Fig. 1

mTOR biosensor design and peptide selection. a Schematic representations of the conformational changes of AIMTOR induced upon mTOR activation (adapted from [19]). The biosensor contains a phosphorylable T757 peptide derived from the mTOR substrate, ULK1, a flexible linker and a WW domain able to recognize phosphorylated serine or threonine residues. These elements lay between a nanoluciferase donor entity emitting light at 460 nm after furimazine addition and a YPET acceptor protein emitting light at 530 nm. Upon mTOR phosphorylation, the WW domain will bind to the phosphorylated peptide increasing the proximity between nanoluciferase and YPET, which results in a BRET increase measured in live cells using a luminometer or a microscopic device (BRET ratio = Em530nm/Em460nm). b mTOR biosensor optimization. BRET measurements in live H1299 adherent cell population transfected with ULK1 peptides (S757 or T757)-containing biosensors. Cells were incubated in different media: “Fasting” medium (devoid of glutamine and serum), “Fasting + GLN” (fasting medium + glutamine), and “rich” medium (supplemented with glutamine and 10% serum). Each dot on the graph represents the mean of the BRET intensity recorded over 20 min for T757 (circles) and S757 (squared) expressing cells, respectively. The horizontal black bar shows the mean of BRET intensities of 3 independent experiments. One-way ANOVA with post hoc using the Tukey multiple comparison test. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the “Fasting” condition. ^##p < 0.01 compared to the “Fasting + GLN” condition. c Kinetics of net BRET intensity in H1299 suspension live cells transfected with AIMTOR or AIMTOR-T757A mutant plasmids. Mutating the mTOR-targeted threonine residue in AIMTOR into a non-phosphorylable alanine strongly reduces BRET intensity