Subcellular localization of collapsin response mediator proteins to lipid rafts.
Collapsin response mediator proteins (CRMPs) are involved in signal transduction after exposure of neural cells to the axon guidance molecule Semaphorin 3A/collapsin. All five known CRMPs are expressed in the developing cerebral cortex and neocortical neurons are responsive to Semaphorin 3A. Here, we examine the expression and subcellular localization of CRMPs in neocortical neurons and in neonatal rat brain. In neocortical neurons CRMP-4 was detected in the perikaryon with a diffuse cytosolic distribution. In neurites and at growth cones punctate staining patterns were observed. Extraction of neuron cultures with methyl-beta-cyclodextrin to deplete cholesterol caused rapid redistribution of the punctate CRMP-4 staining into larger patches and abundant growth cone collapse. Western blotting of brain extracts demonstrated for all CRMPs the existence of soluble, detergent-extractable, and Triton X-100-resistant forms. Furthermore, sucrose density gradient centrifugation after solubilization of brain membranes with Triton X-100 revealed that CRMP-1, -3, -5, and to a lower extent CRMP-4 are associated with a detergent-resistant fraction with low buoyant density, but CRMP-2 was not detectable in this fraction. Thus, we propose that lipid rafts form sites for the compartmentalization of signaling events involving specific CRMPs and that the integrity of these membrane microdomains is essential for the maintenance of growth cones.
Institut für Physiologische Chemie, Rheinische Friedrich-Wilhelms-Universität Bonn, Nussallee 11, 53115, Bonn, Germany.
Volker
V
Rosslenbroich
Lingsong
L
Dai
Sebastian
S
Franken
Marion
M
Gehrke
Ulrich
U
Junghans
Volkmar
V
Gieselmann
Joachim
J
Kappler
D008841:Actin Cytoskeleton / Q000648:ultrastructure; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000166:cytology / Q000254:growth & development; D001923:Brain Chemistry; D002460:Cell Line; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D003505:Cyclodextrins / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D021962:Membrane Microdomains / Q000737:chemistry*; D051379:Mice; D019579:Neocortex / Q000737:chemistry / Q000166:cytology; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000276:immunology; D009474:Neurons / Q000737:chemistry* / Q000648:ultrastructure; D051381:Rats; D017208:Rats, Wistar; D047392:beta-Cyclodextrins
D003505:Cyclodextrins; C449760:Dpysl3 protein, rat; D009419:Nerve Tissue Proteins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin