Rtnl1 loss decreases mitochondrial Ca ²⁺ handling in Type Is boutons. (Extended data from Fig. 8.) (A) Loss of Rtnl1 does not affect resting CEPIA3mt fluorescence. Although Rtnl1¹⁸/Rtnl1¹ flies show a marginally significant decrease in CEPIA3mt fluorescence, this effect is not found in a homozygous Rtnl1¹⁸ background, indicating that loss of Rtnl1 does not affect resting mitochondrial Ca²⁺. Plot shows individual larval datapoints and median ± interquartile ranges; sample size (larvae) is indicated within the plot for each genotype. Comparisons were performed using Kruskal–Wallis tests. (B) Impact of Rtnl1 loss-of-function on peak evoked mitochondrial Ca²⁺. Plots show all single time traces and mean ± SEM time traces in every genotype for four stimulation frequencies tested. Sample size (larvae) is indicated within the plot for each genotype. (C) Impact of Rtnl1 loss-of-function on peak evoked mitochondrial Ca²⁺. Plots show mean ± SEM of every genotype for each stimulation frequency tested; sample size (larvae) is indicated within the plot for each genotype. For each larva, responses from a 50-s recording from one muscle 1 NMJ between A4-A6 segments were analyzed. Comparisons were analyzed with a mixed-effects two-way ANOVA. (D) Rtnl1 loss did not affect time to peak mitochondria Ca²⁺. Data analyzed as in C. (E) Impact of Rtnl1 loss on time to 50% recovery of mitochondria Ca²⁺. Rtnl1 loss in both Rtnl1¹⁸ and Rtnl1¹⁸/Rtnl1¹ backgrounds slightly decreases time to 50% recovery when compared to WT and WT/Rtnl1¹⁸ backgrounds, indicating that mitochondria in Rtnl1 mutants lose their Ca²⁺ slightly faster than in WT. Data analyzed as in C. In D and E, datapoints represent the time to peak ΔF/F and 50% or 100% recovery. Genotypes are Is-GAL4, UAS-CEPIA3mt/UAS-tdTom::Sec61β, in either a WT, or Rtnl1¹⁸, Rtnl1¹⁸/Rtnl1¹, or WT/Rtnl1¹⁸ background.