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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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Post-Golgi transport of axonal and dendrite carriers in hippocampal neurons visualized by CLSM. (a) Sorting of membrane protein–GFP fusion proteins under specific cell manipulation. VSVtsO45-G::GFP localizes in the ER at 39.5°C and moves to the TGN after a 30-min incubation at 19.5°C. Its post-Golgi transport starts when the temperature is shifted to 37°C. Note that VSV-G::GFP carriers are predominantly transported to one neurite (arrows) among the others. Kv2.1::GFP distributes diffusely in the somatodendritic area when expressed overnight in the presence of 1 μM brefeldin A. Kv2.1::GFP accumulates at the Golgi region 1 h after the brefeldin A washout, and starts post-Golgi transport. Note that Kv2.1::GFP was transported to all the dendrites 3 h after wash. (b) Characterization of VSV-G::G(Y)FP probes in neurons. (b1) Neurons were infected with Ad(VSVtsO45-G::GFP) and incubated overnight at 39.5°C, for 30 min at 19.5°C, and for 1 h at 37°C in the presence of 1 mg/ml Texas-red dextran (MW3000). Note that post-Golgi VSV-G carriers in the axon (arrows, green) are not labeled with the endocytotic marker (red). (b2 and b3) Double-label of hippocampal neuron with VSV-G::YFP and Golgi–CFP. At 19.5°C (b2), VSV-G::YFP (b2, green) was colocalized with the Golgi complex marker (b2, red) in the cell body. When the temperature is shifted to 37°C (b3), VSV-G::YFP moves from the Golgi complex area (yellow area at the upper right of b3, due to the overlap of VSV-G::YFP [green] and Golgi–CFP [red]) to the axon (b3, arrows). (c) VSV-G::GFP was dominantly transported from the TGN to the axon (arrow). Neurons were infected with Ad(VSVtsO45-G::GFP) and incubated overnight at 39.5°C, for 30 min at 19.5°C, and for 1 h at 37°C to visualize post-Golgi membrane transport (green). After fixation, the dendrites were stained with the anti-MAP2 antibody (red). Bar, 50 μm. Videos 1 and 2 are available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1. (d) Post-Golgi axonal carriers of VSV-G::GFP transport various membrane proteins. Axonally transported tubulovesicular organelles were simultaneously double-labeled with CFP- and YFP-tagged proteins, and time-lapse data were collected by sequential activation with 442 and 488 nm lasers by CLSM. (d1) VSV-G::CFP::CFP (red) and GAP-43::YFP (green). (d2) VSV-G::CFP::CFP (red) and β-APP::YFP (green). (d3) VSV-G::YFP (green) and Vamp2::CFP (red). (d4) Vamp2::CFP (red) and GAP-43::YFP (green). (d5) Vamp2::CFP (red) and β-APP::YFP (green). In each set, interval between the right and left panel is 10 s. Slight gaps between the CFP and YFP images along the longitudinal axis of vesicles are due to the time lag (∼0.7 s) between the sequential data acquisition. (e and f) Dominant negative kinesin (S205A,H206A; stained with the H2 antibody; red) inhibits polarized axonal transport of VSV-G::GFP (e, green), whereas it does not inhibit significantly dendrite transport of Kv2.1::GFP (f, green). Bars, 10 μm. (g) Inhibition of polarized axonal transport by dominant negative kinesins. Black bars indicate percentage of neurons with polarized VSV-G::GFP transport, and white bars indicate percentage of neurons exhibiting accumulation of VSV-G::GFP at TGN. Data were collected from four independent cultures.
