Expression patterns and proximal proteins of Drice in the adult brain.

(A) Schematic diagram of Drosophila apoptosis signaling.

(B) Western blot of expression of C-terminally V5::TurboID knocked-in tagged caspases in adult male heads. Biotinylated proteins are detected by streptavidin (SA).

(C) Representative images of adult male brains. Expression patterns of each C-terminally V5::TurboID knocked-in tagged caspase are visualized using SA (magenta). Nuclei are visualized by Hoechst33342 (green). Arrows indicate the mushroom body (MB), antennal lobe (AL), optic lobe (OL), and suboesophageal ganglion (SOG), respectively. Scale bar: 50 µm.

(D) Representative images of ALs in the adult male brains. Expression patterns of Drice are visualized using SA (magenta). Nuclei are visualized by Hoechst33342 (green). Scale bar: 50 µm.

(E) Western blot of biotinylated proteins labelled by Drice::V5::TurboID extracted from adult male brains. Biotinylated proteins are detected by streptavidin (SA).

(F) Schematic diagram of the TurboID-mediated identification of proximal proteins. Drice proximal proteins are promiscuously labeled in vivo with the administration of biotin. Then, biotinylated proteins are purified using NeutrAvidin magnetic beads and are subsequently analyzed by mass spectrometry.

(G) A summary of mass spectrometry analysis of proteins of the adult male brains. Among 643 proteins identified, 158 proteins were detected as highly specific proteins to Drice::V5::TurboID flies compared to wild-type flies (FC (Drice::V5::TurboID/w1118) > 10).

(H) Gene Ontology analysis (Cellular Compartment) of 158 Drice proximal proteins (FC (Drice::V5::TurboID/w1118) > 10). Cyan: neuronal-related fraction, Red: membrane fraction.

A specific isoform of Fasciclin 3 is in proximity to Drice.

(A) Representative expression patterns of C-terminally V5::TurboID knocked-in tagged Drice (Streptavidin (SA); magenta) and Fasciclin 3 (Fas3, anti-Fasciclin 3 antibody staining; green) in the adult male brains. Arrows indicate the antennal lobe (AL), optic lobe (OL), and suboesophageal ganglion (SOG), respectively. Scale bar: 50 µm.

(B) Representative expression patterns of C-terminally V5::TurboID knocked-in tagged Drice (SA; magenta) and Fas3 (anti-GFP antibody staining; green) in the antennal lobes (ALs) of the adult male brains. Scale bar: 50 µm.

(C) Schematic protein structures of Fas3 isoforms. Intracellular regions differ from each other. A peptide region used to raise anti-Fas3G antibody is shown. Signal peptides (SPs), immunoglobulin-like (Ig-like) domains, and transmembrane (TM) domains are shown in boxes.

(D) IDR predictions of intracellular regions of each protein isoform. Regions wherein the score is more than 0.5 are predicted to be disordered.

(E) Western blot of proteins extracted from adult heads overexpressing each 3xFLAG-tagged Fas3 isoform. Anti-Fas3G antibody specifically detects Fas3 isoform G.

(F) Western blot of endogenous Fas3 expressed in adult male heads. Drice proximal proteins biotinylated by C-terminally knocked-in tagged V5::TurboID are purified using NeturAvidin

(G) Western blot of 3xFLAG-tagged Fas3 isoforms overexpressed in adult head. Drice proximal proteins biotinylated by C-terminally knocked-in tagged V5::TurboID are purified using NeturAvidin.

A Gal4-Manipulated Area Specific CaspaseTracker/CasExpress (MASCaT).

(A) A schematic diagram of MASCaT expressed using the Gal4/UAS system with FLP-mediated recombination. Caspase sensitive-QF2 is activated by caspase-mediated cleavage at the plasma membrane. Cleaved QF2 translocates to the nucleus to induce reporter protein expression downstream of QUAS sequence.

(B) Representative images of Drosophila S2 cells expressing caspase sensitive-QF2 probes by the Gal4/UAS system with FLP-mediated recombination. Scale bar: 20 µm.

(C) Representative images of adult male brains of young (1 week old) and old (3 weeks old) flies raised at 29℃ expressing MASCaT probe (mCD8; green). Caspase activation is visualized by MASCaT-induced tdTomato (magenta) expression. Arrowheads indicate optic lobes (OLs). Magnified images (white rectangle) show antennal lobes (ALs; white circles). Arrows indicate age-dependent caspase activation at the ALs. Scale bar: 100 µm.

(D) Representative images of adult male brains of young (1 week old raised at 25℃ or 29℃) and old (5 weeks old raised at 25℃ or 3 weeks old raised at 29℃) expressing MASCaT probe (mCD8; green). Caspase activation is visualized by MASCaT-induced tdTomato (magenta) expression. White circles show ALs. Arrows indicate age-dependent caspase activation at the ALs. Scale bar: 50 µm.

Fasciclin 3 isoform G overexpression enhances non-lethal caspase activation.

(A) Representative images of the adult male antennae of 1 weeks old flies raised at 29℃. Cell bodies are visualized using RedStinger (red). Scale bar: 20 µm.

(B) Quantification of cell number of (A). Data are presented as mean ± SEM. P-values were calculated using one-way analysis of variance (ANOVA) with Bonferroni’s correction for selected pairs. NS: P > 0.05, †: P < 0.05. Sample sizes: V5::TbID (N = 15), DroncCG:::V5::TbID (N = 16), Dronc:::V5::TbID (N = 23), Dcp-1CG::V5::TbID (N = 15), Dcp-1::V5::TbID (N = 16), DriceCG::V5::TbID (N = 15), Drice::V5::TbID (N = 15).

(C) Representative images of the adult male antennae of 1 week old flies raised at 29℃. Cell bodies are visualized using RedStinger (red). Scale bar: 20 µm.

(D) Quantification of cell number of (C). Data are presented as mean ± SEM. Light blue dots represent Fas3G overexpression conditions while red bars represent Drice overexpression conditions. P-values were calculated using one-way ANOVA with Bonferroni’s correction for every pair. NS: P > 0.05. Sample sizes: mNeonGreen (N = 44), Fasciclin 3 isoform G (Fas3G; N = 15), Drice (N = 18), Fas3G + Drice (N = 15).

(E) Representative images of the antennal lobes (ALs) of male brains of 1 week old flies raised at 29℃ expressing MASCaT probe (mCD8; magenta). Expression of the probes are restricted to the Or42b-Gal4 positive (mNeonGreen; green) regions. Scale bar: 30 µm.

(F) Representative images of the ALs of brains of 1 week old flies raised at 29℃ with caspase activation visualized by MASCaT-induced tdTomato (magenta) expression. Expression of mNeonGreen (green) or 3xFLAG-tagged Fas3G (cyan) are simultaneously visualized. Scale bar: 30 µm.

(G) Quantifications of caspase activity detected by MASCaT of (F). P-values were calculated using chi-square test with Bonferroni’s correction using mNeonGreen as control. NS: P > 0.05, †: P < 0.05. Sample sizes are shown in the graph.

(H) Representative images of the ALs of brains of 1 week old flies raised at 29℃ with caspase activation visualized by MASCaT-induced tdTomato (magenta) expression. Expression of mNeonGreen (green) or 3xFLAG-tagged Fas3G (cyan) are simultaneously visualized. Scale bar: 30 µm.

(I) Quantifications of caspase activity detected by MASCaT of (H). P-values were calculated using chi-square test with Bonferroni’s correction using mNeonGreen and Fas3G as controls, respectively. NS: P > 0.05, †: P < 0.05. Sample sizes are shown in the graph.

Fasciclin 3 isoform G overexpression enhances non-lethal caspase activation in a Dronc-dependent manner.

(A) Representative images of adult male brains. Expression patterns of C-terminally V5::TurboID knocked-in tagged Dronc are visualized using Streptavidin (SA; magenta) with or without the expression of 3xFLAG-tagged Fasciclin isoform G (Fas3G; green). Magnified images (white rectangle) show antennal lobes (ALs). Scale bar: 50 µm.

(B) Western blot of adult male heads. Dronc proximal proteins biotinylated by C-terminally knocked-in tagged V5::TurboID are purified using NeturAvidin.

(C) Representative images of the AL of brains of 1 week old flies raised at 29℃ with caspase activation visualized by MASCaT-induced tdTomato (magenta) expression. Expression of mNeonGreen (green) or 3xFLAG-tagged Fas3G (cyan) are simultaneously visualized. Scale bar: 30 µm.

(D) Quantifications of caspase activity detected using MASCaT of (C). P-values were calculated using chi-square test with Bonferroni’s correction using mNeonGreen and Fas3G as controls, respectively. NS: P > 0.05, †: P < 0.05. Sample sizes are shown in the graph.

(E) Western blot of adult male heads. Drice proximal proteins biotinylated by C-terminally knocked-in tagged V5::TurboID are purified using NeturAvidin.

(F) A schematic model of subcellularly restricted non-lethal caspase activation at the plasma membrane (PM). Inactive Drice is in proximity to Fas3G in wildtype condition. Fas3G overexpression induces Dronc expression. Then, Dronc comes close to Fas3G and activates Drice. Activated Drice non-lethally cleaves substrate near PM including MASCaT probes.

Fasciclin 3 isoform G overexpression-facilitated caspase activation regulates attraction behavior.

