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Microtubules provide directional cues for polarized axonal transport through interaction with kinesin motor head.

Nakata T, Hirokawa N - J. Cell Biol. (2003)

Bottom Line: Post-Golgi carriers of various newly synthesized axonal membrane proteins, which possess kinesin (KIF5)-driven highly processive motility, were transported from the TGN directly to axons.We found that KIF5 has a preference to the microtubules in the initial segment of axon.These findings revealed unique features of the microtubule cytoskeletons in the initial segment, and suggested that they provide directional information for polarized axonal transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Tokyo, Japan 113-0033.

ABSTRACT
Post-Golgi carriers of various newly synthesized axonal membrane proteins, which possess kinesin (KIF5)-driven highly processive motility, were transported from the TGN directly to axons. We found that KIF5 has a preference to the microtubules in the initial segment of axon. Low dose paclitaxel treatment caused missorting of KIF5, as well as axonal membrane proteins to the tips of dendrites. Microtubules in the initial segment of axons showed a remarkably high affinity to EB1-YFP, which was known to bind the tips of growing microtubules. These findings revealed unique features of the microtubule cytoskeletons in the initial segment, and suggested that they provide directional information for polarized axonal transport.

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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.
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fig3: 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.

Mentions: Because our observation suggested that VSV-G carriers are driven by a number of active KIF5 motors, we tested whether KIF5 itself has a property to find its preferential way to the axons. When the full-length KIF5::GFP fusion protein was expressed in hippocampal neurons, a considerable amount of KIF5::GFP was localized in the cell body and dendrites (Fig. 3 a; see Fig. S1 for double label image with axonal marker, available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1), consistent with the immunocytochemical localization of kinesin (Kanai et al., 2000). Simple localization of kinesin does not reveal the actual site of action of kinesin because recent studies showed that most of the kinesin remains inhibited from binding to MTs by the association of its motor domain with its tail domain (Coy et al., 1999; Hackney and Stock, 2000), and unbound kinesin will be subject to simple diffusion. We eliminated the effect of tail inhibition as well as cargo binding, by deleting the tail and COOH-terminal region of the rod domain (Seiler et al., 2000). The localization of tailless motor proteins can be attributed simply to their motor activity. In fact, these motors are shown to accumulate at the tips of processes (Seiler et al., 2000). Although MT polarity is mixed in the proximal area of dendrites, it is plus end–directed in both the axons and distal parts of dendrites (Baas et al., 1988). If motor proteins do not discriminate MTs, they will run on both axonal and dendrite MTs toward the plus ends, and should accumulate at both axon and dendrite tips. When the tailless KIF5B::GFP fusion protein was expressed, the protein accumulated at the tips of axons, with a marked decrease in its level in dendrites (71 of 84 transfected neurons; Fig. 3 b; see Fig. S1 b for double label image with CFP). Total amount of GFP motor in axonal tips shown in Fig. 3 b was 3.14-fold higher that in the whole dendrites. This was confirmed in all the members of the KIF5 subfamily (KIF5A-C) in mouse. Various KIFs proteins are involved in axonal versus dendrite transport (Miki et al., 2001). We found that KIF17, an MT-plus end–directed motor transporting glutamate receptor NR2B in dendrites (Setou et al., 2000), accumulated at the tips of both dendrites and axons when their tail domains were deleted (41 of 47 tailless KIF17 transfected neurons; Fig. 3 c; see Fig. S1 c for double label image with dendrite marker). The accumulated KIFs were extracted by mild saponin extraction in the presence of paclitaxel without ATP (unpublished data), indicating that their accumulation is not due to strong binding to MTs in the distal (Nakata and Hirokawa, 1995). Chimera proteins with the KIF5 head and KIF17 neck and rod accumulated at the tips of axons (Fig. 3, d and j), whereas chimera proteins with KIF17 head and KIF5 neck and rod accumulated in both axons and dendrites (Fig. 3, e and j), indicating that the motor domain is a key to determine the KIF5 preference to axons. Motility of these chimera proteins was verified by in vitro motility assay (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1).


