Results

Identification of proteins expressed during autophagic cell death of salivary glands

A rise in the steroid hormone 20-hydroxyecdysone (ecdysone) triggers larval salivary gland autophagic programmed cell death.^{9, 22} Although proteins associated with apoptosis, including caspases, are induced and participate in the destruction of salivary glands, the inhibition of caspases does not completely prevent changes associated with dying cells.^{6, 19, 20} To identify proteins that are expressed in salivary glands, soluble proteins were extracted from salivary glands dissected from wild-type animals that were staged 6 and 13 h after puparium formation; stages before and immediately following the rise in ecdysone that triggers cell death. Two independent samples of Trypsin-digested proteins from each stage were separated by capillary isoelectric focusing (IEF), and 12 different fractions were separated further by capillary reverse-phase liquid chromatography. These fractions were analyzed using a ThermoFinigan (Waltham, MA, USA) LCQ ion trap mass spectrometer (MS). Three MS/MS scans were performed and raw data files were processed and submitted for database searching using MASCOT (Matrix Science, Boston, MA, USA). We identified 3352 proteins in the 6 h samples with greater than 95% confidence, and 1111 of these proteins contained two or more distinct peptide sequences (Table S1). Similarly, 4523 proteins were identified in the 13 h sample with greater than 95% confidence, and 1907 of these proteins were identified based on two or more distinct peptide sequences. The total number of proteins identified in both samples is 5661 with 2214 proteins common to both 6- and 13 h stages.

Proteins that are known to be involved in salivary gland programmed cell death were identified using this proteomic approach. The ecdysone regulated proteins Eip74EF (E74), Eip93F (E93), and the nuclear hormone receptors Usp and E75B were all detected using this approach (Table S1), and this is consistent with DNA microarray studies¹⁸ and previous studies of these transcription regulators.^{17, 23, 24, 25} Caspase proteases function in salivary gland cell death,^{6, 7, 16, 26} and several proteins that function during apoptosis were detected including the caspase activator Ark, and the caspases Strica/Dream and Nc/Dronc, consistent with DNA microarray studies.¹⁸ These data provide confidence that proteins that are known to be involved in salivary gland programmed cell death can be identified using this shotgun proteomics approach, and that this can provide a valuable overview of the proteome in dying cells.

Identification of new salivary gland proteins

Although the identification of several known salivary gland proteins provided evidence that this novel proteome technology is sensitive, the validation of the presence of newly discovered proteins is critical to endear confidence before investing effort in reverse genetic studies. We analyzed our data for previously studied proteins so that we could use available antibodies to validate our salivary gland proteome data using immunohistochemical staining. Salivary glands were isolated from wild-type Canton S animals staged to 6 and 13 h after puparium formation, fixed and stained with available antibodies against the Drosophila insulin receptor (DInR), Kelch (Kel), Crumbs (Crb), Cactus (Cact), Quail (Qua), Highwire (Hiw), and Osa.^{27, 28, 29, 30, 31, 32, 33} We had previously shown that nuclear Lamin DmO and -spectrin are expressed in dying salivary glands,⁷ and these antibodies were used as positive controls.

Whole-genome DNA microarray studies indicated that several of the identified salivary gland proteins are transcribed in these cells,¹⁸ and we intitiated our validation by evaluating the presence of these proteins (Figure 1). DInR was identified based on the detection of multiple distinct peptides and the presence of RNA using DNA microarrays, and DInR antigen is present in salivary gland cells. The actin-binding protein Kel and the cell polarity protein Crb were both detected using antibodies as expected based on the identification of multiple distinct peptides from each of these proteins, as well as detection of their RNAs in previous microarray studies. We only detected a single peptide from the IkB protein Cact, and the presence of this nuclear factor kappa B regulator was validated using antibodies as expected based on the presence of Cact RNA in microarray studies. We then addressed if the actin binding protein Qua was present in these cells, as quail RNA was present at low levels in DNA microarray studies,¹⁸ and we only detected a single Qua peptide in our proteomic studies. The presence of Qua antigen in salivary glands provided greater confidence that the detection of single peptides using this approach can lead to the identification of proteins.

