Fig. 4

Phase shifts of the fibroblast clock by the arrestin-biased JellyOp F112A mutant. a Immunocytochemistry photomicrographs showing detection of JellyOp variants stably expressed in rat1 fibroblasts and labelled with a 1D4 antibody (pseudo coloured red in the merged image on the left; monochromatic image on the right). Cells are also stained with DAPI (blue). Scale bar = 20 μm. b The circadian per2 rhythm of JellyOp F112A-expressing per2::luc rat1 fibroblasts exposed to 4 h intermittent white light (28.40 mW cm^–2) at various phases of the per2 rhythm (indicated by arrows at CT12.5, CT2.6, CT17.9). For phase analysis, baseline corrected bioluminescence rhythms prior to light treatment were modelled by a sine wave (grey dotted line). Time of first trough in per2:luc rhythm aligned for all traces to facilitate comparisons. c Double-plotted phase response profile for JellyOp (black circles, replotted from Fig. 2c) and JellyOp F112A (red squares) expressing per2::luc rat1 fibroblasts, illustrates the relationship between the timing of light onset and magnitude of phase shifts in per2 rhythm. Data represent extent of phase shifts captured from individual light pulsed cultures and plotted on a y-axis where positive and negative values denote phase advances and delays, respectively. Linear regression fits with R² of 0.88 and 0.91 for JellyOp and JellyOp F112A, respectively (values in Additional file 1). d Acute light-dependent changes in transcription of core clock genes of JellyOp (d) and JellyOp F112A (e). mRNA was extracted from fibroblasts exposed to 30 min or 2 h intermittent white light (28.40 mW cm^–2), at the circadian time when the biggest phase shift was produced for each optogenetic tool, namely at CT2 and CT20 for JellyOp (d) and JellyOp F112A (e), respectively. per2 and cry2 are upregulated in JellyOp wt and both have cAMP responsive element (CRE) in their promoters that regulates their expression. All results were normalised to actin expression. All data are presented as the relative gene expression mean ± SEM (*P < 0.05, **P < 0.01, One-sample t test, n = 4; values in Additional file 1)