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fig1: Post-Golgi transport of axonal and dendrite carriers in hippocampal neurons visualized by CLSM. (a) Sorting of membrane protein–GFP fusion proteins under specific cell manipulation. VSVtsO45-G::GFP localizes in the ER at 39.5°C and moves to the TGN after a 30-min incubation at 19.5°C. Its post-Golgi transport starts when the temperature is shifted to 37°C. Note that VSV-G::GFP carriers are predominantly transported to one neurite (arrows) among the others. Kv2.1::GFP distributes diffusely in the somatodendritic area when expressed overnight in the presence of 1 μM brefeldin A. Kv2.1::GFP accumulates at the Golgi region 1 h after the brefeldin A washout, and starts post-Golgi transport. Note that Kv2.1::GFP was transported to all the dendrites 3 h after wash. (b) Characterization of VSV-G::G(Y)FP probes in neurons. (b1) Neurons were infected with Ad(VSVtsO45-G::GFP) and incubated overnight at 39.5°C, for 30 min at 19.5°C, and for 1 h at 37°C in the presence of 1 mg/ml Texas-red dextran (MW3000). Note that post-Golgi VSV-G carriers in the axon (arrows, green) are not labeled with the endocytotic marker (red). (b2 and b3) Double-label of hippocampal neuron with VSV-G::YFP and Golgi–CFP. At 19.5°C (b2), VSV-G::YFP (b2, green) was colocalized with the Golgi complex marker (b2, red) in the cell body. When the temperature is shifted to 37°C (b3), VSV-G::YFP moves from the Golgi complex area (yellow area at the upper right of b3, due to the overlap of VSV-G::YFP [green] and Golgi–CFP [red]) to the axon (b3, arrows). (c) VSV-G::GFP was dominantly transported from the TGN to the axon (arrow). Neurons were infected with Ad(VSVtsO45-G::GFP) and incubated overnight at 39.5°C, for 30 min at 19.5°C, and for 1 h at 37°C to visualize post-Golgi membrane transport (green). After fixation, the dendrites were stained with the anti-MAP2 antibody (red). Bar, 50 μm. Videos 1 and 2 are available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1. (d) Post-Golgi axonal carriers of VSV-G::GFP transport various membrane proteins. Axonally transported tubulovesicular organelles were simultaneously double-labeled with CFP- and YFP-tagged proteins, and time-lapse data were collected by sequential activation with 442 and 488 nm lasers by CLSM. (d1) VSV-G::CFP::CFP (red) and GAP-43::YFP (green). (d2) VSV-G::CFP::CFP (red) and β-APP::YFP (green). (d3) VSV-G::YFP (green) and Vamp2::CFP (red). (d4) Vamp2::CFP (red) and GAP-43::YFP (green). (d5) Vamp2::CFP (red) and β-APP::YFP (green). In each set, interval between the right and left panel is 10 s. Slight gaps between the CFP and YFP images along the longitudinal axis of vesicles are due to the time lag (∼0.7 s) between the sequential data acquisition. (e and f) Dominant negative kinesin (S205A,H206A; stained with the H2 antibody; red) inhibits polarized axonal transport of VSV-G::GFP (e, green), whereas it does not inhibit significantly dendrite transport of Kv2.1::GFP (f, green). Bars, 10 μm. (g) Inhibition of polarized axonal transport by dominant negative kinesins. Black bars indicate percentage of neurons with polarized VSV-G::GFP transport, and white bars indicate percentage of neurons exhibiting accumulation of VSV-G::GFP at TGN. Data were collected from four independent cultures.