Figure 1.

Verification of proteome results by immunohistochemistry. Salivary glands of staged wild-type Canton S animals were stained with antibodies against either DInR (green) and nuclear Lamin DmO (red), or - Spectrin (green) and experimental antibodies (in red) against Kel, Crb, Cact, Qua, Hiw, Osa

Full figure and legend (180K)

The identification of low abundance proteins, and proteins that may not be regulated at the level of RNA transcription, provides one of the greatest incentives for the development of sensitive high-throughput proteomics technologies. To test if we could detect proteins that were absent in our whole-genome microarray studies,¹⁸ but were present in our proteomics data, we stained salivary glands with antibodies against Hiw and Osa (Figure 1). Hiw encodes a putative Ubiquitin ligase that regulates synaptic growth,³⁴ and the identification of multiple distinct peptides was validated based on Hiw localization in the cytoplasm of salivary gland cells. Similar to Hiw, the transcription regulator Osa was not detected in our previous DNA microarray studies, but Osa protein was present in the nuclei of salivary gland cells as expected based on the identification of multiple Osa peptides. Although our previous use of this proteomics approach to identify yeast proteins indicated the robust nature of this technology,³⁵ these data provide further evidence of its strength by identification of numerous proteins from a complex animal with a larger genome.

We identified many interesting proteins using this novel high-throughput technology (Table S1), thus our next interest was to investigate what signaling pathways and cell processes may be involved in the autophagic cell death of salivary glands. Transcription is known to play an important role in this steroid-triggered cell death, and the identification of several new proteins in this category including DHR96, Cactus, Bunched, Hsf and the Mediator/Trap proteins MED1, MED14, and MED17 (Table 1) reflect the importance of the regulation of RNA levels in these dying cells. Little is known about the transcription coactivators and corepressors that are involved in programmed cell death, and the identification of several proteins in these categories, including Hcf, Nej, Tai, Brm, Gug, H, Hira, and Smarter (Smr), provides a starting point for investigating their function in autophagic programmed cell death (Table 1).

Table 1 - Summary of new transcription regulators that were identified in dying salivary glands.

Full table

Salivary glands rapidly degrade during autophagic programmed cell death,⁶ and we hypothesize that many categories of proteins may contribute to this process. Supporting this idea, we identified proteins involved in apoptosis, the ubiquitin proteasome system, autophagy, lysosome biogenesis, and noncaspase proteases (Table 2, Table S2). Several proteins that function during apoptosis were detected including the Bcl-2 family members Debcl and Buffy, the ubiquitin ligase Morgue, the caspase activator Ark, and the caspases Dream/Strica and Nc/Dronc. The ubiquitin proteasome system serves an important role in the degradation of short-lived proteins including caspase regulators. In addition to the identification of several proteasome components (Table S2), numerous proteins that are involved in the regulation of ubiquitination were detected including Bruce and Ago, as well as many others (Table 2). Autophagic vacuoles form following the initiation of salivary gland cell death. Although the mechanism(s) by which autophagy is regulated is not fully understood in these cells, the presence of the autophagy proteins Atg1, Atg2, Atg4, Atg6, Atg9, and the Atg18-like protein CG8678 are consistent with this catabolic process being involved in salivary gland cell death (Table 2). Autophagy requires lysosomes for the degradation of cargo, and it is intriguing that several proteins involved in lysosome biogenesis and organization, such as orange, ruby, and garnet that were originally isolated based on defects in eye pigmentation, were identified (Table 2). Given this result, we tested if lysosomes are more abundant in later stages of salivary gland cell death by using a fusion between the lysosomal associated membrane protein 1 (LAMP1) and horse radish peroxidase (HRP) that has been previously used to detect lysosomes in Drosophila.³⁶. Salivary glands isolated from animals 12 h after puparium formation have large vacuoles in every cell and little LAMP1–HRP staining (Figure 2). By contrast, salivary glands dissected 14 h after puparium formation possess increased LAMP1-HRP staining that is localized to perinuclear lysosome membranes (Figure 2). These results are consistent with the timing of the induction of autophagy, and Atg RNA and proteins, that immediately precede cell degradation^{6, 18} (Table 2), and suggest that lysosome biogenesis may be important for completion of salivary gland degradation.

