Fig. 1

Different treatments enhance substrate availability and luminescence detection in Nluc-expressing nematodes. A Schematic representation of constructs used in this study. For luminescence detection and subsequent BRET analyses, a Nanoluciferase (Nluc) fused to either NPR-11 or LAT-1, respectively, was expressed in the respective knockout nematodes. A GFP was fused to the second intracellular loop of Nluc::LAT-1 for monitoring correct localization of the protein at the cell membrane. Synchronized young adult nematodes (L4 + 1 day) were left intact or cut, and luminescence was directly measured after coelenterazine H addition. B In intact wild-type nematodes, basal luminescence is 19.5 ± 2.2 AU, while it is 2-fold higher in Nluc::npr-11 worms. Luminescence of Nluc::lat-1 is 14-fold higher than in Nluc::npr-11 worms. C Luminescence of Nluc::NPR-11 in intact worms is not increasing when raising the animal counts per well. D Nluc::Npr-11 worms homogenized with beads and harsh shaking provide a strong luminescence signal compared to intact animals. Luminescence of nematodes with an incision is comparable to homogenized samples. E Luminescence in cut worms incubated with coelenterazine decreases over time (− 90% from 0 min to 25 min) while furimazine provides stable values over the entire time period. F Luminescence in cut worms either expressing Nluc::npr-11 or npr-11::Nluc incubated with furimazine. Both strains show luminescence. Data in B-F are shown as mean ± SEM; n ≥ 3, N = 50 with 3 technical replicates; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; two-sided t-test; AU = arbitrary units. Schematic pictures were created with BioRender.com