Abstract
The neuronal Growth Associated Protein 43 (GAP43), also known as B-50 or neuromodulin, is involved in mechanisms controlling pathfinding and branching of neurons during development and regeneration. For many years this protein was classified as neuron-specific, but recent evidences suggest that a) GAP43 is expressed in the nervous system not only in neurons, but also in glial cells, and b) probably it is present also in other tissues. In particular, its expression was revealed in muscles from patients affected by various myopathies, indicating that GAP43 can no-longer considered only as a neuron-specific molecule. We have investigated the expression and subcellular localization of GAP43 in mouse satellite cells, myotubes, and adult muscle (extensor digitorum longus or EDL) using Western blotting, immuno-fluorescence combined to confocal microscopy and electron microscopy. Our in vitro results indicated that GAP43 is indeed expressed in both myoblasts and differentiating myotubes, and its cellular localization changes dramatically during maturation: in myoblasts the localization appeared to be mostly nuclear, whereas with differentiation the protein started to display a sarcomeric-like pattern. In adult fibers, GAP43 expression was evident with the protein labeling forming (in longitudinal views) a double cross striation reminiscent of the staining pattern of other organelles, such as calcium release units (CRUs) and mitochondria. Double immuno-staining and experiments done in EDL muscles fixed at different sarcomere lengths, allowed us to determine the localization, from the sarcomere Z-line, of GAP43 positive foci, falling between that of CRUs and of mitochondria. Staining of cross sections added a detail to the puzzle: GAP43 labeling formed a reticular pattern surrounding individual myofibrils, but excluding contractile elements. This work leads the way to further investigation about the possible physiological and structural role of GAP43 protein in adult fiber function and disease.
Highlights
The neuronal Growth Associated Protein 43 (GAP43), known as neuromodulin, was first isolated about 30 years ago from synaptosomal plasma membranes of rat brain and identified as a B-50 phosphoprotein band [1,2]
In the present work we studied the expression and intracellular localization of GAP43 in mouse satellite cells, myotubes and adult skeletal fibers from mouse extensor digitorum longus (EDL) muscle, using Western blotting, immuno-fluorescence combined to confocal microscopy and electron microscopy (EM)
Expression of GAP43 in Skeletal Muscle Models The presence of GAP43 was verified in different sample preparations obtained from: proliferating mouse satellite cells, myotubes, and EDL muscle fibers, as well as from mouse brain used as positive control (Figure 1)
Summary
The neuronal Growth Associated Protein 43 (GAP43), known as neuromodulin, was first isolated about 30 years ago from synaptosomal plasma membranes of rat brain and identified as a B-50 phosphoprotein band [1,2] This protein is highly conserved, expressed in different species, and regulated by post-translational mechanisms, which modulate its intracellular functions [3]. Knockout of GAP43 in mice results in considerable reduction in dimensions of cerebellum, possibly due to the impaired neuronal branching and pathfinding, and leads to high rate mice lethality (90–95%) within 2 days after birth [8,9,10] These data are supported by data obtained in transgenic mice: overexpression of GAP43 leads to the spontaneous formation of new synapses and enhanced sprouting after injury [11]. Whereas transgenic mice expressing a mutant GAP43, that cannot be phosphorylated by protein kinase C (PKC), presented a reduced sprout-promoting activity, demonstrating that phosphorilation is crucial for GAP43 effect on neurite outgrowth [11]
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