Microtubules provide directional cues for polarized axonal transport through interaction with kinesin motor head.

Nakata T, Hirokawa N - J. Cell Biol. (2003)

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.
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Related In: Results  -  Collection

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fig3: 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.
Mentions: Because our observation suggested that VSV-G carriers are driven by a number of active KIF5 motors, we tested whether KIF5 itself has a property to find its preferential way to the axons. When the full-length KIF5::GFP fusion protein was expressed in hippocampal neurons, a considerable amount of KIF5::GFP was localized in the cell body and dendrites (Fig. 3 a; see Fig. S1 for double label image with axonal marker, available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1), consistent with the immunocytochemical localization of kinesin (Kanai et al., 2000). Simple localization of kinesin does not reveal the actual site of action of kinesin because recent studies showed that most of the kinesin remains inhibited from binding to MTs by the association of its motor domain with its tail domain (Coy et al., 1999; Hackney and Stock, 2000), and unbound kinesin will be subject to simple diffusion. We eliminated the effect of tail inhibition as well as cargo binding, by deleting the tail and COOH-terminal region of the rod domain (Seiler et al., 2000). The localization of tailless motor proteins can be attributed simply to their motor activity. In fact, these motors are shown to accumulate at the tips of processes (Seiler et al., 2000). Although MT polarity is mixed in the proximal area of dendrites, it is plus end–directed in both the axons and distal parts of dendrites (Baas et al., 1988). If motor proteins do not discriminate MTs, they will run on both axonal and dendrite MTs toward the plus ends, and should accumulate at both axon and dendrite tips. When the tailless KIF5B::GFP fusion protein was expressed, the protein accumulated at the tips of axons, with a marked decrease in its level in dendrites (71 of 84 transfected neurons; Fig. 3 b; see Fig. S1 b for double label image with CFP). Total amount of GFP motor in axonal tips shown in Fig. 3 b was 3.14-fold higher that in the whole dendrites. This was confirmed in all the members of the KIF5 subfamily (KIF5A-C) in mouse. Various KIFs proteins are involved in axonal versus dendrite transport (Miki et al., 2001). We found that KIF17, an MT-plus end–directed motor transporting glutamate receptor NR2B in dendrites (Setou et al., 2000), accumulated at the tips of both dendrites and axons when their tail domains were deleted (41 of 47 tailless KIF17 transfected neurons; Fig. 3 c; see Fig. S1 c for double label image with dendrite marker). The accumulated KIFs were extracted by mild saponin extraction in the presence of paclitaxel without ATP (unpublished data), indicating that their accumulation is not due to strong binding to MTs in the distal (Nakata and Hirokawa, 1995). Chimera proteins with the KIF5 head and KIF17 neck and rod accumulated at the tips of axons (Fig. 3, d and j), whereas chimera proteins with KIF17 head and KIF5 neck and rod accumulated in both axons and dendrites (Fig. 3, e and j), indicating that the motor domain is a key to determine the KIF5 preference to axons. Motility of these chimera proteins was verified by in vitro motility assay (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1).

Bottom Line: Post-Golgi carriers of various newly synthesized axonal membrane proteins, which possess kinesin (KIF5)-driven highly processive motility, were transported from the TGN directly to axons.We found that KIF5 has a preference to the microtubules in the initial segment of axon.These findings revealed unique features of the microtubule cytoskeletons in the initial segment, and suggested that they provide directional information for polarized axonal transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Tokyo, Japan 113-0033.

ABSTRACT
Post-Golgi carriers of various newly synthesized axonal membrane proteins, which possess kinesin (KIF5)-driven highly processive motility, were transported from the TGN directly to axons. We found that KIF5 has a preference to the microtubules in the initial segment of axon. Low dose paclitaxel treatment caused missorting of KIF5, as well as axonal membrane proteins to the tips of dendrites. Microtubules in the initial segment of axons showed a remarkably high affinity to EB1-YFP, which was known to bind the tips of growing microtubules. These findings revealed unique features of the microtubule cytoskeletons in the initial segment, and suggested that they provide directional information for polarized axonal transport.

Show MeSH