Figure 5.

Loss of Rtnl1 decreases evoked cytosolic Ca²⁺ responses in presynaptic NMJs. Cytosolic Ca²⁺ responses to 2 s of 30-Hz stimulation were measured at Type Is and Type Ib termini at muscle 1 in segment A4-A6, using myr::GCaMP. (A) Panels show GCaMP fluorescence at rest (F), maximum fluorescence (Max F), and maximum relative change in fluorescence (Max ΔF/F) in representative examples of WT and Rtnl1¹⁸ presynaptic terminals. (B) The ratiometric cytosolic Ca²⁺ sensor tdTom-p2a-GCaMP56 detected no difference in resting cytosolic Ca²⁺ in Type Ib Rtnl1¹⁸ boutons compared to WT. Graphs show individual larval datapoints for the ratio of GCaMP to Tomato fluorescence, with one Type Ib muscle 1 NMJ per larva, averaged across the responding area. Genotypes were compared using a Mann–Whitney U test, with median ± interquartile range shown. (C) Timecourses of evoked cytosolic GCaMP responses to 50 Hz stimulation (mean ± SEM of larval datapoints). (D) Loss of Rtnl1 significantly decreases cytosolic Ca²⁺ responses across a range of stimulation frequencies. Graph shows mean ± SEM of maximum evoked ΔF/F values (larval datapoints), with comparisons using mixed-effects model repeated-measures ANOVA. (A–D) Genotypes: Is-GAL4 or Ib-GAL4, UAS-myr::GCaMP6s/UAS-tdTom::Sec61β in either a WT or Rtnl1¹⁸ mutant background. (E) Expression of UAS-Rtnl1::HA or UAS-STIM::mCherry using Ib-GAL4 rescues evoked cytosolic Ca²⁺ response in mutant larvae. Data were normalized to the WT mean of maximum cytosolic GCaMP responses (ΔF/F) evoked by 50 Hz stimulation, from two different microscope setups. Graph shows individual larval datapoints, with one NMJ per larva, averaged across the responding area. Genotypes: Ib-GAL4, UAS-myr::GCaMP6s/attP2⁺, or Ib-GAL4, UAS-myr::GCaMP6s/UAS-Rtnl1::HA, or Ib-GAL4, UAS-myr::GCaMP6s/UAS-STIM::mCherry, in either a WT or Rtnl1¹⁸ mutant background; the attP2 landing site acts as a WT control for insertion of UAS-Rtnl1::HA at attP2.