Serum response factor (SRF) regulates the expression of immediate-early genes as well as the expression of many muscle-specific genes. Differential regulation of the expression is achieved by exclusive binding of specific coactivators. MAL and MKL2, two coactivators of the myocardin family link SRF activity to a Rho-actin signalling pathway. Their activity is controlled through regulated interaction with G-actin, which is mediated by an RPEL domain. Rho-activation and G-actin depletion leads to accumulation of MAL in the nucleus where it activates transcription. Recent results show, that actin binding controls MAL activity at multiple levels. Actin binding inhibits MAL nuclear import, promotes MAL nuclear export and inhibits transcriptional activation by MAL. A major unresolved question is the mechanism by which actin binding regulates these events. In the proposed project, I will address this question by developing a system to track MAL in real time and study the interactions between MAL and actin and between MAL and SRF in live cells. I plan to use FRET/FLIM to assess the real time dynamics and changes in the localisation of MAL-actin interactions and the interaction of isolated RPEL domain with actin. I will investigate the stoichiometry of MAL-actin interaction, in vivo as well as in vitro. I will use FRAP analysis to assess changes in the mobility of MAL following stimulation, and compare the kinetics with that previous obtained for the MAL-actin interaction by FRET, and perform detailed in vitro actin binding studies. Additionally, I plan to elucidate actin’s interaction with members of the other family of RPEL motif-containing proteins, the Phactr/Scapinin family. By applying this wide variety of biophysical techniques I hope to understand the mechanism of the actin-dependent regulation of MAL activity in more detail.