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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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Analysis of axonal and dendrite carrier movement by CAFM. CAFM images of the post-Golgi complex carriers to the axon (a: VSV-G::GFP) and to the dendrites (b: Kv2.1::GFP). Two images at 10-s intervals were merged into one image to show the displacements of individual carriers for 10 s (red images are obtained 10 s later than the green images). Axonal tubulovesicular organelles showed high motility and dominantly transported to the axon (arrows in a indicate three moving carriers). In contrast, post-Golgi carriers for Kv2.1 were vesicular in shape and do not show directional preference in motility (inset in b shows a high magnification of the area indicated by an arrow. Three moving carriers are indicated by arrows in the inset. Note carriers with displacements less than their diameter show overlapping yellow region of two merged images). Corresponding videos are available as Videos 3–5. (c–e) Quantitative analysis of the dynamics of axonal versus dendrite carriers. (c) Percentage of the carriers which shows 0 μm, <1 μm, >1 μm displacements in 10 s in the cell body. Black bar, Kv2.1 carriers; white bar, VSV-G carriers. Data were obtained from three independent cultures. 200 carriers were counted in each culture. (d) Number of the post-Golgi carriers remaining within the cell body. Carriers were counted at 1 h after the start of the post-Golgi transport. Black bar, Kv2.1 carriers; white bar, VSV-G carriers. (e) Histogram of the run-length of individual 100 post-Golgi carriers of Kv2.1 and VSV-G. Carriers, which showed no displacement, were omitted from the histogram. Carriers, which move into neurites and get out of the observing field, were included in the group of >5 μm run-length. (f) Simultaneous double labeling with Kv2.1::YFP (green) and VSV-G::CFP::CFP (red). Note that they colocalize in the Golgi complex area. Kv2.1 carriers distributed in the somatodendritic area, but VSV-G carriers move to the IS (arrow). Bars, 10 μm.
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fig2: Analysis of axonal and dendrite carrier movement by CAFM. CAFM images of the post-Golgi complex carriers to the axon (a: VSV-G::GFP) and to the dendrites (b: Kv2.1::GFP). Two images at 10-s intervals were merged into one image to show the displacements of individual carriers for 10 s (red images are obtained 10 s later than the green images). Axonal tubulovesicular organelles showed high motility and dominantly transported to the axon (arrows in a indicate three moving carriers). In contrast, post-Golgi carriers for Kv2.1 were vesicular in shape and do not show directional preference in motility (inset in b shows a high magnification of the area indicated by an arrow. Three moving carriers are indicated by arrows in the inset. Note carriers with displacements less than their diameter show overlapping yellow region of two merged images). Corresponding videos are available as Videos 3–5. (c–e) Quantitative analysis of the dynamics of axonal versus dendrite carriers. (c) Percentage of the carriers which shows 0 μm, <1 μm, >1 μm displacements in 10 s in the cell body. Black bar, Kv2.1 carriers; white bar, VSV-G carriers. Data were obtained from three independent cultures. 200 carriers were counted in each culture. (d) Number of the post-Golgi carriers remaining within the cell body. Carriers were counted at 1 h after the start of the post-Golgi transport. Black bar, Kv2.1 carriers; white bar, VSV-G carriers. (e) Histogram of the run-length of individual 100 post-Golgi carriers of Kv2.1 and VSV-G. Carriers, which showed no displacement, were omitted from the histogram. Carriers, which move into neurites and get out of the observing field, were included in the group of >5 μm run-length. (f) Simultaneous double labeling with Kv2.1::YFP (green) and VSV-G::CFP::CFP (red). Note that they colocalize in the Golgi complex area. Kv2.1 carriers distributed in the somatodendritic area, but VSV-G carriers move to the IS (arrow). Bars, 10 μm.

