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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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MT dynamics in the IS. (a) Maximal Z-projection of small pinhole size CLSM images of neuron transfected with EB1::YFP (green) was stained with anti-MAP2 antibody (red). EB1 extensively labeled the IS (arrow). (b and c) CAFM images of EB1::YFP expressing neurons. (b) Individual EB1 dots, which decorate the growing tips of MTs are clearly observed in dendrites, cell body, and axon in a low level of EB1::YFP expression. Note that individual EB1 dots are resolved in the IS (b, arrows; Video 8). (c) In a higher level of expression, EB1::YFP extensively labeled the MTs in the IS (c, arrow), whereas it labeled only tips of MTs in the cell body and dendrites (c, arrowheads; Video 9). (d) CAFM image of MTs stained with antitubulin antibody. Note that MTs in dendrites as well as cell bodies are visualized by CAFM. Arrow indicates MTs in the cell body. Bars, 10 μm.
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fig6: MT dynamics in the IS. (a) Maximal Z-projection of small pinhole size CLSM images of neuron transfected with EB1::YFP (green) was stained with anti-MAP2 antibody (red). EB1 extensively labeled the IS (arrow). (b and c) CAFM images of EB1::YFP expressing neurons. (b) Individual EB1 dots, which decorate the growing tips of MTs are clearly observed in dendrites, cell body, and axon in a low level of EB1::YFP expression. Note that individual EB1 dots are resolved in the IS (b, arrows; Video 8). (c) In a higher level of expression, EB1::YFP extensively labeled the MTs in the IS (c, arrow), whereas it labeled only tips of MTs in the cell body and dendrites (c, arrowheads; Video 9). (d) CAFM image of MTs stained with antitubulin antibody. Note that MTs in dendrites as well as cell bodies are visualized by CAFM. Arrow indicates MTs in the cell body. Bars, 10 μm.

Mentions: Our paclitaxel experiments prompted us to see the MT dynamics around the cell body. For this purpose, we used EB1:: YFP, which is known to bind to the growing tips of MTs (Tirnauer and Bierer, 2000). Although electron microscopists have long used the difference of MT organization in order to identify the IS around the cell body, their chemical property is not elucidated yet. Localization of conventional MAPs cannot explain this because, for example, although MAP2 and tau have been used as markers for dendrites and axons, MAP2 localizes in dendrites as well as in the IS, and the phosphorylated form of tau only localizes in the axons considerably distal to the IS. When we expressed EB1::YFP, in cultured hippocampal neurons, we found intense accumulation of EB1::YFP to the IS compared with the cell body and dendrites by CLSM at high Z-resolution (Fig. 6 a; see Fig. S5 a for wider field of view, available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1; fluorescence ratio of axon to dendrites was 172 ± 31.6% [n = 20]). However, CLSM cannot visualize individual growing tips of MTs in the cell body. By using CAFM, we could visualize the growing tips of MTs as EB1 dots around the cell body. In a low level of EB1::YFP expression, we could observe the movement of individual EB1 dots in both axon ISs and dendrites (Fig. 6 b; Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1). Number of EB1 dots per 10 μm2 per 1 min by CAFM was 4.87 ± 2.23 in the ISs, 2.16 ± 1.59 in the dendrites, and 0.77 ± 0.13 in the cell body (n = 19), which was markedly reduced to 0.58 ± 0.08, 0.20 ± 0.18, 0.13 ± 0.06, respectively after a 10-min treatment of 10 nM paclitaxel. The inhibition of MTs dynamics by the paclitaxel also decreased the speed of EB1 dots from 4.6 ± 0.9 μm/min to 1.8 ± 1.1 μm/min. We noticed that, in a higher level of EB1::YFP expression, MTs in the IS were fully decorated by EB1::YFP, whereas at the same time EB1::YFP labels only the tips of MTs as dots in the cell body and dendrites of the same neuron (Fig. 6 c, arrow; Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1), which makes EB1::YFP an excellent marker for the axon around the cell body (Fig. 6, a and c). Control CAFM image of tubulin staining showed bright dendrite staining (Fig. 6 d). Fig. S5 shows gallery of EB1 staining (Fig. S5 b) and immuno-electron microscopy of EB1::YFP in dendrites (Fig. S5 c) and axons (Fig. S5 d). Such spatial difference of EB1 behavior within a cell was never observed in nonneuronal cells at any level of EB1 expression. Our results demonstrate unique properties of MTs in the IS compared with those in the cell body and dendrites.


