A llama-derived gelsolin single-domain antibody blocks gelsolin-G-actin interaction.
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.
Department of Medical Protein Research, VIB, 9000 Ghent, Belgium.
Anske
A
Van den Abbeele
Sarah
S
De Clercq
Ariane
A
De Ganck
Veerle
V
De Corte
Berlinda
B
Van Loo
Sameh Hamdy
SH
Soror
Vasundara
V
Srinivasan
Jan
J
Steyaert
Joël
J
Vandekerckhove
Jan
J
Gettemans
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002161:Camelids, New World / Q000276:immunology*; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology; D018360:Crystallography, X-Ray; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism; D018260:Gelsolin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008928:Mitochondria / Q000378:metabolism / Q000648:ultrastructure; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D000939:Epitopes; D018260:Gelsolin; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D002118:Calcium