Collapsin response mediator protein-4 regulates F-actin bundling |
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Authors: | Rosslenbroich Volker Dai Lingsong Baader Stephan L Noegel Angelika A Gieselmann Volkmar Kappler Joachim |
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Institution: | Institut für Physiologische Chemie, Rheinische Friedrich-Wilhelms-Universit?t Bonn, Nussallee 11, D-53115 Bonn, Germany. |
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Abstract: | 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. |
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Keywords: | ADF actin depolymerizing factor arp2/3 actin related protein 2/3 CB cytochalasin B CRMP collapsin response mediator protein GSK-3 glycogen synthase kinase 3 ICC immunocytochemistry LatA latrunculin A WB Western blotting |
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