Figure 3. Expansion of SSR- and PSD-localized proteins at leon NMJs.

(A) Images show SSR-localized proteins Dlg, Cact and Dl in immunostaining of boutons in w¹¹¹⁸, leon²/19-2 and leon¹/19-2. Co-staining of Dlg (green) and HRP (red) is shown (left panels). (B) Images show PSD-localized dPak, GluRIIA and GluRIIB immunostaining at synapses of w¹¹¹⁸, leon²/19-2 and leon¹/19-2. Arrowheads indicate overlapping signals of expanded GluRIIA (green) and GluRIIB (red) clusters in middle panels. GluRIIA images are shown with magnification (right panels). (C) Bar graphs show means ± SEM of Dlg-positive zone thickness, dPak intensity and GluRIIA intensity and diameter in w¹¹¹⁸, leon²/19-2 and leon¹/19-2. (D) Images show Dlg, dPak and GluRIIA immunostaining at NMJs, with postsynaptic 24B-GAL4-driven UAS-leon or UAS-ED-leon expression in leon¹/19-2. (E) Bar graphs show means ± SEM of Dlg-positive zone thickness, dPak intensity and GluRIIA intensity and diameter. All scale bars represent 5 μm except in magnified GluRIIA images, which is 1 μm. All data were compared to controls unless specifically indicated by brackets with n.s. indicating no significance, ** for p<0.01 and *** for p<0.001 according to Student’s t tests. The detail statistic numbers also see Supplementary file 1.

DOI: http://dx.doi.org/10.7554/eLife.26886.005

Figure 3—figure supplement 1. Localization of presynaptic proteins and expansion of GluRIIC clusters at leon NMJs.

(A) Images show immunostaining for Brp (green) and dPak (red), and GluRIIC (right panels) at NMJs of w¹¹¹⁸, leon²/19-2 and leon¹/19-2. Scale bar, 5 μm. (B) Bar graphs show means ± SEM of Brp density, GluRIIB intensity and GluRIIC intensity. All data were compared to controls with n.s. indicating no significance, * for p<0.05 and *** for p<0.001 according to Student’s t tests. The detail statistic numbers also see Supplementary file 1. (C) Images show NMJ immunostaining for presynaptic proteins Synapsin (left), CSP and HRP (green and red, respectively, at middle), and Futsch and HRP (red and green, respectively, at right) in w¹¹¹⁸, leon²/19-2 and leon¹/19-2. Scale bar, 5 μm (left) and 20 μm (right).

DOI: http://dx.doi.org/10.7554/eLife.26886.006

Figure 3—figure supplement 2. Postsynaptic defects of leon mutants in earlier larval stages.

(A and B) Images show Cact and GluRIIA immunostaining of NMJs in w¹¹¹⁸, leon²/19-2 and leon¹/19-2 at 72, 96 and 120 hr AEL. (C) Line graphs show means ± SEM of Cact-positive zone thickness and GluRIIA intensity and diameter at 72, 96 and 120 hr AEL. All data were compared to controls with * for p<0.05, ** for p<0.01, *** for p<0.001 according to Student’s t tests. The detail statistic numbers also see Supplementary file 1.

DOI: http://dx.doi.org/10.7554/eLife.26886.007

Figure 3—figure supplement 3. Presynaptic leon is not required for postsynaptic formation.

(A) Images show immunostaining for Dlg (green, co-stained with HRP in red), dPak, and GluRIIA in presynaptic D42-GAL4-driven UAS-leon or UAS-ED-leon expression that failed to suppress Dlg, dPak and GluRIIA expansion in leon¹/19-2. Scale bar, 5 μm. (B) Bar graphs show means ± SEM of Dlg-positive zone thickness, dPak intensity and GluRIIA intensity and diameter. No rescue effect was shown by n.s. (no significance) when compared to D42-GAL4, leon¹/19-2 according to Student’s t tests. The detail statistic numbers also see Supplementary file 1.

DOI: http://dx.doi.org/10.7554/eLife.26886.008

Figure 3—figure supplement 4. Postsynaptic leon knockdown exhibits postsynaptic defects.

(A,B) Images show Dlg, dPak and GluRIIA immunostaining at NMJs. Expansions of Dlg, dPak, and GluRIIA were shown in UAS-leonRNAi driven by postsynaptic 24B-GAL4 (A), but not by presynaptic D42-GAL4 (B). Scale bars, 5 μm. (C) Bar graphs show means ± SEM of Dlg-positive zone thickness, dPak intensity, GluRIIA intensity and diameter in 24B-GAL4, 24B>leonRNAi, D42-GAL4, and D42>leonRNAi. Pairwise comparisons are shown by brackets with n.s. indicating no significance and * for p<0.05, ** for p<0.01, *** for p<0.001 according to Student’s t tests. The detail statistic numbers also see Supplementary file 1.

DOI: http://dx.doi.org/10.7554/eLife.26886.009