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First published online 15 March 2006
doi: 10.1242/dev.02332

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Autophagy occurs upstream or parallel to the apoptosome during histolytic cell death

Fatih Akdemir¹, Robert Farka², Po Chen¹, Gabor Juhasz³, Lucia Medved'ová^2,4, Miklos Sass⁵, Lai Wang⁶, Xiaodong Wang⁶, Suganthi Chittaranjan⁷, Sharon M. Gorski⁷, Antony Rodriguez⁸ and John M. Abrams^1,*

¹ Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX 75390, USA.
² Institute of Experimental Endocrinology, Slovak Academy of Sciences, Vlarska 3, 83306 Bratislava-Kramare, Slovakia.
³ Department of Genetics, Cell Biology and Development, University of Minnesota, 6-160 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA.
⁴ Department of Genetics, Faculty of Science, Comenius University, 84215 Bratislava, Slovakia.
⁵ Department of General Zoology, Lorand Eotvos University, Pazmany setany 1/C, H-1117 Budapest, Hungary.
⁶ Department of Biochemistry, UT Southwestern Medical Center, Dallas, TX 75390, USA.
⁷ Genome Sciences Centre, BC Cancer Research Centre, 675 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada.
⁸ Wellcome Trust, Sanger Institute, Genome Campus, Cambridge CB10 1SA, UK.

View larger version (14K): [in a new window]   Fig. 1. Generation of a dark82 null mutation. (A) Schematized view of the genomic structure of the dark locus, relevant alleles and the dark82 null mutation. The dark transcript spans 6.6 kb. dark82 is a 6324 bp deletion (dashed line) generated by imprecise excision of the indicated P-element in the darkCD4 strain (Rodriguez et al., 1999). The allele was mapped by sequencing a 1.3 kb genomic PCR fragment (see B) using a primer pair (designated 1 and 2) spanning the junctional interval. In dark82, sequences from -1277 bp (upstream of the translation start codon) to 19 bp downstream of the stop codon are absent such that the entire dark ORF and part of the untranslated first exon are missing. Note that 396 bp of sequence from the CD4 transposon remain at this junction. (C) RT-PCR with primer pair 3 and 4, using total RNA from prepupae, confirms complete loss of the dark transcript in the dark82 allele. Two different isolates of dark82 from the screen were assayed here, 82 (1) and 82 (2). rp49 is a control.

View larger version (52K): [in a new window]   Fig. 2. dark is essential for programmed and unprogrammed apoptosis. (A-D) Maternal and zygotic sources of dark were removed using a Dominant Female Sterile strategy (see Materials and methods). The resulting embryos lacked nearly all PCD, shown here by Acridine Orange (AO) staining (green). A and B show mid-staged embryos eliminated for maternal dark but heterozygous for zygotic dark; C and D show comparably staged embryos lacking both maternal and zygotic dark. Note that without a source of dark, embryos are head involution defective with only few AO-positive cells (C,D). (E-G) Requirement for dark in models of stress-induced cell death. Hemocyte aspirates from dark82 and wild-type (wt) wandering third instar larvae were treated with chemical stressors ex vivo and stained with CellTracker (see Materials and methods). Induction of apoptosis in wild-type (E) but not dark82 (F) hemocytes is exemplified here with micrographs taken 6 hours after Cycloheximide (CHX) treatment. (G) Quantification of apoptosis 6 hours after challenge with either CHX or a Smac mimetic (Li et al., 2004) are plotted as the incidence of cell death in percentages. Error bars indicate s.d.

View larger version (48K): [in a new window]   Fig. 3. Alteration of a caspase cleavage site produces a hypermorphic Dark variant. (A) Recombinant Dark protein (lane 1) was incubated with cytosolic S20 fractions prepared from control S2 cells (lane 2) or cycloheximide (CHX)-treated S2 cells (lane 3). Asterisk denotes the small Dark C-terminal fragment after cleavage. (B) Consistent with in vitro studies (A), stimulus-dependent cleavage of Dark is detected here in Drosophila S2 cells. Samples from unchallenged (Ctrl) S2 cells or cells treated with 20 µM Cycloheximide (CHX) or 200 mJ/cm2 UV were harvested after 4 hours. (C) Cleavage of Dark as seen in panel B with CHX treatment, is reversed by the caspase inhibitor z-VAD (100 µM), shown here 5 hours post-treatment. In A,B and C, Dark was visualized with an anti-Dark polyclonal antibody. (D) The cleavage site, detected in vitro at residue 1292, is shown (arrow) in the schematized domain structure of the Dark protein. (E) Illustration of the defective anatomy of dark82 flies rescued by leaky expression of UAS-darkV, which mutates the caspase site mapped in D. The notum of a dark82 homozygote rescued to viability by UAS-darkV, shown here next to a wild-type fly notum (left), exhibits a `split thorax' phenotype and bristle abnormalities. (F) Levels of transgenic Dark protein in various UAS-dark transgenic lines in the absence of any driver or under Tubulin-Gal4 were examined by immunoblot using an anti-Myc antibody. Arrowhead denotes Dark-myc; asterisk indicates an irrelevant cross-reacting band showing equal loading on each lane. Note that the levels of wild-type Dark and DarkV are comparable when expressed from the Tubulin-Gal4 driver or when examined for basal expression. (G) Hemocyte aspirates from dark82; Hml-Gal4:UAS-darkWT (Hml:darkWT) and dark82; Hml-Gal4:UAS-darkV (Hml:darkV) L3 larvae were treated with DMSO or the Smac mimetic (Li et al., 2004), a potent apoptotic inducer. Expression of UAS-darkWT in dark82 hemocytes only mildly restored apoptosis after Smac mimetic treatment. However, UAS-darkV almost completely restored this apoptotic response to dark82 hemocytes.

