Cellular localization of activated N-WASP using a conformation-sensitive antibody

The main regulators of Arp2/3 activity appear to be N‐WASP and the other members of the Scar/WAVE family of proteins. We show here that after EGF stimulation, N‐WASP is recruited to the nucleation zone of the dynamic leading edge compartment of carcinoma cells, with maximal recruitment of N‐WASP wit...

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Published in:	Cell motility and the cytoskeleton Vol. 59; no. 2; pp. 141 - 152
Main Authors:	Sukumvanich, P., DesMarais, V., Sarmiento, C.V., Wang, Y., Ichetovkin, I., Mouneimne, G., Almo, S., Condeelis, J.
Format:	Journal Article
Language:	English
Published:	Hoboken Wiley Subscription Services, Inc., A Wiley Company 01-10-2004
Subjects:	actin Actin Cytoskeleton - metabolism Animals Antibodies - immunology cdc42 GTP-Binding Protein - metabolism Cell Shape - drug effects Cell Shape - physiology cytoskeleton Cytoskeleton - metabolism EGF stimulation Epidermal Growth Factor - pharmacology filopodia Immunoprecipitation lamellipod extension Molecular Conformation Nerve Tissue Proteins - metabolism Pseudopodia - metabolism Rats Tumor Cells, Cultured Wiskott-Aldrich Syndrome Protein, Neuronal
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Summary:	The main regulators of Arp2/3 activity appear to be N‐WASP and the other members of the Scar/WAVE family of proteins. We show here that after EGF stimulation, N‐WASP is recruited to the nucleation zone of the dynamic leading edge compartment of carcinoma cells, with maximal recruitment of N‐WASP within 1 min after EGF stimulation. The timing of N‐WASP recruitment mirrors the timing of barbed‐end formation at the leading edge. To determine the cellular activation of N‐WASP after EGF stimulation, we made a conformation‐sensitive antibody (CSA) against the CRIB domain of N‐WASP that is predicted to recognize N‐WASP in its open, active conformation, but not in its closed, inactive conformation. The ability of CSA to detect only active N‐WASP was demonstrated by in vitro experiments using immunoprecipitation of active N‐WASP from EGF‐stimulated cells and Cdc42 activation of N‐WASP activity. In cell staining experiments, N‐WASP is maximally accessible to CSA 40 sec after EGF stimulation and this activated N‐WASP is in the nucleation zone. These results indicate that active N‐WASP is present at the leading edge of lamellipods, an unexpected finding given its reported involvement in filopod formation. This work establishes the feasibility of using antibodies directed against specific conformations or epitopes with changing accessibilities as a window on the status and localization of activity. Cell Motil. Cytoskeleton 59:141–152, 2004. © 2004 Wiley‐Liss, Inc.
Bibliography:	NIH - No. 38511 ark:/67375/WNG-VLHJ05S6-R ArticleID:CM20030 istex:7CFA25F0D8F4B7D85A38F9B4144675AA4091F703 The first two authors contributed equally to this work. ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0886-1544 1097-0169
DOI:	10.1002/cm.20030