Extended Data Fig. 8: Loss of Tollo does not affect Cirl expression levels or localization.
From: Molecular sensing of mechano- and ligand-dependent adhesion GPCR dissociation
a, Schematic illustration of the experimental set-up for affinity-immunoprecipitation of Cirl ligands. b, Tollo-GAL4 and Cirl-NRS-LexA co-labelling shows co-expression of Cirl-NRS-LexA+ (magenta) and Tollo-GAL4+ (green) in specific areas of the brain hemispheres and VNC (inset). Strong Cirl-NRS-LexA>lexAop-myr-mCherry activity in the central brain is found in the mushroom body (asterisk) and in a reticular pattern in the cortex (arrows). Scale bar, 25 µm. Inset: some cells in the VNC display Tollo-GAL4+/Cirl-NRS-LexA+ co-labelling (closed arrowheads) while others are either Tollo-GAL4+ or Cirl-NRS-LexA+ (open arrowheads). Scale bar, 10 µm. Experiment independently repeated 6x with similar results. c, Principle of synaptic interaction screen between Tollo-GAL4+ and Cirl-NRS-LexA+ cells through t-GRASP. d,e, t-GRASP signals in L3 brain hemispheres enhanced by an anti-GFP immunostaining; neuroblasts visualized using anti-Mir antibody. Scale bar = 50 µm. Representative t-GRASP signals upon co-expression by Tollo-GAL4 and Cirl-NRS-LexA are abundant (e), but hardly detectable in control flies lacking the drivers (d). t-GRASP signals appear to line cell boundaries (arrowheads). Scale bar, 10 µm. f, Quantification of t-GRASP signals in the brain indicates that Tollo-GAL4+ and Cirl-NRS-LexA+ cells are contacting each other. (i) and (ii) relate to images in d and e, respectively. pre-t-GRASP/+; post-t-GRASP/+ (n = 4 independent flies), Tollo-GAL4>pre-t-GRASP; Cirl-NRS-LexA>post-t-GRASP (n = 5 independent flies). Data are presented in a box-whisker plot (all data points plotted; horizontal line represents median, boxes the 25th and 75th percentiles, whiskers minimum and maximum values). Data were compared with a two-tailed unpaired t-test (confidence interval = 95 %). See also Source Data. g, Illustration of C-terminally V5-tagged Cirl. h, Western blot analysis showing similar Cirl expression levels in the presence and absence of Tollo. α-tubulin served as loading control. Experiment independently repeated 2x with similar results. For gel source data, see Supplementary Fig. 1h. i, Confocal images of Cirl expression in larval brains appears unaltered in TolloKO. Scale bar 100 µm. Experiment independently repeated 3x with similar results.