Mentions: Next, we compared the polarized axonal transport with dendrite transport within the cell body. Because the construct with longer spacers is reported to reduce the effect of GFP tagging on VSV-G sorting in MDCK cells (Keller et al., 2001), we tried the same construct to test whether it could be a marker for dendrite transport. However, its effect was insufficient in the neurons, and a considerable amount of the protein was still sorted to axons (unpublished data). We used Kv2.1::YFP, a potassium channel that is sorted to somatodendritic plasma membrane (Lim et al., 2000), in order to visualize individual post-Golgi dendrite carriers. Among the number of channels and receptors we tried, most of which showed considerable amount of ER retention, which obscured the observation of the post-Golgi dendrite transport, Kv2.1::YFP was accumulated in the Golgi region by brefeldin A washout treatment (Fig. 1 a), which enabled us to follow the subsequent post-Golgi transport. Brefeldin A washout procedure did not affect on sorting as well as the time course of distribution of Kv2.1::YFP within dendrites and used in Fig. 1 a and Fig. 2 (a–e). Simultaneous expression of VSV-G::CFP::CFP and Kv2.1::YFP showed each markers are properly targeted to axonal and dendrite carriers while they are colocalized in the Golgi region (Fig. 2 f). We used CAFM (see Materials and methods), which enabled us to visualize small dim dendrite vesicles that our confocal laser scan microscopy (CLSM) system could not visualize. Our CAFM image of biased axonal transport of VSV-G was fully consistent with the CLSM image. Intense staining in the center of the cell body in Fig. 2 (a and b) are the basal surface of the Golgi region demonstrating the depth of CAFM image from the coverslip. We found that VSV-G carriers were tubular and vesicular in shape, highly motile with long processivity, and preferentially transported to axons (Fig. 2 a; Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1), whereas Kv2.1 carriers were vesicular in shape, less motile with short processivity, and evenly distributed within the cell body (Fig. 2 b; Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1). Comparison of percentage of the carriers with >1 μm displacements in 10 s showed that VSV-G carriers have much higher motile activity than Kv2.1 carriers (Fig. 2 c), which results in the smaller number of VSV-G carriers remaining in the cell body (Fig. 2 d). These differences are not due to the time-lapse imaging at a 5-s interval, as the time-lapse video with a 0.5-s interval (Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1) showed the same tendency of Kv2.1 carrier movements. Next, we compared the run-length of each 100 individual carriers before they stop or change the direction of movements (Fig. 2 e). We found ∼50% of VSV-G carriers show >5 μm processive movements, whereas >50% of Kv2.1 carriers show <1 μm processivity. Given the average run-length of single kinesin motor proteins ∼0.6 μm (Vale et al., 1996), the data indicate that multiple active motors are associated with a single VSV-G carrier. This motile property of VSV-G carriers will be suitable for the polarized axonal transport because once the carriers choose the axonal MTs, they will continue to translocate along them until they get out of the cell body.


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

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

Analysis of axonal and dendrite carrier movement by CAFM. CAFM images of the post-Golgi complex carriers to the axon (a: VSV-G::GFP) and to the dendrites (b: Kv2.1::GFP). Two images at 10-s intervals were merged into one image to show the displacements of individual carriers for 10 s (red images are obtained 10 s later than the green images). Axonal tubulovesicular organelles showed high motility and dominantly transported to the axon (arrows in a indicate three moving carriers). In contrast, post-Golgi carriers for Kv2.1 were vesicular in shape and do not show directional preference in motility (inset in b shows a high magnification of the area indicated by an arrow. Three moving carriers are indicated by arrows in the inset. Note carriers with displacements less than their diameter show overlapping yellow region of two merged images). Corresponding videos are available as Videos 3–5. (c–e) Quantitative analysis of the dynamics of axonal versus dendrite carriers. (c) Percentage of the carriers which shows 0 μm, <1 μm, >1 μm displacements in 10 s in the cell body. Black bar, Kv2.1 carriers; white bar, VSV-G carriers. Data were obtained from three independent cultures. 200 carriers were counted in each culture. (d) Number of the post-Golgi carriers remaining within the cell body. Carriers were counted at 1 h after the start of the post-Golgi transport. Black bar, Kv2.1 carriers; white bar, VSV-G carriers. (e) Histogram of the run-length of individual 100 post-Golgi carriers of Kv2.1 and VSV-G. Carriers, which showed no displacement, were omitted from the histogram. Carriers, which move into neurites and get out of the observing field, were included in the group of >5 μm run-length. (f) Simultaneous double labeling with Kv2.1::YFP (green) and VSV-G::CFP::CFP (red). Note that they colocalize in the Golgi complex area. Kv2.1 carriers distributed in the somatodendritic area, but VSV-G carriers move to the IS (arrow). Bars, 10 μm.
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Related In: Results  -  Collection