Mentions: Previously, axonally transported vesicles were considered to be transported nonselectively into both dendrites and axons because vesicles, which carry axonal membrane protein–GFP fusion proteins often accumulated in both dendrites and axons (Jareb and Banker, 1998). This may be due to a small amount of missorted proteins, which finally accumulated at the time of observation in the somatodendritic area with a limited volume because they cannot be exocytosed to dendrite membranes. To eliminate these effects, we used temperature-sensitive vesicular stomatitis virus G-protein (VSV-G tsO45; Hirschberg et al., 1998; Toomre et al., 1999). We expressed VSV-GtsO45::GFP at 39.5°C overnight, allowed it to accumulate in the Golgi apparatus by decreasing the temperature to 19.5°C, and monitored the post-Golgi transport at 37°C (Fig. 1 a; Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1). Double label with Golgi–CFP (CLONTECH Laboratories, Inc.) and VSV-G::YFP revealed that VSV-G::YFP colocalized with the Golgi complex–marker at 19.5°C (Fig. 1, b2), and post-Golgi VSV-G::YFP carriers moved predominantly to one neurite from the Golgi region after temperature shift to 37°C (Fig. 1, b3, arrows). This temperature shift protocol was used for VSV-G throughout the experiment. These carriers were not labeled with endocytotic marker Texas red dextran (Fig. 1, b1, arrows). This result, together with previous papers, indicates that these carriers are not derived from endosomes (Nakata et al., 1998; Ahmari et al., 2000). Although VSV-G itself is sorted to dendrites (Dotti and Simons, 1990), we found that VSV-G tsO45 was sorted to axons in hippocampal neurons when tagged with GFP in its COOH terminus as shown by staining of dendrites with anti–MT-associated protein (MAP) 2 antibody (Fig. 1 c). Thus, we used VSV-G::GFP as an axonal transport marker. Simultaneous expression of CFP and YFP fusion proteins revealed that VSV-G::GFP was transported by the tubulovesicular organelles, which transport a number of newly synthesized axonal membrane proteins such as β-APP, GAP-43, and vamp-2 (Fig. 1 d), indicating that VSV-G tsO45::GFP labels major post-Golgi carriers for various axonal membrane proteins (Nakata et al., 1998). Number of vesicles entered axons is 3.8 times more than those to any dendrites in average. We judged the transport is polarized if it is more than twice as much as those to any dendrites. We found that VSV-G::GFP was transported in a polarized manner in ∼60% of neurons (Fig. 1 g and Fig. 5 j), indicating that there is a mechanism for polarized vectorial axonal transport.


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

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

Post-Golgi transport of axonal and dendrite carriers in hippocampal neurons visualized by CLSM. (a) Sorting of membrane protein–GFP fusion proteins under specific cell manipulation. VSVtsO45-G::GFP localizes in the ER at 39.5°C and moves to the TGN after a 30-min incubation at 19.5°C. Its post-Golgi transport starts when the temperature is shifted to 37°C. Note that VSV-G::GFP carriers are predominantly transported to one neurite (arrows) among the others. Kv2.1::GFP distributes diffusely in the somatodendritic area when expressed overnight in the presence of 1 μM brefeldin A. Kv2.1::GFP accumulates at the Golgi region 1 h after the brefeldin A washout, and starts post-Golgi transport. Note that Kv2.1::GFP was transported to all the dendrites 3 h after wash. (b) Characterization of VSV-G::G(Y)FP probes in neurons. (b1) Neurons were infected with Ad(VSVtsO45-G::GFP) and incubated overnight at 39.5°C, for 30 min at 19.5°C, and for 1 h at 37°C in the presence of 1 mg/ml Texas-red dextran (MW3000). Note that post-Golgi VSV-G carriers in the axon (arrows, green) are not labeled with the endocytotic marker (red). (b2 and b3) Double-label of hippocampal neuron with VSV-G::YFP and Golgi–CFP. At 19.5°C (b2), VSV-G::YFP (b2, green) was colocalized with the Golgi