Fig. 1

Figure 1. (A) Ribbon diagram of the cAMP sensor composed of CNBD-GFP protein that is unstable without cAMP but stabilized upon cAMP binding: solution structure of a bacterial cyclic nucleotide-activated K+ channel binding domain in cAMP-free form on the left (PBD 2KXL) and bound to cAMP on the right (PBD 2K0G). (B) NIH 3T3 cells stably expressing CNBD-GFP derived from error-prone PCR were treated with 1% serum or 20 μM forskolin for 17 h. (C) Synthetic mRNA encoding the sensor variants N41 and N49 CNBD and the corresponding cAMP-insensitive controls (R307Q). (D) mRNA was injected into zebrafish embryos at the one-cell stage. Embryos were treated at 4–5 hpf with FSK or DMSO for 20 h, then imaged at 24–25 hpf. (E) Representative images showing 24 hpf embryos injected with N41-GFP and N41R307Q-GFP mRNA at one-cell stage and treated with DMSO or 20 μM FSK starting at 4 hpf. (F) Each dot represents mean GFP intensity of one embryo. Error bars indicate SD: *p < 0.5 by one-way ANOVA (with Šídák’s multiple comparisons), n = 13–22 embryos each condition; ns = not significant. AU = arbitrary unit.