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Fig. 1 | BMC Biology

Fig. 1

From: Optogenetic interrogation reveals separable G-protein-dependent and -independent signalling linking G-protein-coupled receptors to the circadian oscillator

Fig. 1

Comparison of JellyOp and Opto-hβ2AR for optogenetic control over Gαs signalling. a Baseline (dark) luminescence of cells expressing JellyOp, Opto-hβ2AR or the GloSensor reporter alone. Opto-hβ2AR causes a significant increase in baseline luminescence compared to the GloSensor alone condition (**P < 0.01, one-way ANOVA with Dunnett’s multiple comparison, mean ± SEM, n = 3; values are reported in Additional file 1). b Time course showing changes in cAMP reporter luminescence in cells expressing either JellyOp or Opto-hβ2AR held in the dark and given a 5 second light flash (λmax = 470 nm, 3 mW cm–2) at 2 min, as indicated by a grey arrow (data expressed as raw luminescence normalised to mean luminescence over baseline recording, mean ± SEM, n = 5; values in Additional file 1). c Irradiance response curves for the peak increase in cAMP reporter luminescence (normalised to baseline) produced by the two photopigments (light: 2 s, λmax = 470 nm). Data fitted with standard sigmoidal dose response curves; mean ± SEM, n = 3. d Irradiance response profiles from (c) expressed as a percentage of maximum luminescence response for each photopigment, fitted with sigmoidal dose response curves (dotted lines mark irradiance at 50% max response for JellyOp and Opto-hβ2AR, mean ± SEM, n = 3; values in Additional file 1). e Comparison of peak increase in cAMP reporter luminescence (normalised to baseline) following a saturating light pulse (as in (b)) with and without pertussis toxin (10 μM PTX). The increase in response amplitude in Opto-hβ2AR expressing cells following PTX administration implies that significant Gαi/o activation by this receptor had suppressed the response in the absence of PTX (P < 0.001, max response amplitude 19.65 ± 0.97 vs. 34.8 ± 2.64, without/with PTX; Student’s t test, n = 5). This effect is absent in JellyOp expressing cells (P = 0.31, 341.7 ± 31.6 vs. 392.96 ± 34.5 without/with PTX, max response amplitude, Student’s t test n = 5; values in Additional file 1). All data mean ± SEM unless stated

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