complex marker (b2, red) in the cell body. When the temperature is shifted to 37°C (b3), VSV-G::YFP moves from the Golgi complex area (yellow area at the upper right of b3, due to the overlap of VSV-G::YFP [green] and Golgi–CFP [red]) to the axon (b3, arrows). (c) VSV-G::GFP was dominantly transported from the TGN to the axon (arrow). Neurons were infected with Ad(VSVtsO45-G::GFP) and incubated overnight at 39.5°C, for 30 min at 19.5°C, and for 1 h at 37°C to visualize post-Golgi membrane transport (green). After fixation, the dendrites were stained with the anti-MAP2 antibody (red). Bar, 50 μm. Videos 1 and 2 are available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1. (d) Post-Golgi axonal carriers of VSV-G::GFP transport various membrane proteins. Axonally transported tubulovesicular organelles were simultaneously double-labeled with CFP- and YFP-tagged proteins, and time-lapse data were collected by sequential activation with 442 and 488 nm lasers by CLSM. (d1) VSV-G::CFP::CFP (red) and GAP-43::YFP (green). (d2) VSV-G::CFP::CFP (red) and β-APP::YFP (green). (d3) VSV-G::YFP (green) and Vamp2::CFP (red). (d4) Vamp2::CFP (red) and GAP-43::YFP (green). (d5) Vamp2::CFP (red) and β-APP::YFP (green). In each set, interval between the right and left panel is 10 s. Slight gaps between the CFP and YFP images along the longitudinal axis of vesicles are due to the time lag (∼0.7 s) between the sequential data acquisition. (e and f) Dominant negative kinesin (S205A,H206A; stained with the H2 antibody; red) inhibits polarized axonal transport of VSV-G::GFP (e, green), whereas it does not inhibit significantly dendrite transport of Kv2.1::GFP (f, green). Bars, 10 μm. (g) Inhibition of polarized axonal transport by dominant negative kinesins. Black bars indicate percentage of neurons with polarized VSV-G::GFP transport, and white bars indicate percentage of neurons exhibiting accumulation of VSV-G::GFP at TGN. Data were collected from four independent cultures.
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fig1: Post-Golgi transport of axonal and dendrite carriers in hippocampal neurons visualized by CLSM. (a) Sorting of membrane protein–GFP fusion proteins under specific cell manipulation. VSVtsO45-G::GFP localizes in the ER at 39.5°C and moves to the TGN after a 30-min incubation at 19.5°C. Its post-Golgi transport starts when the temperature is shifted to 37°C. Note that VSV-G::GFP carriers are predominantly transported to one neurite (arrows) among the others. Kv2.1::GFP distributes diffusely in the somatodendritic area when expressed overnight in the presence of 1 μM brefeldin A. Kv2.1::GFP accumulates at the Golgi region 1 h after the brefeldin A washout, and starts post-Golgi transport. Note that Kv2.1::GFP was transported to all the dendrites 3 h after wash. (b) Characterization of VSV-G::G(Y)FP probes in neurons. (b1) Neurons were infected with Ad(VSVtsO45-G::GFP) and incubated overnight at 39.5°C, for 30 min at 19.5°C, and for 1 h at 37°C in the presence of 1 mg/ml Texas-red dextran (MW3000). Note that post-Golgi VSV-G carriers in the axon (arrows, green) are not labeled with the endocytotic marker (red). (b2 and b3) Double-label of hippocampal neuron with VSV-G::YFP and Golgi–CFP. At 19.5°C (b2), VSV-G::YFP (b2, green) was colocalized with the Golgi complex marker (b2, red) in the cell body. When the temperature is shifted to 37°C (b3), VSV-G::YFP moves from the Golgi complex area (yellow area at the upper right of b3, due to the overlap of VSV-G::YFP [green] and Golgi–CFP [red]) to the axon (b3, arrows). (c) VSV-G::GFP was dominantly transported from the TGN to the axon (arrow). Neurons were infected with Ad(VSVtsO45-G::GFP) and incubated overnight at 39.5°C, for 30 min at 19.5°C, and for 1 h at 37°C to visualize post-Golgi membrane transport (green). After fixation, the dendrites were stained with the anti-MAP2 antibody (red). Bar, 50 μm. Videos 1 and 2 are available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1. (d) Post-Golgi