Fly strains and expression vectors
Flies containing the sal-gal4
driver (provided by K. Basler, University of Zurich, Zurich, Switzerland) were crossed to flies with the reporter transgene CPV
(Williams et al., 2006
), and the progeny were used as wild-type controls. The fly mutant alleles used in this study are droncI24
(Xu et al., 2005
(Muro et al., 2006
(Laundrie et al., 2003
(Akdemir et al., 2006
), and strica4
(Baum et al., 2007
). The UAS-CPV
transgenes were recombined to the drice
mutant alleles. The following UAS
fly lines were also used: UAS-p35
(provided by H. Steller, Howard Hughes Medical Institute, Strang Laboratory of Cancer Research, The Rockefeller University, New York, NY), UAS-pro-dronc
(provided by H.D. Ryoo, New York University School of Medicine, New York, NY), and UAS-diap1
(provided by T. Volk, Weizmann Institute of Science, Rehovot, Israel). The dronc
-RNAi line was obtained from the Vienna Drosophila RNAi Center (transformant ID no. 23035), and diap1
-RNAi was a gift from P. Meier (Institute of Cancer Research, London, England, UK).
To generate the UAS-CD8::parpDEVG::venus (UAS-CPGV) construct, the CD8::parp::venus was subcloned into pBluescript II SK plasmid, and then a point mutation was introduced using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) with the primers forward 5′-GGCGATGAGGTGGGTGGAGTGGATGAA-3′ and reverse 5′-TTCATCCACTCCACCCACCTCATCGC-3′, which changed the adenine residue (A722) into guanine, leading to a change of the conserved aspartic acid at position P1 into glycine.
The rescue transgenic lines drice:drice
were generated as follows: first, a 564-bp fragment from the drice
genome encompassing its promoter and 5′ UTR was PCR amplified using the forward primer 5′-GGCAATTGCCTCTTTGAGAGTGTGACCG-3′ and reverse primer 5′-CCAAGATCTGGCTAAGTTCTCTCCTTGAG-3′ with added MfeI and BglII restriction sites, respectively. Second, a 708-bp fragment of drice
3′ UTR was also amplified by genomic PCR using the forward primer 5′-CCGGGCGGCCGCTGGCTAATGGTATGGATCAA-3′ and reverse primer 5′-GCGGTACCAGGGTCAACAGCAAACAGCCAA-3′ with added NotI and Acc65I restriction sites, respectively. Both the promoter/5′ UTR and 3′ UTR fragments were respectively cloned in a sequential order into the EcoRI + BglII and NotI + Acc65I sites of the pattB plasmid (a gift from J. Bischof, University of Zurich, Zurich, Switzerland), giving rise to the pattB-drice
-5′-3′ plasmid. The drice
coding region was amplified from a cDNA clone (a gift from H. Steller) using the forward primer 5′-CCAAGATCTATGGACGCCACTAACAATGG-3′ and reverse primer 5′-GGGGGGCCCTCAAACCCGTCCGGCTGGTG-3′ with added BglII and PspOMI restriction sites and was subsequently respectively cloned into the BglII and NotI restriction sites of the pattB-drice
-5′-3′ plasmid. The dcp-1
coding region was amplified from a cDNA clone (a gift from H. Steller) using the forward primer 5′-CGGGGATCCATGACCGACGAGTGCGTAAC-3′ and reverse primer 5′-CGGCGCGGCCGCCTAGCCAGCCTTATTGCCGT-3′ with added BglII and NotI restriction sites and was subsequently respectively cloned into the BamHI and NotI restriction sites of the pattB-drice
-5′-3′ plasmid. The two transgenic fly lines, drice:drice
, were generated using the ϕC31-mediated site-specific transgenesis technique, which allows insertion of transgenes into known sites of the Drosophila
genome (Bischof et al., 2007
; Fish et al., 2007
). Specifically, these transgenes were inserted into the attP18 site on the X chromosome. Transcriptional expression of the transgenes was confirmed by RT-qPCR analysis on RNA from WDs of the two transgenic fly lines.
