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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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Paclitaxel at a low dose reverts the polarized sorting of axonal motor proteins as well as membrane proteins. Data were obtained by CLSM with small pinhole and maximal Z-projection (b, k, l, m, and n) and with pinhole fully open (a, c, d, e, f, g, and h). Arrows indicate axons. MAP2 (red) was stained in a, b, e, l, and m. (a) Tailless KIF5::YFP (green) was sorted to dendrite tips in the presence of 10 nM paclitaxel. (b) Rigor-KIF5::YFP (green) failed to accumulate in the IS in the presence of 10 nM paclitaxel. (c and d) VSV-G::GFP was initially observed 1 h after the temperature shift to 30°C from 19.5°C without paclitaxel (c), and 10 nM paclitaxel (d) was added to the medium. Axonal transport from the IS was unchanged. However, as a result of the decrease in the supply of vesicles from TGN, the amount of vesicles in the IS markedly decreased 1 h later (d, arrow). Note that dendrite staining is elevated in d. (e and f) β-APP::YFP was expressed overnight without (e) or with paclitaxel (f). (g and h) Kv2.1::YFP was expressed overnight without (g) or with paclitaxel (h). Kv2.1 (green) is a potassium channel sorted to the cell body and dendrites in the absence (g) and in the presence of paclitaxel (h). MAP2 was stained with red. Bars, 10 μm. (i) MT polarity is mixed in proximal dendrites after incubation with 100 nM paclitaxel overnight. (inset) Axonal MTs. Polarity of MTs is determined by the curvature of hooks in the electron micrographs (arrows). Bar, 100 nm. Electron micrograph, which shows MT organization in paclitaxel treated neurons, is available as Fig. S3. (j) Dose dependence of membrane phenotype and that of motor phenotype are similar. For membrane phenotype, percentage of neurons, which show polarized axonal transport of VSV-G::GFP are presented (n = 3, each 50 cells counted). For motor phenotype, percentage of neurons, which show preferential binding of rigor-KIF5 to the IS are presented (n = 2, each 50 cells counted). (k–n) In vitro reconstitution of preferential association of KIF5 to the MTs in the IS. Hippocampal neuronal cytoskeleton was prepared by permeabilization with 0.1% Triton X-100, and recombinant tailless-KIF5::YFP was added to the cytoskeleton (k and m). Subsequently, the cells were fixed and stained with anti-MAP2 antibody. In l and n, MAP2 staining (red) is superimposed on the KIF5 images in k and m (green). In control neurons, intense binding of KIF5 to the IS was observed (k and l, arrows), compared with other neurites. In contrast, when neurons were pretreated with 10 nM paclitaxel before permeabilization, binding of KIF5 to the axonal IS was similar to other neurites (m and n, arrows).
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fig5: Paclitaxel at a low dose reverts the polarized sorting of axonal motor proteins as well as membrane proteins. Data were obtained by CLSM with small pinhole and maximal Z-projection (b, k, l, m, and n) and with pinhole fully open (a, c, d, e, f, g, and h). Arrows indicate axons. MAP2 (red) was stained in a, b, e, l, and m. (a) Tailless KIF5::YFP (green) was sorted to dendrite tips in the presence of 10 nM paclitaxel. (b) Rigor-KIF5::YFP (green) failed to accumulate in the IS in the presence of 10 nM paclitaxel. (c and d) VSV-G::GFP was initially observed 1 h after the temperature shift to 30°C from 19.5°C without paclitaxel (c), and 10 nM paclitaxel (d) was added to the medium. Axonal transport from the IS was unchanged. However, as a result of the decrease in the supply of vesicles from TGN, the amount of vesicles in the IS markedly decreased 1 h later (d, arrow). Note that dendrite staining is elevated in d. (e and f) β-APP::YFP was expressed overnight without (e) or with paclitaxel (f). (g and h) Kv2.1::YFP was expressed overnight without (g) or with paclitaxel (h). Kv2.1 (green) is a potassium channel sorted to the cell body and dendrites in the absence (g) and in the presence of paclitaxel (h). MAP2 was stained with red. Bars, 10 μm. (i) MT polarity is mixed in proximal dendrites after incubation with 100 nM paclitaxel overnight. (inset) Axonal MTs. Polarity of MTs is determined by the curvature of hooks in the electron micrographs (arrows). Bar, 100 nm. Electron micrograph, which shows MT organization in paclitaxel treated neurons, is available as Fig. S3. (j) Dose dependence of membrane phenotype and that of motor phenotype are similar. For membrane phenotype, percentage of neurons, which show polarized axonal transport of VSV-G::GFP are presented (n = 3, each 50 cells counted). For motor phenotype, percentage of neurons, which show preferential binding of rigor-KIF5 to the IS are presented (n = 2, each 50 cells counted). (k–n) In vitro reconstitution of preferential association of KIF5 to the MTs in the IS. Hippocampal neuronal cytoskeleton was prepared by permeabilization with 0.1% Triton X-100, and recombinant tailless-KIF5::YFP was added to the cytoskeleton (k and m). Subsequently, the cells were fixed and stained with anti-MAP2 antibody. In l and n, MAP2 staining (red) is superimposed on the KIF5 images in k and m (green). In control neurons, intense binding of KIF5 to the IS was observed (k and l, arrows), compared with other neurites. In contrast, when neurons were pretreated with 10 nM paclitaxel before permeabilization, binding of KIF5 to the axonal IS was similar to other neurites (m and n, arrows).