axonal carriers of VSV-G::GFP transport various membrane proteins. Axonally transported tubulovesicular organelles were simultaneously double-labeled with CFP- and YFP-tagged proteins, and time-lapse data were collected by sequential activation with 442 and 488 nm lasers by CLSM. (d1) VSV-G::CFP::CFP (red) and GAP-43::YFP (green). (d2) VSV-G::CFP::CFP (red) and β-APP::YFP (green). (d3) VSV-G::YFP (green) and Vamp2::CFP (red). (d4) Vamp2::CFP (red) and GAP-43::YFP (green). (d5) Vamp2::CFP (red) and β-APP::YFP (green). In each set, interval between the right and left panel is 10 s. Slight gaps between the CFP and YFP images along the longitudinal axis of vesicles are due to the time lag (∼0.7 s) between the sequential data acquisition. (e and f) Dominant negative kinesin (S205A,H206A; stained with the H2 antibody; red) inhibits polarized axonal transport of VSV-G::GFP (e, green), whereas it does not inhibit significantly dendrite transport of Kv2.1::GFP (f, green). Bars, 10 μm. (g) Inhibition of polarized axonal transport by dominant negative kinesins. Black bars indicate percentage of neurons with polarized VSV-G::GFP transport, and white bars indicate percentage of neurons exhibiting accumulation of VSV-G::GFP at TGN. Data were collected from four independent cultures.
Mentions: Previously, axonally transported vesicles were considered to be transported nonselectively into both dendrites and axons because vesicles, which carry axonal membrane protein–GFP fusion proteins often accumulated in both dendrites and axons (Jareb and Banker, 1998). This may be due to a small amount of missorted proteins, which finally accumulated at the time of observation in the somatodendritic area with a limited volume because they cannot be exocytosed to dendrite membranes. To eliminate these effects, we used temperature-sensitive vesicular stomatitis virus G-protein (VSV-G tsO45; Hirschberg et al., 1998; Toomre et al., 1999). We expressed VSV-GtsO45::GFP at 39.5°C overnight, allowed it to accumulate in the Golgi apparatus by decreasing the temperature to 19.5°C, and monitored the post-Golgi transport at 37°C (Fig. 1 a; Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1). Double label with Golgi–CFP (CLONTECH Laboratories, Inc.) and VSV-G::YFP revealed that VSV-G::YFP colocalized with the Golgi complex–marker at 19.5°C (Fig. 1, b2), and post-Golgi VSV-G::YFP carriers moved predominantly to one neurite from the Golgi region after temperature shift to 37°C (Fig. 1, b3, arrows). This temperature shift protocol was used for VSV-G throughout the experiment. These carriers were not labeled with endocytotic marker Texas red dextran (Fig. 1, b1, arrows). This result, together with previous papers, indicates that these carriers are not derived from endosomes (Nakata et al., 1998; Ahmari et al., 2000). Although VSV-G itself is sorted to dendrites (Dotti and Simons, 1990), we found that VSV-G tsO45 was sorted to axons in hippocampal neurons when tagged with GFP in its COOH terminus as shown by staining of dendrites with anti–MT-associated protein (MAP) 2 antibody (Fig. 1 c). Thus, we used VSV-G::GFP as an axonal transport marker. Simultaneous expression of CFP and YFP fusion proteins revealed that VSV-G::GFP was transported by the tubulovesicular organelles, which transport a number of newly synthesized axonal membrane proteins such as β-APP, GAP-43, and vamp-2 (Fig. 1 d), indicating that VSV-G tsO45::GFP labels major post-Golgi carriers for various axonal membrane proteins (Nakata et al., 1998). Number of vesicles entered axons is 3.8 times more than those to any dendrites in average. We judged the transport is polarized if it is more than twice as much as those to any dendrites. We found that VSV-G::GFP was transported in a polarized manner in ∼60% of neurons (Fig. 1 g and Fig. 5 j), indicating that there is a mechanism for polarized vectorial axonal transport.

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
Related in: MedlinePlus