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

Abstract

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.

Interphase microtubule disassembly is a signaling cue that drives cell rounding at mitotic entry

“…ERMs exist at the plasma membrane under a closed-inactive and an open-active conformation 12 . Under their closed conformation, their N-terminal four-point-one, ezrin, radixin, moesin (FERM) domain binds to their C-terminal actin-binding domain (C-ERMAD) and ERMs cannot link actin filaments (F-actin) to the plasma membrane 15,16 .…”

“…In order to better understand the regulation of ERMs, we recently designed ERM bioluminescence resonance energy transfer (BRET) based biosensors 12 . Briefly, we fused the bioluminescent donor ( Renilla luciferase, rLucII) to the C-terminus of individual ERMs.…”

“…MyrPB-Ezrin-rLucII, Ezrin-rLucII and rGFP-CAAX constructs were previously described 12 . Ezrin KK211,212MM -rLucII was obtained by inverse PCR from Ezrin-rLucII using the following primers: forward) 5’-ATGATGGGAACAGACCTTTGGCTTGGAGTTGATGCCCTTGG-3’ and (reverse) 5’-GTTTTTTATCTCGAAATAGTTGATTCCATACATTTCCAGGTCC-3’.…”

“…Human HEK293T, HeLa, A375, SW620 cells and murine MC38 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; 4.5g/L D-Glucose, L-Glutamine, 110mg/L sodium pyruvate; Invitrogen #11995073) supplemented with 10% fetal bovine serum (FBS; Life Invitrogen #12483020) and 1% penicillin-streptomycin antibiotics (ThermoFisher #15070063) at 37°C with 5% CO 2 . Transfections for BRET experiments were performed as previously described 12 . Briefly, HEK293T cells were transfected with 1µg of total DNA (50ng rLucII construct, 300ng rGFP-CAAX and 650ng salmon sperm DNA) using linear polyethyleneimine (PEI, Alfa Aesar #43896) as transfecting agent (PEI:DNA ratio of 3:1).…”

“…While ERMs drive metaphase cell rounding, the cell-cycle signals that prompt their conformational activation in mitosis are unknown 11 . We screened a library of small molecules using novel ERM biosensors 12 and we unexpectedly found that drugs that disassemble microtubules promote ERM activation. Remarkably, cells disassemble their interphase microtubules while entering mitosis 13 .…”