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

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

MT dynamics in the IS. (a) Maximal Z-projection of small pinhole size CLSM images of neuron transfected with EB1::YFP (green) was stained with anti-MAP2 antibody (red). EB1 extensively labeled the IS (arrow). (b and c) CAFM images of EB1::YFP expressing neurons. (b) Individual EB1 dots, which decorate the growing tips of MTs are clearly observed in dendrites, cell body, and axon in a low level of EB1::YFP expression. Note that individual EB1 dots are resolved in the IS (b, arrows; Video 8). (c) In a higher level of expression, EB1::YFP extensively labeled the MTs in the IS (c, arrow), whereas it labeled only tips of MTs in the cell body and dendrites (c, arrowheads; Video 9). (d) CAFM image of MTs stained with antitubulin antibody. Note that MTs in dendrites as well as cell bodies are visualized by CAFM. Arrow indicates MTs in the cell body. Bars, 10 μm.
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Related In: Results  -  Collection

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fig6: MT dynamics in the IS. (a) Maximal Z-projection of small pinhole size CLSM images of neuron transfected with EB1::YFP (green) was stained with anti-MAP2 antibody (red). EB1 extensively labeled the IS (arrow). (b and c) CAFM images of EB1::YFP expressing neurons. (b) Individual EB1 dots, which decorate the growing tips of MTs are clearly observed in dendrites, cell body, and axon in a low level of EB1::YFP expression. Note that individual EB1 dots are resolved in the IS (b, arrows; Video 8). (c) In a higher level of expression, EB1::YFP extensively labeled the MTs in the IS (c, arrow), whereas it labeled only tips of MTs in the cell body and dendrites (c, arrowheads; Video 9). (d) CAFM image of MTs stained with antitubulin antibody. Note that MTs in dendrites as well as cell bodies are visualized by CAFM. Arrow indicates MTs in the cell body. Bars, 10 μm.
Mentions: Our paclitaxel experiments prompted us to see the MT dynamics around the cell body. For this purpose, we used EB1:: YFP, which is known to bind to the growing tips of MTs (Tirnauer and Bierer, 2000). Although electron microscopists have long used the difference of MT organization in order to identify the IS around the cell body, their chemical property is not elucidated yet. Localization of conventional MAPs cannot explain this because, for example, although MAP2 and tau have been used as markers for dendrites and axons, MAP2 localizes in dendrites as well as in the IS, and the phosphorylated form of tau only localizes in the axons considerably distal to the IS. When we expressed EB1::YFP, in cultured hippocampal neurons, we found intense accumulation of EB1::YFP to the IS compared with the cell body and dendrites by CLSM at high Z-resolution (Fig. 6 a; see Fig. S5 a for wider field of view, available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1; fluorescence ratio of axon to dendrites was 172 ± 31.6% [n = 20]). However, CLSM cannot visualize individual growing tips of MTs in the cell body. By using CAFM, we could visualize the growing tips of MTs as EB1 dots around the cell body. In a low level of EB1::YFP expression, we could observe the movement of individual EB1 dots in both axon ISs and dendrites (Fig. 6 b; Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1). Number of EB1 dots per 10 μm2 per 1 min by CAFM was 4.87 ± 2.23 in the ISs, 2.16 ± 1.59 in the dendrites, and 0.77 ± 0.13 in the cell body (n = 19), which was markedly reduced to 0.58 ± 0.08, 0.20 ± 0.18, 0.13 ± 0.06, respectively after a 10-min treatment of 10 nM paclitaxel. The inhibition of MTs dynamics by the paclitaxel also decreased the speed of EB1 dots from 4.6 ± 0.9 μm/min to 1.8 ± 1.1 μm/min. We noticed that, in a higher level of EB1::YFP expression, MTs in the IS were fully decorated by EB1::YFP, whereas at the same time EB1::YFP labels only the tips of MTs as dots in the cell body and dendrites of the same neuron (Fig. 6 c, arrow; Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1), which makes EB1::YFP an excellent marker for the axon around the cell body (Fig. 6, a and c). Control CAFM image of tubulin staining showed bright dendrite staining (Fig. 6 d). Fig. S5 shows gallery of EB1 staining (Fig. S5 b) and immuno-electron microscopy of EB1::YFP in dendrites (Fig. S5 c) and axons (Fig. S5 d). Such spatial difference of EB1 behavior within a cell was never observed in nonneuronal cells at any level of EB1 expression. Our results demonstrate unique properties of MTs in the IS compared with those in the cell body and dendrites.

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