Figure 2.

Lysosomes are induced just before salivary gland cell death. Salivary glands of animals that express the lysosome protein Lamp1 fused to HRP were staged 12 h (a) and 14 h (b–d) after puparium formation to visualize lysosomes by either light (a and b) or transmission electron microscopy (c and d). (a) Twelve-hour salivary glands have large vacuoles in every cell (asterisks) and little HRP staining. (b–d) Salivary glands dissected 14 h after puparium formation have either few or no large vacuoles (asterisks), and these cells possess increased HRP staining that is localized to perinuclear lysosome membranes (arrows). Scale bars in c and d, 1 m

Full figure and legend (181K)

Table 2 - Summary of new proteins that function in degradation that were identified in dying salivary glands.

Full table

Dying salivary glands exhibit dynamic changes in structure including the loss and formation of different types of vacuoles⁶ (Figure 2), and the actin cytoskeleton is reorganized independent of caspase activation.⁷ Although some of these changes could be caused by the cleavage of proteins by proteases, other mechanisms may regulate reorganization of the cytoskeleton and movement of structures during autophagic cell death. Consistent with this hypothesis, we identified a large number of proteins that participate in changes in cell organization including GTPases, ARFs, ARPs, formin homology proteins, and motors (Table S3).

An intricate relationship exists between cell growth and proliferation, and programmed cell death during animal development. Growth and proliferation arrest are often associated with the activation of apoptosis, but the relationship between growth and autophagic cell death is unclear. The insulin receptor (InR) signaling pathway is an important mechanism for the regulation of growth. InR signaling may be relevant to autophagic cell death, as autophagy is influenced by the activity of several proteins within this pathway.³⁷ We identified many components of this pathway including InR, the class I phosphotidylinositol 3 kinase (PI3 K) PI3K92E, the tuberous sclerosis protein TSC1, the target of rapamycin (Tor), as well as 2 other InR-like proteins (Table 3). In addition to our identification of the class I PI3 K, we also identified a protein encoding the class II PI3K68D (Table 3).

Table 3 - Summary of growth regulators that were identified in dying salivary glands.

Full table

Wts functions in salivary gland cell death

Mutations in the serine/threonine kinase warts (Wts) result in overgrowth of adult tissues,³⁸ and it was of interest that we identified two distinct Wts peptides in 13 h salivary glands (Table S1). Wts is among the novel proteins identified in this study that were not identified using DNA microarrays.¹⁸ As Wts has been previously linked to the regulation of cell-cycle arrest and apoptosis, and encodes a tumor suppressor, we tested if wts function is required for autophagic cell death of salivary glands.

Strong loss-of-function wts mutants are lethal before puparium formation. Therefore, we analyzed the weak wts^P2 allele for defects in salivary gland cell death.³⁸ Homozygous wts^P2 and wts^P2 /wild-type control pupae were aged 12 h after future adult head eversion which is 8 h after larval salivary gland cell death is complete in wild-type flies.⁶ These pupae were embedded in paraffin, sectioned, and stained for examination of cell death defects. In sections of control wts^P2 /wild-type, salivary glands are destroyed (Figure 3). By contrast, 100% of homozygous wts pupae had defects in salivary gland destruction (Figure 3), with 32% having intact salivary glands and 68% having fragmented salivary glands that are not properly degraded. Although homozygous wts pupae often exhibited over growth of imaginal tissues, this did not prevent either head eversion or the normal development of other structures. As Wts functions in a pathway with Hippo (Hpo), we also tested if expression of a previously studied hippoRNAi construct (hpoIR)³⁹ in salivary glands prevented salivary gland cell death by analysis of paraffin sections of pupae that were aged 12 h after future adult head eversion. Control animals, either UAS-hpoIR/wild type or fkh-GAL4/wild type, did not possess salivary glands (Figure 3). Animals that express hpoIR in salivary glands had fragmented remnants of salivary glands in 40% of the specimens (Figure 3). These results indicate that wts and hpo function in autophagic cell death of salivary glands.

