HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Supplemental Data Buy All Versions of this Article: fj.09-134304v1 24/1/105    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Delanote, V. Articles by Gettemans, J. Search for Related Content PubMed PubMed Citation Articles by Delanote, V. Articles by Gettemans, J.

An alpaca single-domain antibody blocks filopodia formation by obstructing L-plastin-mediated F-actin bundling

Veerle Delanote^*,,1, Berlinda Vanloo^*,, Marie Catillon, Evelyne Friederich, Joël Vandekerckhove^*, and Jan Gettemans^*,,2

^* Department of Medical Protein Research, Flanders Institute for Biotechnology (VIB), Ghent, Belgium;

Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium; and

Life Sciences Research Unit, Faculty of Sciences, Technology, and Communication, University of Luxembourg, Luxembourg

² Correspondence: Department of Biochemistry, Ghent University, Albert Baertsoenkaai 3, B-9000 Ghent, Belgium. E-mail: jan.gettemans{at}ugent.be

L-plastin, a conserved modular F-actin bundling protein, is ectopically expressed in tumor cells and contributes to cell malignancy and invasion. The underlying molecular mechanisms involved remain unclear, in part, because specific inhibitors of L-plastin are lacking. We used recombinant alpaca-derived L-plastin single-domain antibodies (nanobodies) as effector of L-plastin function in cells. Key findings were compared with L-plastin down-regulation by RNAi. We show that nanobodies strongly interact with L-plastin by targeting discrete conformational epitopes with nanomolar affinity. A nanobody that selectively interacts with the tandem ABDs in L-plastin completely inhibits F-actin bundling at equimolar ratios, in contrast to a control green fluorescent protein (GFP) nanobody. This "knockout" nanobody inhibits filopodia formation, motility, and invasion when expressed in PC-3 cells. L-plastin RNA interference showed no significant effect on filopodial integrity and only marginally restrained the motile properties of cells. L-plastin nanobodies uniquely expose a fundamental role for this protein in filopodia formation and cell migration. Therefore, these molecules represent a potent instrument to ablate functions of structural proteins without manipulating gene expression. In addition, we show that they can be instrumental in uncovering new functions of proteins that remain obscured by RNAi.—Delanote, V., Vanloo, B., Catillon, M., Friederich, E., Vandekerckhove, J., Gettemans, J. An alpaca single-domain antibody blocks filopodia formation by obstructing L-plastin-mediated F-actin bundling.

Key Words: immunomodulation • cytoskeleton • fimbrin • cancer cell • invasion