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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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Plasma membrane association decreases diffusion of GAP43 in the cell body and neuritic shaft. (A) Time-lapse microscopy of PC12 cells transfected with GAP43wt-PAGFP after fluorescence photoactivation in the cell body (top) or in the middle of a process (bottom). The position of photoactivation is indicated by a red circle in the preactivation image (pre). Arrowheads mark regions where plasma membrane association is evident. Scale bar, 10 μm. (B) FDAP of cells expressing GAP43 constructs (top) or reference constructs (bottom) after photoactivation in the cell body. The plots represent the fraction of fluorescence measured in the activated region and total fluorescence of cells. FDAP is fastest with GAP43S41A-PAGFP and 3×PAGFP, indicating quick dissipation of the cytsosolic constructs. FDAP is slowest with GAP43S41D-PAGFP and PAGFP-F, which exhibit plasma membrane association. GAP43wt-PAGFP exhibits intermediate FDAP, reflecting partial binding to the plasma membrane. Mean ± SEM, n = 10–39. (C) Determination of diffusion constants after modeling of FDAP curves as shown in B. Diffusion in the cell body was determined only for the plasma membrane–associated proteins since the geometry of activation did not permit modeling of the dissipation of cytosolic proteins. Mean ± SEM. *,***Significantly lower values compared with the cytosolic reference protein (3×PAGFP); osignificantly lower value compared with the diffusion of the same construct in the process. Statistical analysis was performed using Student's t test. p values are as follows: *(o), p < 0.05 ***, p < 0.001.
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Figure 4: Plasma membrane association decreases diffusion of GAP43 in the cell body and neuritic shaft. (A) Time-lapse microscopy of PC12 cells transfected with GAP43wt-PAGFP after fluorescence photoactivation in the cell body (top) or in the middle of a process (bottom). The position of photoactivation is indicated by a red circle in the preactivation image (pre). Arrowheads mark regions where plasma membrane association is evident. Scale bar, 10 μm. (B) FDAP of cells expressing GAP43 constructs (top) or reference constructs (bottom) after photoactivation in the cell body. The plots represent the fraction of fluorescence measured in the activated region and total fluorescence of cells. FDAP is fastest with GAP43S41A-PAGFP and 3×PAGFP, indicating quick dissipation of the cytsosolic constructs. FDAP is slowest with GAP43S41D-PAGFP and PAGFP-F, which exhibit plasma membrane association. GAP43wt-PAGFP exhibits intermediate FDAP, reflecting partial binding to the plasma membrane. Mean ± SEM, n = 10–39. (C) Determination of diffusion constants after modeling of FDAP curves as shown in B. Diffusion in the cell body was determined only for the plasma membrane–associated proteins since the geometry of activation did not permit modeling of the dissipation of cytosolic proteins. Mean ± SEM. *,***Significantly lower values compared with the cytosolic reference protein (3×PAGFP); osignificantly lower value compared with the diffusion of the same construct in the process. Statistical analysis was performed using Student's t test. p values are as follows: *(o), p < 0.05 ***, p < 0.001.

Mentions: To determine whether plasma membrane association affects the intracellular mobility of GAP43, we performed photoactivation experiments in the cell body and in processes of neuronally differentiated cells. Photoactivation was performed by a laser flash at 405 nm in a 5-μm spot, and GFP fluorescence was followed by time-lapse imaging at an excitation wavelength of 488 nm as described previously (Weissmann et al., 2009). Photoactivated GAP43wt-PAGFP quickly dissipated within the cell and showed partial association with the periphery both in the cell body and the process, indicative for plasma membrane association of a subpopulation (Figure 4A, arrowheads). To quantitatively analyze protein mobility, we recorded fluorescence decay in activated regions in the cell body and middle of neurites over time and plotted the result. Total fluorescence did not decrease with time, confirming high photostability of PAGFP at the conditions of imaging (example shows recording in the cell body; Figure 4B). Thus fluorescence decay after photoactivation (FDAP) directly reflected differences in the mobility of the activated proteins.


