A FRET Activation Sensor for Arf6

Abstract

The involvement of the small GTPase Arf6 in Rac activation, cell migration and cancer invasiveness suggests that it is activated in a spatially and temporally regulated manner. Small GTPase activation has been imaged in cells using probes in which the GTPase and a fragment of a downstream effector protein are fused to fluorescent reporter proteins that constitute a FRET donor/acceptor pair. Unlike other Ras family GTPases, the N-terminus of Arf6 is critical for membrane targeting, thus, cannot be modified by fusion to a fluorescent protein. We found that the previously described C-terminal GFP derivative also shows diminished membrane targeting. We therefore inserted a fluorescent protein into an inert loop within the Arf6 sequence. This fusion showed normal membrane targeting, nucleotide-dependent interaction with the downstream effector GGA3 and normal regulation by a GAP and a GEF. Using the recently developed CyPET/YPET fluorescent proteins as a FRET pair, we found that Arf6-CyPET underwent efficient energy transfer when bound to YPET-GGA3 effector domain in intact cells. Addition of PDGF to fibroblasts triggered a rapid and transient increase in FRET, indicative of Arf6 activation. These reagents should be useful for investigations of Arf6 activation and function.