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Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43.

Gauthier-Kemper A, Igaev M, Sündermann F, Janning D, Brühmann J, Moschner K, Reyher HJ, Junge W, Glebov K, Walter J, Bakota L, Brandt R - Mol. Biol. Cell (2014)

Bottom Line: Plasma membrane association decreased the diffusion constant fourfold in neuritic shafts.Simulations confirmed that a combination of diffusion, dynamic plasma membrane interaction and active transport of a small fraction of GAP43 suffices for efficient sorting to growth cones.Our data demonstrate a complex interplay between phosphorylation and lipidation in mediating the localization of GAP43 in neuronal cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Osnabrück, 49076 Osnabrück, Germany.

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Directed transport of GAP43 by transient piggybacking on exocytic vesicles. (A) Flux analysis of photoactivated protein in processes of transfected PC12 cells. Top, schematic showing the region of photoactivation (gray box) and the position of the recording regions distal and proximal from the center of activation. Bottom, ratios of distal to proximal fluorescence at different times after activation. Mean ± SEM, n = 7–9. The percentage of processes that show higher distal than proximal fluorescence is given in the boxes. *Significantly higher values compared with 1.0. (B) TIRF image of a part of a cell body of a living PC12 cell expressing GAP43-HaloTag (labeled with HTL-OG; green) and synaptophysin-mCherry (red). The focal plane was adjusted in such a way as to visualize a region above the cellular contact site, and the nucleus is visible at the right. Note the presence of green and red structures with vesicular appearance. Note that some structures show yellow fluorescence (arrowheads) indicative of colocalization of GAP43 with vesicular structures. Scale bar, 10 μm. Bottom, time series of TIRF micrographs at the transition of a neurite shaft (n.s.) to the growth cone (g.c.). An example of a particle showing colocalization of GAP43 and synaptophysin, which can be tracked for several seconds, is shown. Scale bar, 10 μm (left), 5 μm (right). The table gives numbers for individual tracking events with colocalization of HTL-OG–labeled GAP43-HaloTag and synaptophysin-mCherry. Note that double-labeled particles move with an average speed of 1.5 ± 0.2 μm/s (n = 10), similar to the speed of fast axonal transport. Statistical analysis was performed using Student's t test. *, p < 0.05.
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Figure 7: Directed transport of GAP43 by transient piggybacking on exocytic vesicles. (A) Flux analysis of photoactivated protein in processes of transfected PC12 cells. Top, schematic showing the region of photoactivation (gray box) and the position of the recording regions distal and proximal from the center of activation. Bottom, ratios of distal to proximal fluorescence at different times after activation. Mean ± SEM, n = 7–9. The percentage of processes that show higher distal than proximal fluorescence is given in the boxes. *Significantly higher values compared with 1.0. (B) TIRF image of a part of a cell body of a living PC12 cell expressing GAP43-HaloTag (labeled with HTL-OG; green) and synaptophysin-mCherry (red). The focal plane was adjusted in such a way as to visualize a region above the cellular contact site, and the nucleus is visible at the right. Note the presence of green and red structures with vesicular appearance. Note that some structures show yellow fluorescence (arrowheads) indicative of colocalization of GAP43 with vesicular structures. Scale bar, 10 μm. Bottom, time series of TIRF micrographs at the transition of a neurite shaft (n.s.) to the growth cone (g.c.). An example of a particle showing colocalization of GAP43 and synaptophysin, which can be tracked for several seconds, is shown. Scale bar, 10 μm (left), 5 μm (right). The table gives numbers for individual tracking events with colocalization of HTL-OG–labeled GAP43-HaloTag and synaptophysin-mCherry. Note that double-labeled particles move with an average speed of 1.5 ± 0.2 μm/s (n = 10), similar to the speed of fast axonal transport. Statistical analysis was performed using Student's t test. *, p < 0.05.

Mentions: Pharmacologic inhibition of transport indicated that a fraction of GAP43 is anterogradely transported. If true, a flux of GAP43 toward the tip of the process should be observed. To test this hypothesis, we photoactivated a population of GAP43 in the middle of the process and recorded changes in the fluorescence distal to and proximal from the center of activation (Figure 7A, top). Flux should then become evident by a ratio of distal/proximal fluorescence higher than one in the majority of the processes. Indeed, we observed increased distal/proximal ratios for GAP43wt, which became significant at 25 min (Figure 7A, middle). No flux was observed with the cytosolic control protein (3×PAGFP). Treatment with brefeldin A abolished distal flux, supporting a requirement for vesicle transport in trafficking of GAP43. Distal flux was also observed with the phosphoblocking GAP43 construct (GAP43S41A), supporting that phosphorylation-mediated membrane reaction is not required for transport of GAP43 (Figure 7A, bottom, right).


Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43.