(A) Images and schematic diagram of two choice preference assay. Flies are starved for 4 or 16 hours before the assay. One week-old young flies are left for 2 hours in the bottle with 2 traps. After the assay, preference index is calculated by counting the number of flies left in the trap with apple cider vinegar (ACV) as X, in the trap with MilliQ (MQ) water as Y, and other (field) compartments as Z.

(B) Preference index of 1 week old male flies raised at 29℃. Flies are starved for 16 or 4 hours and are subsequently tested in response to 25% or 1% ACV in two choice preference assay. Data are presented as mean ± SEM. P-values were calculated using unpaired t-test. †: P < 0.05. Sample sizes: 16 hr Stv/25% AC (N = 13 (915 flies)), 4 hr Stv/1% ACV (N = 13 (945 flies)).

(C) Preference index of 1 week old male flies raised at 29℃. Flies are starved for 4 hours and are subsequently tested in response to 1% ACV in two choice preference assay. Data are presented as mean ± SEM. Light blue dots represent Fas3G overexpression conditions while a red bar indicates caspase-activated condition. P-values were calculated using one-way ANOVA followed by Dunnets’ multiple comparison test using mNeonGreen and Fas3G as controls. NS: P > 0.05, †: P < 0.05. Sample sizes: mNeonGreen (N = 16 (1159 flies)), DroncDN (N = 15 (1099 flies)), Fas3G (N = 20 (1419 flies)), Fas3G + DroncDN (N = 20 (1318 flies)).

Expression patterns of Drice in the adult brain in males and females.

(A) Representative images of adult male and female brains. Expression patterns of C-terminally V5::TurboID knocked-in tagged Drice are visualized using Streptavidin (SA, magenta). Nuclei are visualized by Hoechst33342 (green). Scale bar: 50 µm.

(B) Representative images of antennal lobes in the adult female brains. Expression patterns of Drice are visualized using SA (magenta). Nuclei are visualized by Hoechst33342 (green). Scale bar: 50 µm.

Predicted protein complexes of Drice and each Fas3 isoform

(A) Predicted aligned error (PAE) plots of protein complex of Drice and each Fasciclin 3 (Fas3) isoform generated by AlphaFold2-Multimer. In Y axis, A represents Drice and B represents each Fas3 isoform. Numbers in X axis represent amino acid position. The intracellular region of each Fas3 isoform is represented by the orange bar. Potential interaction sites predicted by low PAE scores in Drice-Fas3A/G are highlighted by blue rectangles.

Biochemical property of Fas3s with caspases.

(A) Co-immunoprecipitation of 3xFLAG-tagged Fasciclin 3s (Fas3s) with V5-tagged Drice extracted from adult male heads. 3xFLAG-tagged Fas3s are immunoprecipitated using an anti-FLAG antibody. Biotinylated proteins labelled by Drice::V5::TurboID are detected by streptavidin (SA).

(B) Western blot for 3xFLAG-tagged Fas3s expressed in Drosophila S2 cells. Apoptosis was induced by treating Drosophila S2 cells with cycloheximide (CHX). Induction of caspase activation is confirmed by the cleavage of endogenous Lamin B.

Overexpression of mCD8::DQVD::QF2 probes in Drosophila S2 cells.

(A) A schematic diagram of MASCaT expressed directly using the Gal4/UAS system. Caspase sensitive-QF2 is activated by caspase-mediated cleavage at the plasma membrane. In theory, only cleaved QF2 translocates to the nucleus to induce reporter protein expression downstream of QUAS sequence.

(B) Representative images of Drosophila S2 cells expressing caspase sensitive-QF2 probes directly by the Gal4/UAS system. A pan-caspase inhibitior, zVAD-fmk, is treated to inhibit caspase activity. Scale bar: 20 µm.

Loss of function analysis of Fas3G.

(A) Gene structure of Fasciclin 3 (Fas3). CDS (orange) and UTR (grey) are shown in boxes. Target sequence of Fas3G-shRNA is shown in red.

(B) Western blot for 3xFLAG-tagged Fas3s expressed in Drosophila S2 cells with Fas3G-shRNA.

(C) Representative images of the antennal lobes (ALs) of brains of 1 week old flies raised at 29℃ with caspase activation visualized by MASCaT probe (mCD8; green)-induced tdTomato (magenta) expression. Scale bar: 30 µm.

(D) Quantifications of caspase activity detected by MASCaT of (C). P-values were calculated using chi-square test with Bonferroni’s correction using LacZ-RNAi as control. NS: P > 0.05. Sample sizes are shown in the graph.

(E) Representative images of the ALs of brains of 1 week old flies expressing mNeonGreen-tagged Drice (green) and 3xFLAG-tagged Fas3G (magenta). Scale bar: 50 µm.