The rescue transgenic lines dcp-1:drice and dcp-1:dcp-1 were generated as follows: first, a 703-bp fragment from the dcp-1 genome encompassing its promoter and 5′ UTR was amplified using the forward primer 5′-AACAGATCTCTGTTTTTAATGTTAGATTAG-3′ and reverse primer 5′-GCGAATTCCTTGCGCCCCTTTTCTTGC-3′ with added BglII and EcoRI restriction sites, respectively. Second, a 1,090-bp fragment of dcp-1 3′ UTR was also amplified by genomic PCR using the forward primer 5′-AACTCGAGGAAGAGATCTCCCTTCGAAG-3′ and reverse primer 5′-AATCTAGAGTAAGCGGCTCCATCCATGGG-3′ with added XhoI and XbaI restriction sites, respectively. Both the promoter/5′ UTR and 3′ UTR fragments were respectively cloned in a sequential order into the BamHI + EcoRI and XhoI + XbaI sites of the pattB plasmid, giving rise to the pattB-dcp-1-5′-3′ plasmid. The dcp-1 and drice coding regions were each amplified from the cDNA clones as described above but this time using primers with added NotI and SalI restriction sites. Each fragment was then cloned into the NotI and XhoI restriction sites in the pattB-dcp-1-5′-3′ plasmid. These transgenes were inserted into the attP40 site on chromosome 2L using the ϕC31 system as before.
Immunofluorescence staining, TUNEL, and AO labeling and antibodies
Third instar larvae were subjected to γ-irradiation (50- or 20-Gy doses) and allowed to recover for several hours at room temperature, and their WDs were dissected and stained for either cleaved human PARP, cleaved caspase-3, TUNEL (see below), or AO (see below) using standard protocols (Arama et al., 2003
; Arama and Steller, 2006
). The corresponding antibodies used were rabbit polyclonal anticleaved human PARP (1:500, Ab2317; Abcam), rabbit polyclonal anticleaved caspase-3 (1:75, Asp175; Cell Signaling Technology), and rabbit polyclonal anticleaved caspase-3 (1:500, Ab13847; Abcam).
For TUNEL labeling, WDs were dissected and fixed for 20 min at room temperature in 4% PFA in PBS. The samples were then rinsed in PBS, washed twice, 10 min per wash, in 1× BSS (5× BSS: 270 mM NaCl, 200 mM KCl, 37 mM MgSO4, 12 mM CaCl2, 2 mM H2O, 24 mM tricine, 1.8% glucose, and 8.5% sucrose), and washed three times, 5 min per wash, with PBTw (0.1% Tween 20 in PBS). The samples were refixed in 4% PFA for 20 min, washed in PBTw five times, 5 min per wash, incubated in equilibration buffer (ApopTag kit; Millipore) for 1 h, and incubated again in reaction buffer (TdT enzyme; ratio 7:3; ApopTag kit) at 37°C overnight. On the next day, the TdT reaction mix was replaced with stop buffer (diluted 1:34 in dH2O; ApopTag kit) and incubated at 37°C for 3–4 h. Then, the samples were washed three times, 5 min per wash, blocked in BTN solution (1× BSS, 0.3% Triton X-100, and 5% normal goat serum) at room temperature for 1 h, and incubated with antidigoxigenin antibody solution (diluted 47:53 in blocking solution; ApopTag kit) overnight in the dark at 4°C. On the following day, the samples were washed four times in 1× BSS, 20 min per wash, and mounted in Fluoromount-G (SouthernBiotech).
For AO staining, WDs were incubated in a 0.6-µg/ml solution of AO (diluted in PBS) for 5 min, washed briefly in PBS, and mounted in a drop of PBS. Images were taken on a confocal microscope (LSM510 Meta Inverted Axio Observer; Carl Zeiss) using an EC Plan Neofluar 20×/0.50 M27 lens. Fluoromount-G was used as the imaging medium. All images were captured using the LSM510 operating software (Carl Zeiss).
