Dronc-independent basal executioner caspase activity sustains Drosophila imaginal tissue growth

Abstract

Caspase is best known as an enzyme involved in programmed cell death, which is conserved among multicellular organisms. In addition to its role in cell death, caspase is emerging as an indispensable enzyme in a wide range of cellular functions, which have recently been termed caspase-dependent nonlethal cellular processes (CDPs). In this study, we examined the involvement of cell death signaling in tissue-size determination using Drosophila wing as a model. We found that the Drosophila executioner caspases Dcp-1 and Decay, but not Drice, promoted wing growth independently of apoptosis. Most of the reports on CDPs argue the importance of the spatiotemporal regulation of the initiator caspase, Dronc; however, this sublethal caspase function was independent of Dronc, suggesting a more diverse array of CDP regulatory mechanisms. Tagging of TurboID, an improved promiscuous biotin ligase that biotinylates neighboring proteins, to the C terminus of caspases revealed the differences among the neighbors of executioner caspases. Furthermore, we found that the cleavage of Acinus, a substrate of the executioner caspase, was important in promoting wing growth. These results demonstrate the importance of executioner caspase-mediated basal proteolytic cleavage of substrates in sustaining tissue growth. Given the existence of caspase-like DEVDase activity in a unicellular alga, our results likely highlight the original function of caspase-not cell death, but basal proteolytic cleavages for cell vigor.

Keywords: TurboID; caspase-dependent nonlethal cellular processes; executioner caspase; fluctuating asymmetry; tissue-size regulation.

Fig. 1.

Cell death signaling inhibition reduces Drosophila imaginal tissue size. (A) Schematic diagram of Drosophila apoptosis signaling. (B) Wing sizes under control (C10 > LacZ; n = 32) and caspase-inhibited conditions (C10 > Dronc^DN, n = 11; C10 > p35, n = 28). (C and D) Cell sizes (C) and cell numbers (D) of B. (E) Representative image of a bilaterally asymmetric Drosophila wing pair of C10 > p35. Green, right wing; magenta, left wing. (F) Wing size differences between right and left wings within the same flies in control (+ > p35, n = 60) and caspase-inhibited conditions (C10 > p35, n = 56). Red bars indicate the median of each group. (G) Femur, (G′) tibia, and (G″) tarsal segment 1 lengths in the control (C10 > GFP, n = 19) and caspase-inhibited conditions (C10 > Dronc^DN, n = 20; C10 > p35, n = 23). For all graphs except F, data are mean ± SD. Statistical analyses were performed with Tukey’s multiple comparison test after 1-way ANOVA (B, G, G′, and G″), unpaired Student’s t test (C and D), or the Mann–Whitney U test (F). n.s., P > 0.05; **P < 0.01; ****P < 0.0001. n.s., not significant.

Fig. 2.

Enhanced cell death signaling increases Drosophila wing size. (A) Wing size in control (WP > mCD8::GFP, n = 30; th⁴, WP > mCD8::GFP, n = 30) and wing size-reduced conditions (WP > InR^DN, n = 30; th⁴, WP > InR^DN, n = 29). (B and C) Cell sizes (B) and cell numbers (C) of A. For all graphs, data are mean ± SD. Statistical analyses were performed with unpaired Student’s t test. n.s., P > 0.05; ***P < 0.001; ****P < 0.0001. n.s., not significant.

Fig. 3.

Dcp-1 and Decay, but not Dronc or Drice, are required for promoting wing growth. (A) Wing size in No-Gal4 controls (+ > +, n = 20; + > LacZ-i, n = 19; + > dark-i^V100405, n = 19; + >dronc-i^V23035, n = 20; + > drice-i^V28065, n = 22; + > dcp-1-i^V107560, n = 20; + > decay-i^V43028, n = 20; + > decay-i^V100168, n = 20) and RNAi groups (C10 > +, n = 20; C10 > LacZ-i, n = 22; C10 > dark-i^V100405, n = 20; C10 >dronc-i^V23035, n = 16; C10 > drice-i^V28065, n = 19; C10 > dcp-1-i^V107560, n = 20; C10 > decay-i^V43028, n = 20; C10 > decay-i^V100168, n = 20) for screening results validation. (B) Relative wing sizes of A normalized to the mean of corresponding No-Gal4 control. For graphs A and B, data are mean ± SD. Statistical analyses were performed with unpaired Student’s t test with Bonferroni correction for A and Dunnett’s multiple comparison test against control (C10 > +) after 1-way ANOVA for B. n.s., P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. n.s., not significant.

Fig. 4.

Dronc-independent executioner caspase activity in Drosophila. (A) Schematic diagram of SCAT3 probe. (B) Western blotting against full-length SCAT3 probe (anti-myc antibody, dilution 1:10,000) and cleaved SCAT3 probe (anti-myc antibody, dilution 1:1,000) in the absence of Dronc. Genotypes are described in the figure. Black arrowhead indicates full-length SCAT3; white arrowheads, cleaved SCAT3.

Fig. 5.

The expression patterns of caspases during wing development. (A–C) Schematic diagrams of dcp-1 (A), drice (B), and dronc (C) gene loci. TurboID with V5-tag was knocked in right before the stop codon. (D–I) Expression patterns of Dcp-1 (D and E), Drice (F and G), and Dronc (H and I) in WDs. (Left) Merged image of GFP (green), V5 (magenta), and streptavidin (yellow) staining. (Middle) V5 (gray). (Right) Streptavidin (gray). (Scale bar: 100 µm.) dcp-1::V5::TurboID, ap > mCD8::GFP, LacZ-i (D); dcp-1::V5::TurboID, ap > mCD8::GFP, dcp-1-i^V107560 (E); drice::V5::TurboID, ap > mCD8::GFP, LacZ-i (F), drice::V5::TurboID, ap > mCD8::GFP, drice-i^V28065 (G); dronc::V5::TurboID, ap > mCD8::GFP, LacZ-i (H); dronc::V5::TurboID, ap > mCD8::GFP, dronc-i^V23035 (I). (J) Western blotting against V5-tagged caspases and their potential neighboring (biotinylated) proteins. The white arrowhead indicates Dronc::V5::TurboID; black arrowhead, Dcp-1::V5::TurboID and Drice::V5::TurboID.

Fig. 6.

Caspase-mediated cleavage of Acn increases wing size. (A) Schematic diagram of the acn gene locus. (B) Wing size in acn^WT (acn²⁷/Df(2R)ED1200; acn^WT/+, n = 20; acn²⁷/Df(2R)ED1200; acn^WT/th⁴, n = 22), and acn^D527A (acn²⁷/Df(2R)ED1200; acn^D527A/+, n = 20; acn²⁷/Df(2R)ED1200; acn^D527A/th⁴, n = 25) flies. Data are mean ± SD. Statistical analyses were performed using an unpaired Student’s t test. ***P < 0.001.