Figure 3.

Wts and Hpo function in salivary gland autophagic cell death. Paraffin sections of pupae aged 12 h after head eversion. (a) In control wts^P2/CantonS wild-type pupae salivary glands have degraded (n=13). (b) 100% of wts^P2/wts^P2mutant pupae possess salivary glands 24 h after puparium formation (n=22). (c) Control, either UAS-hpoIR/CantonS or fkh-GAL4/CantonS, pupae lack salivary glands (n=16). (d) Experimental fkh-GAL4 UAS-hpoIR pupae possess partially degraded salivary glands 24 h after puparium formation in 40% of animals (n=15). Red circles mark the absence of salivary glands in controls (a and c), and persistent salivary glands and gland fragments in experimental wts mutant and hpoIR animals (b and d)

Full figure and legend (321K)

Discussion

Although much is known about the mechanisms that regulate apoptosis, little is known about alternative morphological forms of cell killing including autophagic cell death. Here we have used a high-throughput approach to identify proteins in dying salivary glands. This resulted in the identification of numerous factors that we expected based on previous whole-genome microarray and SAGE studies,^{18, 21} but we also identified proteins that were not previously known to be expressed in dying salivary glands. These data indicate that it is important to combine proteomic approaches with genomic technologies designed to detect RNAs to get a complete overview of the possible players involved in a cellular process. The presence of a protein within a dying cell is not evidence that this factor functions in cell death, and caution should be taken before implicating innocent bystanders as being guilty by association. Therefore, such genomic approaches will be most powerful when they are combined with genetic approaches, and we now have a list of cellular processes and signaling pathways that need to be investigated by analyses of mutants.

Previous studies have shown that a rise in the steroid ecdysone triggers a transcription cascade that activates a caspase-dependent autophagic cell death of salivary glands.⁹ This model has strong support, because mutations in primary ecdysone-response transcription regulators prevent salivary gland cell death,⁶ and these mutants also possess decreased levels of caspases and caspase regulators.⁷ However, the inhibition of caspases and caspase mutants do not prevent all of the cellular changes associated with the death of salivary glands,^{6, 7, 19, 40} and suggest that additional noncaspase degradation mechanisms may be involved in the physiological destruction of these cells. Consistent with this hypothesis, our genomic studies indicate that multiple noncaspase degradation systems, including autophagy, the ubiquitin proteasome, and the lysosome, may be involved in the death of salivary glands. Furthermore, the presence and requirement for Wts that is not ecdysone-regulated indicates that post-translational mechanisms also contribute to the death of salivary glands.

Analyses of wts and hpo indicate that at least some of the mechanisms that regulate tissue growth control also function during autophagic cell death, but additional studies are needed to determine the relationship between cell growth and autophagic cell death. Many growth regulators, including several proteins in the Wts and PI3K pathways, depend on phosphorylation to modulate their activities, and studies of post-translational modification of proteins in dying salivary glands should provide insights into the mechanisms that mediate their cell death. The relationship between different signaling pathways, degradation mechanisms, and how these factors contribute to the autophagic cell death morphology is not clear, and it is important to resolve how these mechanisms integrate to result in a physiological cell removal strategy. These studies provide an important foundation for future genetic and biochemical studies of autophagic programmed cell death by providing a comprehensive description of the proteins that are present in dying salivary glands.