Gauthier-Kemper A, Igaev M, Sündermann F, Janning D, Brühmann J, Moschner K, Reyher HJ, Junge W, Glebov K, Walter J, Bakota L, Brandt R - Mol. Biol. Cell (2014)

Directed transport of GAP43 by transient piggybacking on exocytic vesicles. (A) Flux analysis of photoactivated protein in processes of transfected PC12 cells. Top, schematic showing the region of photoactivation (gray box) and the position of the recording regions distal and proximal from the center of activation. Bottom, ratios of distal to proximal fluorescence at different times after activation. Mean ± SEM, n = 7–9. The percentage of processes that show higher distal than proximal fluorescence is given in the boxes. *Significantly higher values compared with 1.0. (B) TIRF image of a part of a cell body of a living PC12 cell expressing GAP43-HaloTag (labeled with HTL-OG; green) and synaptophysin-mCherry (red). The focal plane was adjusted in such a way as to visualize a region above the cellular contact site, and the nucleus is visible at the right. Note the presence of green and red structures with vesicular appearance. Note that some structures show yellow fluorescence (arrowheads) indicative of colocalization of GAP43 with vesicular structures. Scale bar, 10 μm. Bottom, time series of TIRF micrographs at the transition of a neurite shaft (n.s.) to the growth cone (g.c.). An example of a particle showing colocalization of GAP43 and synaptophysin, which can be tracked for several seconds, is shown. Scale bar, 10 μm (left), 5 μm (right). The table gives numbers for individual tracking events with colocalization of HTL-OG–labeled GAP43-HaloTag and synaptophysin-mCherry. Note that double-labeled particles move with an average speed of 1.5 ± 0.2 μm/s (n = 10), similar to the speed of fast axonal transport. Statistical analysis was performed using Student's t test. *, p < 0.05.
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Figure 7: Directed transport of GAP43 by transient piggybacking on exocytic vesicles. (A) Flux analysis of photoactivated protein in processes of transfected PC12 cells. Top, schematic showing the region of photoactivation (gray box) and the position of the recording regions distal and proximal from the center of activation. Bottom, ratios of distal to proximal fluorescence at different times after activation. Mean ± SEM, n = 7–9. The percentage of processes that show higher distal than proximal fluorescence is given in the boxes. *Significantly higher values compared with 1.0. (B) TIRF image of a part of a cell body of a living PC12 cell expressing GAP43-HaloTag (labeled with HTL-OG; green) and synaptophysin-mCherry (red). The focal plane was adjusted in such a way as to visualize a region above the cellular contact site, and the nucleus is visible at the right. Note the presence of green and red structures with vesicular appearance. Note that some structures show yellow fluorescence (arrowheads) indicative of colocalization of GAP43 with vesicular structures. Scale bar, 10 μm. Bottom, time series of TIRF micrographs at the transition of a neurite shaft (n.s.) to the growth cone (g.c.). An example of a particle showing colocalization of GAP43 and synaptophysin, which can be tracked for several seconds, is shown. Scale bar, 10 μm (left), 5 μm (right). The table gives numbers for individual tracking events with colocalization of HTL-OG–labeled GAP43-HaloTag and synaptophysin-mCherry. Note that double-labeled particles move with an average speed of 1.5 ± 0.2 μm/s (n = 10), similar to the speed of fast axonal transport. Statistical analysis was performed using Student's t test. *, p < 0.05.
Mentions: Pharmacologic inhibition of transport indicated that a fraction of GAP43 is anterogradely transported. If true, a flux of GAP43 toward the tip of the process should be observed. To test this hypothesis, we photoactivated a population of GAP43 in the middle of the process and recorded changes in the fluorescence distal to and proximal from the center of activation (Figure 7A, top). Flux should then become evident by a ratio of distal/proximal fluorescence higher than one in the majority of the processes. Indeed, we observed increased distal/proximal ratios for GAP43wt, which became significant at 25 min (Figure 7A, middle). No flux was observed with the cytosolic control protein (3×PAGFP). Treatment with brefeldin A abolished distal flux, supporting a requirement for vesicle transport in trafficking of GAP43. Distal flux was also observed with the phosphoblocking GAP43 construct (GAP43S41A), supporting that phosphorylation-mediated membrane reaction is not required for transport of GAP43 (Figure 7A, bottom, right).

Bottom Line: Plasma membrane association decreased the diffusion constant fourfold in neuritic shafts.Simulations confirmed that a combination of diffusion, dynamic plasma membrane interaction and active transport of a small fraction of GAP43 suffices for efficient sorting to growth cones.Our data demonstrate a complex interplay between phosphorylation and lipidation in mediating the localization of GAP43 in neuronal cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Osnabrück, 49076 Osnabrück, Germany.

Show MeSH
Related in: MedlinePlus