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A llama-derived gelsolin single-domain antibody blocks gelsolin–G-actin interaction

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Abstract

RNA interference has tremendously advanced our understanding of gene function but recent reports have exposed undesirable side-effects. Recombinant Camelid single-domain antibodies (VHHs) provide an attractive means for studying protein function without affecting gene expression. We raised VHHs against gelsolin (GsnVHHs), a multifunctional actin-binding protein that controls cellular actin organization and migration. GsnVHH-induced delocalization of gelsolin to mitochondria or the nucleus in mammalian cells reveals distinct subpopulations including free gelsolin and actin-bound gelsolin complexes. GsnVHH 13 specifically recognizes Ca2+-activated gelsolin (K d ~10 nM) while GsnVHH 11 binds gelsolin irrespective of Ca2+ (K d ~5 nM) but completely blocks its interaction with G-actin. Both GsnVHHs trace gelsolin in membrane ruffles of EGF-stimulated MCF-7 cells and delay cell migration without affecting F-actin severing/cap** or actin nucleation activities by gelsolin. We conclude that VHHs represent a potent way of blocking structural proteins and that actin nucleation by gelsolin is more complex than previously anticipated.

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Acknowledgments

We thank Evelien Martens for technical support. This work was supported by the Fund for Scientific Research-Flanders (FWO-Vlaanderen), the Stichting tegen Kanker, the Vlaamse Liga tegen Kanker, the Concerted Actions Program of Ghent University (GOA), the Interuniversity attraction poles (IUAP06) and the VIB. AVdA is supported by the Vlaamse Liga tegen Kanker through a grant of the Stichting Emmanuel van der Schueren.

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Authors and Affiliations

  1. Department of Medical Protein Research, VIB, 9000, Ghent, Belgium

    Anske Van den Abbeele, Sarah De Clercq, Ariane De Ganck, Veerle De Corte, Berlinda Van Loo, Joël Vandekerckhove & Jan Gettemans

  2. Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Albert Baertsoenkaai 3, 9000, Ghent, Belgium

    Anske Van den Abbeele, Sarah De Clercq, Ariane De Ganck, Veerle De Corte, Berlinda Van Loo, Joël Vandekerckhove & Jan Gettemans

  3. Department of Molecular and Cellular Interactions, VIB, 1050, Brussels, Belgium

    Sameh Hamdy Soror, Vasundara Srinivasan & Jan Steyaert

  4. Structural Biology, Free University of Brussels, Pleinlaan 2, 1050, Brussels, Belgium

    Sameh Hamdy Soror, Vasundara Srinivasan & Jan Steyaert

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  1. Anske Van den Abbeele
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  2. Sarah De Clercq
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Correspondence to Jan Gettemans.

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Van den Abbeele, A., De Clercq, S., De Ganck, A. et al. A llama-derived gelsolin single-domain antibody blocks gelsolin–G-actin interaction. Cell. Mol. Life Sci. 67, 1519–1535 (2010). https://doi.org/10.1007/s00018-010-0266-1

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