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

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 p...