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)

Plasma membrane association decreases diffusion of GAP43 in the cell body and neuritic shaft. (A) Time-lapse microscopy of PC12 cells transfected with GAP43wt-PAGFP after fluorescence photoactivation in the cell body (top) or in the middle of a process (bottom). The position of photoactivation is indicated by a red circle in the preactivation image (pre). Arrowheads mark regions where plasma membrane association is evident. Scale bar, 10 μm. (B) FDAP of cells expressing GAP43 constructs (top) or reference constructs (bottom) after photoactivation in the cell body. The plots represent the fraction of fluorescence measured in the activated region and total fluorescence of cells. FDAP is fastest with GAP43S41A-PAGFP and 3×PAGFP, indicating quick dissipation of the cytsosolic constructs. FDAP is slowest with GAP43S41D-PAGFP and PAGFP-F, which exhibit plasma membrane association. GAP43wt-PAGFP exhibits intermediate FDAP, reflecting partial binding to the plasma membrane. Mean ± SEM, n = 10–39. (C) Determination of diffusion constants after modeling of FDAP curves as shown in B. Diffusion in the cell body was determined only for the plasma membrane–associated proteins since the geometry of activation did not permit modeling of the dissipation of cytosolic proteins. Mean ± SEM. *,***Significantly lower values compared with the cytosolic reference protein (3×PAGFP); osignificantly lower value compared with the diffusion of the same construct in the process. Statistical analysis was performed using Student's t test. p values are as follows: *(o), p < 0.05 ***, p < 0.001.
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Figure 4: Plasma membrane association decreases diffusion of GAP43 in the cell body and neuritic shaft. (A) Time-lapse microscopy of PC12 cells transfected with GAP43wt-PAGFP after fluorescence photoactivation in the cell body (top) or in the middle of a process (bottom). The position of photoactivation is indicated by a red circle in the preactivation image (pre). Arrowheads mark regions where plasma membrane association is evident. Scale bar, 10 μm. (B) FDAP of cells expressing GAP43 constructs (top) or reference constructs (bottom) after photoactivation in the cell body. The plots represent the fraction of fluorescence measured in the activated region and total fluorescence of cells. FDAP is fastest with GAP43S41A-PAGFP and 3×PAGFP, indicating quick dissipation of the cytsosolic constructs. FDAP is slowest with GAP43S41D-PAGFP and PAGFP-F, which exhibit plasma membrane association. GAP43wt-PAGFP exhibits intermediate FDAP, reflecting partial binding to the plasma membrane. Mean ± SEM, n = 10–39. (C) Determination of diffusion constants after modeling of FDAP curves as shown in B. Diffusion in the cell body was determined only for the plasma membrane–associated proteins since the geometry of activation did not permit modeling of the dissipation of cytosolic proteins. Mean ± SEM. *,***Significantly lower values compared with the cytosolic reference protein (3×PAGFP); osignificantly lower value compared with the diffusion of the same construct in the process. Statistical analysis was performed using Student's t test. p values are as follows: *(o), p < 0.05 ***, p < 0.001.
Mentions: To determine whether plasma membrane association affects the intracellular mobility of GAP43, we performed photoactivation experiments in the cell body and in processes of neuronally differentiated cells. Photoactivation was performed by a laser flash at 405 nm in a 5-μm spot, and GFP fluorescence was followed by time-lapse imaging at an excitation wavelength of 488 nm as described previously (Weissmann et al., 2009). Photoactivated GAP43wt-PAGFP quickly dissipated within the cell and showed partial association with the periphery both in the cell body and the process, indicative for plasma membrane association of a subpopulation (Figure 4A, arrowheads). To quantitatively analyze protein mobility, we recorded fluorescence decay in activated regions in the cell body and middle of neurites over time and plotted the result. Total fluorescence did not decrease with time, confirming high photostability of PAGFP at the conditions of imaging (example shows recording in the cell body; Figure 4B). Thus fluorescence decay after photoactivation (FDAP) directly reflected differences in the mobility of the activated proteins.

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