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)

Paclitaxel at a low dose reverts the polarized sorting of axonal motor proteins as well as membrane proteins. Data were obtained by CLSM with small pinhole and maximal Z-projection (b, k, l, m, and n) and with pinhole fully open (a, c, d, e, f, g, and h). Arrows indicate axons. MAP2 (red) was stained in a, b, e, l, and m. (a) Tailless KIF5::YFP (green) was sorted to dendrite tips in the presence of 10 nM paclitaxel. (b) Rigor-KIF5::YFP (green) failed to accumulate in the IS in the presence of 10 nM paclitaxel. (c and d) VSV-G::GFP was initially observed 1 h after the temperature shift to 30°C from 19.5°C without paclitaxel (c), and 10 nM paclitaxel (d) was added to the medium. Axonal transport from the IS was unchanged. However, as a result of the decrease in the supply of vesicles from TGN, the amount of vesicles in the IS markedly decreased 1 h later (d, arrow). Note that dendrite staining is elevated in d. (e and f) β-APP::YFP was expressed overnight without (e) or with paclitaxel (f). (g and h) Kv2.1::YFP was expressed overnight without (g) or with paclitaxel (h). Kv2.1 (green) is a potassium channel sorted to the cell body and dendrites in the absence (g) and in the presence of paclitaxel (h). MAP2 was stained with red. Bars, 10 μm. (i) MT polarity is mixed in proximal dendrites after incubation with 100 nM paclitaxel overnight. (inset) Axonal MTs. Polarity of MTs is determined by the curvature of hooks in the electron micrographs (arrows). Bar, 100 nm. Electron micrograph, which shows MT organization in paclitaxel treated neurons, is available as Fig. S3. (j) Dose dependence of membrane phenotype and that of motor phenotype are similar. For membrane phenotype, percentage of neurons, which show polarized axonal transport of VSV-G::GFP are presented (n = 3, each 50 cells counted). For motor phenotype, percentage of neurons, which show preferential binding of rigor-KIF5 to the IS are presented (n = 2, each 50 cells counted). (k–n) In vitro reconstitution of preferential association of KIF5 to the MTs in the IS. Hippocampal neuronal cytoskeleton was prepared by permeabilization with 0.1% Triton X-100, and recombinant tailless-KIF5::YFP was added to the cytoskeleton (k and m). Subsequently, the cells were fixed and stained with anti-MAP2 antibody. In l and n, MAP2 staining (red) is superimposed on the KIF5 images in k and m (green). In control neurons, intense binding of KIF5 to the IS was observed (k and l, arrows), compared with other neurites. In contrast, when neurons were pretreated with 10 nM paclitaxel before permeabilization, binding of KIF5 to the axonal IS was similar to other neurites (m and n, arrows).
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Related In: Results  -  Collection

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fig5: Paclitaxel at a low dose reverts the polarized sorting of axonal motor proteins as well as membrane proteins. Data were obtained by CLSM with small pinhole and maximal Z-projection (b, k, l, m, and n) and with pinhole fully open (a, c, d, e, f, g, and h). Arrows indicate axons. MAP2 (red) was stained in a, b, e, l, and m. (a) Tailless KIF5::YFP (green) was sorted to dendrite tips in the presence of 10 nM paclitaxel. (b) Rigor-KIF5::YFP (green) failed to accumulate in the IS in the presence of 10 nM paclitaxel. (c and d) VSV-G::GFP was initially observed 1 h after the temperature shift to 30°C from 19.5°C without paclitaxel (c), and 10 nM paclitaxel (d) was added to the medium. Axonal transport from the IS was unchanged. However, as a result of the decrease in the supply of vesicles from TGN, the amount of vesicles in the IS markedly decreased 1 h later (d, arrow). Note that dendrite staining is elevated in d. (e and f) β-APP::YFP was expressed overnight without (e) or with paclitaxel (f). (g and h) Kv2.1::YFP was expressed overnight without (g) or with paclitaxel (h). Kv2.1 (green) is a potassium channel sorted to the cell body and dendrites in the absence (g) and in the presence of paclitaxel (h). MAP2 was stained with red. Bars, 10 μm. (i) MT polarity is mixed in proximal dendrites after incubation with 100 nM paclitaxel overnight. (inset) Axonal MTs. Polarity of MTs is determined by the curvature of hooks in the electron micrographs (arrows). Bar, 100 nm. Electron micrograph, which shows MT organization in paclitaxel treated neurons, is available as Fig. S3. (j) Dose dependence of membrane phenotype and that of motor phenotype are similar. For membrane phenotype, percentage of neurons, which show polarized axonal transport of VSV-G::GFP are presented (n = 3, each 50 cells counted). For motor phenotype, percentage of neurons, which show preferential binding of rigor-KIF5 to the IS are presented (n = 2, each 50 cells counted). (k–n) In vitro reconstitution of preferential association of KIF5 to the MTs in the IS. Hippocampal neuronal cytoskeleton was prepared by permeabilization with 0.1% Triton X-100, and recombinant tailless-KIF5::YFP was added to the cytoskeleton (k and m). Subsequently, the cells were fixed and stained with anti-MAP2 antibody. In l and n, MAP2 staining (red) is superimposed on the KIF5 images in k and m (green). In control neurons, intense binding of KIF5 to the IS was observed (k and l, arrows), compared with other neurites. In contrast, when neurons were pretreated with 10 nM paclitaxel before permeabilization, binding of KIF5 to the axonal IS was similar to other neurites (m and n, arrows).
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