(A–C) Representative images of larval transverse nerves stained with antibodies to Bruchpilot (Brp, green), Synaptotagmin I (Syt I, red), and HRP (blue). Loss of lrp4 (B, lrp4dalek) and srpk79d (C, srpkatc) result in improper axonal accumulations of Brp. This is not a general trafficking defect, as Syt I is absent from focal accumulations. (D) Representative high magnification confocal slice of VA1v ORNs expressing Brp-Short-mStraw and venus-SRPK79D and stained with antibodies to mStraw (red), GFP (green), and N-Cadherin (blue). SRPK79D largely colocalized with Brp-Short-mStraw but Brp-Short-positive / SRPK79D-negative and Brp-Short-negative / SRPK79D-positive puncta were also observed (D’’). (E) Representative confocal slice within a single antennal lobe glomerulus of a brain expressing venus-SRPK79D and LRP4-HA in all ORNs, processed for proExM, and stained with antibodies to venus (green), HA (red), and N-Cadherin (blue). Distinct regions of overlap between venus-SRPK79D and LRP4-HA (E”) are observed, though this represents a subset of venus-SRPK79D localization. (F–G) Representative high magnification confocal maximum intensity projections of VA1v ORN axon terminals expressing venus-SRPK79D in control (F) and lrp4dalek (G) backgrounds and stained with antibodies to GFP (green) and N-Cadherin (blue, inset). Loss of lrp4 results in reduced synaptic SRPK79D. (H) Quantification of venus-SRPK79D (green, left axis) and N-Cadherin fluorescence (blue, right axis). SRPK79D fluorescence is markedly reduced in lrp4dalek animals, but N-Cadherin staining is unaffected, demonstrating specificity. (I–J) Representative high magnification single confocal slices of the antennal lobe where all ORNs are expressing venus-SRPK79D and LRP4-HA via the pebbled-GAL4 driver and the brains subsequently processed using proximity ligation assays to determine whether the two proteins were close enough to interact. The brains were stained with antibodies to venus (green) and HA (blue) and PLA-specific probes (red) to detect proximity ligation events. When PLA-specific probes are not added, no signal is observed (I”) but when present, positive PLA signal (J”) indicates close physical proximity between LRP4-HA and venus-SRPK79D. Positive PLA signal represents a subset of SRPK79D or LRP4 expression, as in (E). **p<0.01; ns, not significant. Statistical comparisons (one-way ANOVA with correction for multiple comparisons) are with control unless otherwise noted. Error bars represent mean ± s.e.m. n (antennal lobes) is noted at the bottom of each column. Scale bars = 10 µm (A–D,I–J), 25 µm (E), 20 µm (F–G), 33 µm (F-G insets).