Collapsin response mediator protein-4 regulates F-actin bundling.
Collapsin response mediator proteins (CRMPs) form a family of cytosolic phosphoproteins which are involved in the signal transduction of semaphorin 3A leading to growth cone collapse. These proteins interact with a variety of cytosolic proteins including tubulin heterodimers. Here, we show that CRMP-4 co-localizes with F-actin in regular rib-like structures within lamellipodia of B35 neuroblastoma cells. Furthermore, depolymerization of actin fibers changed the distribution of GFP-CRMP-4 in vivo. In vitro, recombinant CRMP-4 formed homo-oligomers, bound to F-actin and organized F-actin into tight bundles. Both oligomerization and F-actin bundling depended on the C-terminal part of CRMP-4. The stoichiometry of actin and CRMP-4 in bundles was approximately 1:1 and the apparent equilibrium constant of the microfilament-CRMP-4 interaction was estimated from bundling assays as K(app) = 730 mM(-1). CRMP-4 was abundant in the cytosol of B35 neuroblastoma cells and its concentration was measured as approximately 1.7 microM. Overexpression of CRMP-4 inhibited the migration of B35 neuroblastoma cells, while knockdown of CRMP-4 enhanced cell migration and disturbed rib-like actin-structures in lamellipodia. Taken together, our data indicate that CRMP-4 promotes bundling of F-actin in vitro, that it is an important component of rib-like actin bundles in lamellipodia in vivo and that it functionally regulates the actin cytoskeleton in motile cells. These findings suggest a specific regulatory role of CRMP-4 towards the actin cytoskeleton which may by be relevant for growth cone collapse.
Rosslenbroich
Volker
V
Institut für Physiologische Chemie, Rheinische Friedrich-Wilhelms-Universität Bonn, Nussallee 11, D-53115 Bonn, Germany.
Dai
Lingsong
L
Baader
Stephan L
SL
Noegel
Angelika A
AA
Gieselmann
Volkmar
V
Kappler
Joachim
J
D000199:Actins / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D002465:Cell Movement; D003600:Cytosol / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011554:Pseudopodia / Q000737:chemistry; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
D000199:Actins; C449760:Dpysl3 protein, rat; D009419:Nerve Tissue Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins