Extended Data Fig. 7: Colocalization of Cirl-NRS-LexA and Cirl proteins.
From: Molecular sensing of mechano- and ligand-dependent adhesion GPCR dissociation
a–c, Comparisons of L3 larvae carrying wild-type (a), γ-secretase-resistant (b) or GAIN-domain cleavage-incompetent (c) Cirl-NRS-LexA variants showed that Cirl dissociation is receptor autoproteolysis-dependent in all neurons except in Kenyon cells (chevron) and a few individual neurons throughout the CNS (arrowheads). nls = nuclear localization sequence; Scale bars, 50 µm. d–f, Schematic illustrations of tagged NRS sensor variants. g–i, Single planes of central brain hemispheres from different NRS sensor variants immunostained using anti-HA (in magenta) and anti-V5 (in green) antibodies to visualize the ECR and C termini of NRS sensor variants (right panel). Scale bar = 30 µm. j–l, Insets of merged hemisphere images shown in g–i (dashed rectangles). N- and C-terminal NRS termini colocalize in the membrane in central brain hemisphere cells of third instar larvae (arrowheads). Scale bar = 10 µm. m, L3 larval brain expressing the transcriptional reporter Cirlp-GAL4 (green) and the release sensor Cirl-NRS-LexA (magenta). Scale bar = 50 µm. n–p, Immunohistochemical co-staining of RFP-Cirl (green) and different Cirl-NRS variants (magenta) show colocalization of both proteins in the membrane in central brain hemisphere cells of L3 larvae (arrowheads). Dashed rectangles indicate position of areas magnified in the insets below. Scale bar = 30 µm, inset = 10 µm. All experiments independently repeated 3x with similar results.
