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

P. Sukumvanich, Vera M. Desmarais, C. V. Sarmiento, Yarong Wang, I. Ichetovkin, G. Mouneimne, Steven C. Almo, John S. Condeelis

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)141-152
Number of pages12
JournalCell Motility and the Cytoskeleton
Volume59
Issue number2
DOIs
StatePublished - Oct 2004

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Epidermal Growth Factor
Antibodies
Wiskott-Aldrich Syndrome Protein Family
Immunoprecipitation
Cicatrix
Epitopes
Staining and Labeling
Carcinoma

Keywords

  • Actin
  • Cytoskeleton
  • EGF stimulation
  • Filopodia
  • Lamellipod extension

ASJC Scopus subject areas

  • Cell Biology

Cite this

Cellular localization of activated N-WASP using a conformation-sensitive antibody. / Sukumvanich, P.; Desmarais, Vera M.; Sarmiento, C. V.; Wang, Yarong; Ichetovkin, I.; Mouneimne, G.; Almo, Steven C.; Condeelis, John S.

In: Cell Motility and the Cytoskeleton, Vol. 59, No. 2, 10.2004, p. 141-152.

Research output: Contribution to journalArticle

Sukumvanich, P. ; Desmarais, Vera M. ; Sarmiento, C. V. ; Wang, Yarong ; Ichetovkin, I. ; Mouneimne, G. ; Almo, Steven C. ; Condeelis, John S. / Cellular localization of activated N-WASP using a conformation-sensitive antibody. In: Cell Motility and the Cytoskeleton. 2004 ; Vol. 59, No. 2. pp. 141-152.
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