Development of conformational BRET biosensors that monitor ezrin, radixin and moesin activation in real-time.

Ezrin, Radixin and Moesin compose the family of ERM proteins. They link actin filaments and microtubules to the plasma membrane to control signaling and cell morphogenesis. Importantly, their activity promotes invasive properties of metastatic cells from different cancer origins. Therefore, a precise understanding of how these proteins are regulated is important for the understanding of the mechanism controlling cell shape as well as providing new opportunities for the development of innovative cancer therapies. Here, we developed and characterized novel BRET-based conformational biosensors, compatible with high throughput screening, that monitor Ezrin, Radixin or Moesin individual activation in living cells. We showed that these biosensors faithfully monitor ERM activation and can be used to quantify the impact of small molecules, mutation of regulatory amino acids or depletion of upstream regulators on their activity. The use of these biosensors allowed us to characterize the activation process of ERMs that involves a pool of closed-inactive ERMs stably associated with the plasma membrane. Upon stimulation, we discovered that this pool serves as a cortical reserve that is rapidly activated before the recruitment of cytoplasmic ERMs.