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fig2: Analysis of axonal and dendrite carrier movement by CAFM. CAFM images of the post-Golgi complex carriers to the axon (a: VSV-G::GFP) and to the dendrites (b: Kv2.1::GFP). Two images at 10-s intervals were merged into one image to show the displacements of individual carriers for 10 s (red images are obtained 10 s later than the green images). Axonal tubulovesicular organelles showed high motility and dominantly transported to the axon (arrows in a indicate three moving carriers). In contrast, post-Golgi carriers for Kv2.1 were vesicular in shape and do not show directional preference in motility (inset in b shows a high magnification of the area indicated by an arrow. Three moving carriers are indicated by arrows in the inset. Note carriers with displacements less than their diameter show overlapping yellow region of two merged images). Corresponding videos are available as Videos 3–5. (c–e) Quantitative analysis of the dynamics of axonal versus dendrite carriers. (c) Percentage of the carriers which shows 0 μm, <1 μm, >1 μm displacements in 10 s in the cell body. Black bar, Kv2.1 carriers; white bar, VSV-G carriers. Data were obtained from three independent cultures. 200 carriers were counted in each culture. (d) Number of the post-Golgi carriers remaining within the cell body. Carriers were counted at 1 h after the start of the post-Golgi transport. Black bar, Kv2.1 carriers; white bar, VSV-G carriers. (e) Histogram of the run-length of individual 100 post-Golgi carriers of Kv2.1 and VSV-G. Carriers, which showed no displacement, were omitted from the histogram. Carriers, which move into neurites and get out of the observing field, were included in the group of >5 μm run-length. (f) Simultaneous double labeling with Kv2.1::YFP (green) and VSV-G::CFP::CFP (red). Note that they colocalize in the Golgi complex area. Kv2.1 carriers distributed in the somatodendritic area, but VSV-G carriers move to the IS (arrow). Bars, 10 μm.
Mentions: Next, we compared the polarized axonal transport with dendrite transport within the cell body. Because the construct with longer spacers is reported to reduce the effect of GFP tagging on VSV-G sorting in MDCK cells (Keller et al., 2001), we tried the same construct to test whether it could be a marker for dendrite transport. However, its effect was insufficient in the neurons, and a considerable amount of the protein was still sorted to axons (unpublished data). We used Kv2.1::YFP, a potassium channel that is sorted to somatodendritic plasma membrane (Lim et al., 2000), in order to visualize individual post-Golgi dendrite carriers. Among the number of channels and receptors we tried, most of which showed considerable amount of ER retention, which obscured the observation of the post-Golgi dendrite transport, Kv2.1::YFP was accumulated in the Golgi region by brefeldin A washout treatment (Fig. 1 a), which enabled us to follow the subsequent post-Golgi transport. Brefeldin A washout procedure did not affect on sorting as well as the time course of distribution of Kv2.1::YFP within dendrites and used in Fig. 1 a and Fig. 2 (a–e). Simultaneous expression of VSV-G::CFP::CFP and Kv2.1::YFP showed each markers are properly targeted to axonal and dendrite carriers while they are colocalized in the Golgi region (Fig. 2 f). We used CAFM (see Materials and methods), which enabled us to visualize small dim dendrite vesicles that our confocal laser scan microscopy (CLSM) system could not visualize. Our CAFM image of biased axonal transport of VSV-G was fully consistent with the CLSM image. Intense staining in the center of the cell body in Fig. 2 (a and b) are the basal surface of the Golgi region demonstrating the depth of CAFM image from the coverslip. We found that VSV-G carriers were tubular and vesicular in shape, highly motile with long processivity, and preferentially transported to axons (Fig. 2 a; Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1), whereas Kv2.1 carriers were vesicular in shape, less motile with short processivity, and evenly distributed within the cell body (Fig. 2 b; Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1). Comparison of percentage of the carriers with >1 μm displacements in 10 s showed that VSV-G carriers have much higher motile activity than Kv2.1 carriers (Fig. 2 c), which results in the smaller number of VSV-G carriers remaining in the cell body (Fig. 2 d). These differences are not due to the time-lapse imaging at a 5-s interval, as the time-lapse video with a 0.5-s interval (Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200302175/DC1) showed the same tendency of Kv2.1 carrier movements. Next, we compared the run-length of each 100 individual carriers before they stop or change the direction of movements (Fig. 2 e). We found ∼50% of VSV-G carriers show >5 μm processive movements, whereas >50% of Kv2.1 carriers show <1 μm processivity. Given the average run-length of single kinesin motor proteins ∼0.6 μm (Vale et al., 1996), the data indicate that multiple active motors are associated with a single VSV-G carrier. This motile property of VSV-G carriers will be suitable for the polarized axonal transport because once the carriers choose the axonal MTs, they will continue to translocate along them until they get out of the cell body.

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