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

(A) Schematic diagram of Drosophila apoptosis signaling.

(B) Western blot of expression of caspases in adult male heads.

(C) Representative images of adult male brains. Expression patterns of each caspase are visualized using Streptavidin (SA, magenta). Nuclei are visualized by Hoechst33342 (Green). Scale bar: 50 µm.

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

(E) Western blot of proteins extracted from adult male brains.

(F) Schematic diagram of the TurboID-mediated identification of proximal proteins.

(G) A summary of mass spectrometry analysis of proteins of the adult male brains. Partial list of Drice proximal proteins and their Fold Change (FC) are shown.

(H) Gene Ontology analysis (Cellular Compartment) of 158 Drice proximal proteins (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 Drice (Streptavidin (SA); magenta) and Fasciclin 3 (Fas3, anti-Fasciclin 3 antibody staining; green) in the adult male brains. Scale bar: 50 µm.

(B) Representative expression patterns of 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.

(D) IDR predictions of intracellular regions of each protein isoform.

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

(F) Western blot of adult male heads. Drice proximal proteins were purified using NeturAvidin.

(G) Western blot of adult head proteins. Drice proximal proteins were 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.

(B) Representative images of 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°C. Arrowheads indicate optic lobes. Magnified images (white rectangle) show antennal lobes (ALs; white circles). Arrows indicate age-dependent caspase activation at the ALs. Scale bar: 100 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), DriceCG::V5::TbID (N = 15), Drice::V5::TbID (N = 15), Dcp-1CG::V5::TbID (N = 15), Dcp-1::V5::TbID (N = 16), DroncCG:::V5::TbID (N = 16), Dronc:::V5::TbID (N = 23).

(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. P-values were calculated using one-way ANOVA with Bonferroni’s correction for every pair. NS: P > 0.05, †: P < 0.05. Sample sizes: mNeonGreen (N = 44), 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). 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. 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. 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 Dronc are visualized using Streptavidin (SA, magenta). Magnified images (white rectangle) show antennal lobes (ALs). Scale bar: 50 µm.

(B) Western blot of adult male heads. Dronc proximal proteins were purified using NeturAvidin.

(C) Representative images of the AL of brains of 1 week old flies raised at 29℃ with caspase activation visualized using MASCaT. 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 were purified using NeturAvidin.

(F) A schematic model of subcellularly restricted non-lethal caspase activation upon Fas3G overexpression.

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

(A) Schematic diagram of two choice preference assay. Flies are starved for 4 hours before the assay. Flies are left for 2 hours in the bottle. Preference index is calculated by counting the number of flies left in MQ, ACV and other (field) compartments.

(B) Preference index of 1 week old male flies raised at 29℃ in response to 1% apple cider vinegar in two choice preference assay. Data are presented as mean ± SEM. P-values were calculated using one-way ANOVA followed by Dunnets’ multiple comparison test using mNeonGreen as a control. NS: P > 0.05, †: P < 0.05. Sample sizes: mNG (N = 19 (1327 flies)), Fas3G (N = 17 (1191 flies)), Fas3G + Dronc-RNAi (N = 19 (1295 flies)), Fas3G + DroncDN (N = 20 (1320 flies)).

(C) The ratio of flies in the given conditions in the two choice preference assay of (B). P-values were calculated using chi-square test with Bonferroni’s correction using mNeonGreen as control. †: P < 0.05. Sample sizes: mNG (N = 1327 flies), Fas3G (N = 1191 flies), Fas3G + Dronc-RNAi (N = 1295 flies), Fas3G + DroncDN (N = 1320 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 Drice are visualized using Streptavidin (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 Fas3 isoform generated by AlphaFold2-Multimer. 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 Fas3s with V5-tagged Drice extracted from adult male heads.

(B) Western blot for 3xFLAG-tagged Fas3s expressed in Drosophila S2 cells. Apoptosis was induced by treating S2 cells with cycloheximide (CHX).

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.

(B) Representative images of 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 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 S2 cells with Fas3G-shRNA.

(C) Representative images of the ALs of brains of 1 week old flies raised at 29℃ with caspase activation visualized by MASCaT. 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.