|
Development and in vivo application of ratiometric APLNR-cpGFP-mScarlet-I3 biosensors. a, b Schematic illustration of the ratiometric APLNR conformational biosensors design. mScarlet-I3 and p2A-mScarlet-I3 were integrated C-terminally into the APLNR-cpGFP biosensors, resulting in membrane-bound (a) and cytosolic (b) mScarlet-I3 localization. c HEK293A cells were transiently transfected with the respective ratiometric biosensor plasmid and stimulated with Apelin. Stimulation with Apelin led to a strong increase in cpGFP over mScarlet-I3 fluorescence intensity of each ratiometric biosensor variant. d Quantification of the FRET ratio of the ratiometric biosensor with and without a p2A self-cleaving peptide exhibited no significant differences, indicating that cpGFP and mScarlet-I3 are no FRET partner. e Schematic illustration of the procedure. Plasmids encoding either UAS:APLNR(K235)-cpGFP-mScarlet-I3 or UAS:APLNR(K235)-cpGFP-p2A-mScarlet-I3 were injected into 1-cell stage Tg(fli1a:GAL4FF) zebrafish embryos and imaged at 28 hours post fertilization (hpf). f, g Quantification of APLNR(K235)-cpGFP over mScarlet-I3 fluorescence intensities in ISVs compared to the DA and PCV in UAS:APLNR(K235)-cpGFP-mScarlet-I3 (g) or UAS:APLNR(K235)-cpGFP-p2A-mScarlet-I3 (h) injected embryos at 28 hpf. Data in are presented as mean values ± SEM from three independent experiments conducted in transiently transfected HEK293A cells (c, d) or zebrafish embryos (N number of embryos, n either ISVs or DA/PCV: g DA/PCV N/n: 32/32, ISV N/n: 32/110; h DA/PCV N/n: 25/25, ISV N/n: 25/75). Statistical analysis was performed by using ordinary One-way ANOVA, followed Dunnett’s multiple comparison correction (d) and two-tailed unpaired Student’s t-test with Welch’s correction (g, h). Scale bars 50 µm. ISV intersegmental vessel, DA dorsal aorta, PCV posterior cardinal vein, hpf hours post-fertilization, cpGFP circularly permuted GFP, HEK293 human embryonic kidney 293. Source data are provided as a Source Data file. a, b Created in BioRender. Schihada (2025) https://BioRender.com/odhtcw2. e Created in BioRender. Schihada (2025) https://BioRender.com/mz23zor.
|
