An RPEL-based FRET biosensor for free monomeric actin

Actin is critical for cell integrity and nuclear processes, but methods to visualise how environmental and intracellular signals affect cellular monomeric (G-) actin levels are lacking. We present the GASPs (G-actin sensing proteins), genetically encoded G-actin FRET sensors. Using cytoplasmic- and nuclear-localised GASPs, we show that in fibroblasts integrity of focal adhesions plays a critical role in maintaining low G-actin levels, and that cytoplasmic and nuclear G-actin levels increase at high cell densities and when spreading is restricted. Upon serum stimulation, it is depletion of the cytoplasmic G-actin pool that correlates with rapid nuclear accumulation of the MRTF transcription factors, whose subcellular localisation is controlled by G-actin. Stimuli that induce rapid increases or decreases in cytoplasmic G-actin level induce similar but slower changes in nuclear G-actin level, indicating that fluctuations in nuclear G-actin levels are largely subservient to cytoplasmic actin dynamics.