Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43.
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.
Affiliation: Department of Neurobiology, University of Osnabrück, 49076 Osnabrück, Germany.Show MeSH
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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.
Affiliation: Department of Neurobiology, University of Osnabrück, 49076 Osnabrück, Germany.