RESUMEN
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 Ca(2+)-activated gelsolin (K (d) approximately 10 nM) while GsnVHH 11 binds gelsolin irrespective of Ca(2+) (K (d) approximately 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/capping 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.
Asunto(s)
Actinas/metabolismo , Camélidos del Nuevo Mundo/inmunología , Gelsolina/química , Gelsolina/metabolismo , Estructura Terciaria de Proteína , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/metabolismo , Actinas/genética , Animales , Calcio/metabolismo , Línea Celular , Movimiento Celular/fisiología , Cristalografía por Rayos X , Epítopos/química , Epítopos/metabolismo , Gelsolina/genética , Humanos , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Cadena Única/genéticaRESUMEN
Gelsolin and CapG are both actin binding proteins that modulate a variety of physiological processes by interacting differently with the actin cytoskeleton. Several studies suggest that overexpression of these proteins promotes invasion in vitro. In this study we explored the contribution of these proteins in human cancer cell invasion and motility. We show that down regulation of CapG or gelsolin in several types of cancer cells, including MDA-MB 231 and PC-3 cells, significantly reduces the invasive and motile properties of cells, as well as cell aggregation. These results point to a role for CapG and gelsolin as tumor activator.