructs were destined for the tips of dendrites (green). (f and g) Head exchange affects on polarized axonal transport. (f) Polarized axonal transport of VSV-G::GFP (green) was analyzed in the neurons that overexpress full-length KIF5B (red). VSV-G::GFP was preferentially sorted to the axon (arrow). (g) Preferential sorting of VSV-G::GFP (green, arrows) to the axon was inhibited in the neurons, which overexpress full-length KIF5 whose motor domain was replaced with KIF17 (red; see corresponding Videos 6 and 7). Fixation and immunocytochemistry to detect motor proteins was performed after live-cell imaging of VSV-G::GFP transport. (h and i) Head exchange does not affect on polarized dendrite sorting. NR2B::YFP distribution (green) was examined in the neurons, which overexpress authentic KIF17 (h, red) and full-length KIF17 whose motor domain was replaced with KIF5 (i, red). NR2B (green) was not sorted into axons in both cases. Arrows indicated the most likely candidates for axons. Bars, 10 μm. (j) Schematic diagram of the constructs. Head was exchanged at the border of head and neck-linker region of KIF5, and the corresponding region of KIF17 by the alignment. The colored amino acid sequences in the exchanged area were highly conserved. (k) Inhibition of polarized axonal transport by the head-exchanged KIF5. Bars indicate percentage of neurons with polarized β-APP::YFP transport. Data were collected from three independent cultures.