Detection of effector caspase activity in vivo using the CPV reporter
In living cells, the reporter is localized to the plasma membrane via its CD8 domain. Upon cleavage by caspases, Venus is released to the cytoplasm, and, in principle, this process can be revealed by live imaging in large-diameter cells. However, in tissues of small-diameter cells, it is more difficult to distinguish between the membranal and cytoplasmic Venus, and, thus, the detection requires further staining of the tissue with an anti-cPARP antibody, which specifically detects the cleaved C-terminal fragment of human PARP.
Quantification of images
Quantification of staining was performed by measuring the area of the positively stained pixels (i.e., cPARP or TUNEL) and dividing it in the area of the Venus expression for cPARP or the total disc area for TUNEL using the ImageJ program (National Institutes of Health; Abramoff et al., 2004
). Statistical analysis was performed using a one-way analysis of variance test followed by Fisher’s protected least significant difference posttest for multiple comparisons using the StatView Program (Abacus Concepts). For each experiment, 12–20 WDs were analyzed for each genotype or time point. Significance level was considered as either P < 0.05 or P < 0.001, as indicated above the bars in the figures.
Quantification of drice and dcp-1 mRNA expression levels
WDs from wild-type and mutants flies were dissected, and total RNA was extracted using the RNeasy Micro Kit (QIAGEN). Reverse transcription was performed with 1 µg of total RNA. First-strand synthesis used Oligo(dT) primers (Promega) and SuperScript II Reverse Transcriptase (Promega) in the presence of RNase-free DNase to eliminate DNA contamination and Protector RNase inhibitor (Roche). Measurements were normalized to mitochondrial large-ribosomal RNA (MtlrRNA1: forward 5′-AAAAAGATTGCGACCTCGAT-3′ and reverse 5′-AAACCAACCTGGCTTACACC-3′) or RP49 (Dmel/RpL32: forward 5′-GACCATCCGCCCAGCATAC-3′ and reverse 5′-CCATTTGTGCGACAGCTTAGC-3′). The primers for drice mRNA were forward 5′-CCACTAACAATGGAGAATCCGCC-3′ and reverse 5′-GCCGCTGCTACCCGCTCCTC-3′. The primers for dcp-1 mRNA were forward 5′-ACCGACGAGTGCGTAACCAGAA-3′ and reverse 5′-ACAAGAGACTCCGGCGTACAGC-3′. Products were amplified by the KAPA SYBR FAST quantitative PCR kit (Kapa Biosystems) using the LightCycler 480 quantitative PCR machine (Roche). Results of expression levels were calculated as the mean from three independent experiments with at least two biological repeats with two duplicates each.
Third instar larvae of each genotype were collected in vials with fresh food at 25°C. Eclosing adults were counted, and fly viability was determined as the number of eclosed adults divided in the total larvae collected. Survival rate was normalized to wild-type levels, and the larvae were collected during a period of 3 wk.
50 WDs from larvae with the desired genotype were dissected in PBS and collected into cold lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% NP-40, and a protease inhibitor mix). Then, these WDs were homogenized and incubated for 15 min on ice and centrifuged for 20 min, and the supernatant was collected and mixed with a sample buffer. Gel running and subsequent blotting was performed using standard methods, and the blots were incubated with a guinea pig anti–Dcp-1 antibody (1:1,000; a gift from P. Meier), a mouse anti–lamin Dmo antibody (1:500; a gift from Y. Gruenbaum, The Hebrew University of Jerusalem, Jerusalem, Israel), or a rabbit anti-Drice antibody (1:2,000; a gift from P. Friesen, University of Wisconsin-Madison, Madison, WI).
Online supplemental material
Fig. S1 shows that drice
transcripts are expressed at comparable levels in WDs. Fig. S2 shows that the CPV
reporter does not affect the rate of cell death. Fig. S3 shows that different anticleaved caspase-3 (CM1) antibodies detect both active Drice and Dcp-1, although they are more specific toward active Drice. Fig. S4 shows that Dcp-1 may further activate Drice in a positive amplification loop. Fig. S5 shows that Diap1 does not inhibit Drice and Dcp-1 with different efficiencies. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201107133/DC1