View larger version (51K): [in a new window]   Fig. 4. dark82 salivary glands are defective for histolysis. (A,B) Confocal micrographs of salivary glands from wild-type (A) and dark82 (B) animals at 16 hours APF stained for a cytoplasmic protein, p127 (green), and a nuclear protein, BR-C (red). Head eversion, which marks the prepupal-pupal transition, has occurred in these animals. In wild type, larval salivary glands are completely histolysed, but in dark82 animals the glands persist and structural integrity is maintained. (C,D) Confocal micrographs showing immunohistochemical staining of salivary glands for anti-cleaved caspase 3 (blue), a marker for active DRICE (Yu et al., 2002), together with anti-actin, (red) and OliGreen, a nuclear stain (green). (C) Caspase activity (blue) in wild-type salivary glands is shown here at 12 hours APF, 4 hours before final histolysis. (D) Caspase activity is starkly reduced in salivary glands of dark82 animals, shown here at 16 hours APF. (E-G) Ecdysone signaling and expression of death-related genes are unperturbed in dark mutant salivary glands. Immunohistochemical staining (E,F) shows nuclear accumulation of ecdysone-responsive transcription factors in persisting dark salivary glands at 16 hours APF. The confocal image in E shows coincident nuclear accumulation of Ecdysone Receptor (EcR, red) and BFTZ-F1 (green), counterstained for actin (blue). Overlapping stains for EcR and BFTZ-F1 produces a robust yellow signal in gland cell nuclei. In F, nuclear accumulation of E74A (red) is shown, with counterstaining for actin (blue) and the cytoplasmic protein Rab11 (green). (G) Pre-death expression profiles for the genes indicated were determined using real-time quantitative RT-PCR on RNA prepared from salivary glands dissected from wild-type (OreR) and dark82 animals at 11 hours and 13 hours APF (normalized from 18°C). The gene set analyzed here is a surrogate for profiles of pre-histolytic gene expression (Gorski et al., 2003). Expression levels are represented by Ct values, where Ct=Ct of no template control (set at 38 PCR cycles) - Ct of sample. Ct, or threshold cycle, is the PCR cycle at which a statistically significant increase in fluorescent signal can be detected above background. Drosophila rp49, used here as a control, showed no significant differences in expression. dark transcripts were not detected in mutant salivary glands, but, in all other respects, profiles between wild-type and dark glands were highly comparable.

View larger version (127K): [in a new window]   Fig. 5. Autophagy proceeds normally in dark mutant salivary glands. (A-C) Transmission EM of salivary gland cells. (A) A cytoplasm saturated with small vesicles and an electron dense nucleus (N) are indicative of ongoing cell death in wild-type cells at 14 hours APF. By contrast, salivary gland cells appear healthy in 14-hour APF dark82 (B) and 24-hour APF dark82 (C), showing no sign of cell death (compare the appearance of the nucleus in C with the nucleus in A). Arrows indicate autolysosomes in A-C, demonstrating that dark is not required for autophagy. Insets in panel C show enlargements of representative autophagosomes (top right corner) and autolysosomes (top left corner) seen in mutant glands. N, nucleus; g, secretory granule; asterisks indicate mitochondria. Scale bars: 1 µm; 250 nm for the insets. Arrowheads in C indicate autophagosomes. (D-I) Salivary glands dissected at the indicated time points (25°C) and stained with the acidic marker monodansylcadaverine (MDC) to detect autolysosomes (Munafo and Colombo, 2001). F shows a merged image of MDC staining (red) and detection of GFP-LC3 (green) (Rusten et al., 2004), a transgenic GFP marker for autophagosomes and autolysosomes in wild-type salivary glands (14 hours APF). At this stage, prior to histolysis, the overlap between MDC and GFP-LC3 is extensive, indicating an abundance of autolysosomes. (D-G) Time course of MDC staining in wild-type salivary glands. (D) At 9 hours APF, MDC staining is barely detectable. (E) At 11 hours APF, some punctate MDC-positive staining can be observed. However, by 14 hours APF (F) and in 15-hour APF glands (G), large MDC-positive structures are very conspicuous. Likewise, in comparably staged mutant glands, prominent MDC-positive vesicles are seen, shown here at 12 hours APF (H) and in persisting salivary glands 4 hours later (I).