B-50/GAP-43 potentiates cytoskeletal reorganization in raft domains.
B-50 (GAP-43) is a neural, membrane-associated protein that has been implicated in neurite outgrowth and guidance. Following stable transfection of Rat1 fibroblasts with B-50 cDNA we observed a dispersed distribution of B-50 immunoreactivity in flattened resting cells. In contrast, motile cells exhibited high concentrations of B-50 at the leading edge of ruffling membranes, coinciding with actin polymerization. Time-lapse studies on Rat1 fibroblasts transiently transfected with B-50/EGFP revealed that large vesicles originated from the ruffling membranes. These large vesicles (pinocytes) were found positive for Thy-1, a GPI-anchored protein, but negative for rab-5, an early endosome marker. In primary hippocampal neurons B-50 also colocalized completely with the raft marker Thy-1. Antibody-mediated cross-linking of Thy-1 in hippocampal neurons resulted in a redistribution of the intracellular protein B-50 to Thy-1-immunopositive membrane patches, whereas syntaxin was mainly excluded from the patches, showing that B-50 is associated with rafts. Academic Press.
Aarts
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LH
Rudolf Magnus Institute for Neurosciences, Department of Physiological Chemistry, Utrecht, The Netherlands.
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van Dalen
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van Rozen
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Schotman
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D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000648:ultrastructure; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000502:physiology* / Q000648:ultrastructure*; D018076:DNA, Complementary; D004622:Embryo, Mammalian; D005347:Fibroblasts; D019922:GAP-43 Protein / Q000235:genetics / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology* / Q000378:metabolism; D007150:Immunohistochemistry; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure*; D015388:Organelles / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018800:Thy-1 Antigens / Q000032:analysis; D014162:Transfection
D018076:DNA, Complementary; D019922:GAP-43 Protein; D017261:Glycosylphosphatidylinositols; D011993:Recombinant Fusion Proteins; D018800:Thy-1 Antigens