Extended Data Fig. 3: Development and characterization of a control sensor.
From: A genetically encoded sensor for in vivo imaging of orexin neuropeptides
a, Representative images of HEK cells expressing different mutants of OxLight1, indicated by absolute residue numbering from the N-terminus of OX2R. b, Quantification of maximal ∆F/F0 in response to bath-applied orexin-A and orexin-B (both at 10 µM) for all mutants in a. The OxLight1 E54K + T111A mutant has a significantly decreased response compared to OxLight1: p = 2.174 × 10−21 with n = 32 and 25 cells, respectively. c, The two OxLight1 sites selected for the control sensor are highlighted in magenta in the structural model of OxLight1. Residue sidechains are shown for the two amino acid positions E541.32 and T1112.61. d, Representative images of OxLight-ctr expression in HEK cells and ∆F/F0 after addition of OXA or OXB. e, Identical to d, but in primary cultured neurons. f, Left, membrane ∆F/F0 in OxLight1 or OxLight-ctr expressing HEK cells from time lapse imaging experiments. Bars indicate bath application of orexins (black, both at 10 µM) followed by the OX2R antagonist EMPA (magenta, 10 µM). Right, quantification of maximal ∆F/F0 from time lapse imaging experiments shown on left. n = 16 and 21, cells for OxLight1 OXA HEK compared to OxLight-ctr OXA HEK (p = 1.640 × 10−19). n = 10 and 22 cells for OxLight1 OXB HEK compared to OxLight-ctr OXB HEK (p = 5.926 × 10−11), Data are shown as mean ± SEM and all statistical analysis performed using Two tailed student’s t-test with Welch’s correction from 3 independent experiments. *** p < 0.0001. All scale bars, 10 µm.
