Fluorescence Resonance Energy Transfer (FRET)-based Detection of Profilin–VASP Interaction

Abstract

Profilins belong to a family of small G-actin binding proteins which are thought to assist in F-actin elongation at the leading edge of migrating cells through their interactions with a host of actin-binding proteins including Ena (enabled)/VASP (vasodilator stimulated phosphoprotein). Profilin's interactions with the major actin regulators have been studied almost exclusively using biochemical methods. Therefore spatiotemporal features of these protein-protein interactions have not been resolved so far. In this paper, we for the first time demonstrate the feasibility of GFP-based fluorescence resonance energy transfer (FRET) technique to detect VASP's interaction with profilin-1, a ubiquitously expressed member of profilin family of genes. Specifically, we performed acceptor photobleaching FRET in MDA-MB-231 breast cancer cells to show prominent VASP-Pfn1 interaction at the membrane ruffles near the leading edge.