Materials and Methods

Protein extraction and proteomics

Soluble protein extracts were obtained from wild-type Canton S salivary glands that were dissected from animals staged 6 and 13 h after puparium formation. Salivary glands were homogenized in 40 mM Tris buffer containing 1 mM phenlymethylsulfonyl fluoride, 1 M Pepstatin A, 1 M Leupeptin, and incubated for 5 min at 100°C. Proteins were denatured, reduced and alkylated in 20 mM Tris buffer containing 8 M urea, 0.1 M dithiothreitol (DTT) and 0.1 M iodoacetamide. Denatured, reduced and alkylated proteins were reconstituted in a solution of 10 mM Tris, 0.4 M urea and 10 mM DTT using regenerated cellulose membrane filters (Millipore, Billerica, MA, USA) with a 5000 molecular weight cutoff. Trypsin (1:50, w/w, Promega modified sequencing grade, Madison, WI, USA) was added and the mixture was incubated at 37°C for overnight.

Independent samples of trypsin-digested protein from each sample were separated by capillary isoelectric focusing . Twelve different IEF fractions were separated further by capillary reverse phase liquid chromatography, and analyzed using a ThermoFinigan LCQ ion trap MS. Three MS/MS scans were performed on the top three ions from the preceding MS scan. These data were processed using Distiller (Matrix Science) and submitted for database analyses using MASCOT (Matrix Science). The database that was searched was a non-redundant compilation of the Swiss-plot and TREMBL Drosophila melanogaster data sets available from EBI.

Immunohistochemistry

Wild-type Canton S salivary glands were dissected from animals staged 6 and 13 h after puparium formation at 25°C, fixed in 4% paraformaldehyde/heptane for 20 min at room temperature, blocked in phosphate-buffered saline containing 1% BSA and 0.1%. Triton-X (PBSBT), and incubated with primary antibodies for 16 h at 4°C. Salivary glands were washed for 2 h in PBSBT, incubated with appropriate secondary antibodies for two hours at room temperature and washed for another 2 h in PBSBT at room temperature. Salivary glands were mounted in Vectashield (Vector Laboratories) and examined using a Zeiss Axiovert 100M confocal microscope.

Histology

UAS-HRP-LAMP1 transgenic flies were kindly provided by H. Kramer.³⁶ Salivary glands were dissected from fkh-GAL4; UAS-HRP-LAMP1animals at specific stages during cell death. Salivary glands were fixed for 10 min in 2.5% glutaraldehyde, rinsed in PBS, and incubated in 0.5 mg/ml DAB and 0.03% hydrogen peroxide. Staining of 12 and 14-h salivary glands in DAB was done for the same period of time so that these samples could be compared. For whole mount analyses, salivary glands were then dehydrated, mounted in methyl salicylate, and examined by light microscopy. For examination of thin sections by transmission electron microscopy, DAB-stained salivary glands were fixed for an additional hour in 2.5% glutaraldehyde, postfixed in 4% osmium tetroxide for 1 h, embedded in Spurr's resin, sectioned, and analyzed using a Zeiss EM 10 transmission electron microscope.

For paraffin sections, control (either wts^P2/CantonS, UAS-hpoIR/CantonS, or fkh-GAL4/CantonS) and experimental (either wts^P2/ wts^P2or fkh-GAL4; UAS-hpoIR) animals were maintained at 25°C and aged to 12 h after head eversion. Whole pupae were fixed in 1% glutaraldehyde, 4% formaldehyde and 5% acetic acid in 80% ethanol at 4°C. Fixed pupae were dehydrated through an ethanol series, cleared in xylenes and infiltrated with paraffin. Sections were cut at 7 m, stained with Weigert's hematoxylin and Pollak Trichrome and examined and photographed using a Zeiss Axiophot II microscope.

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Acknowledgements

We thank B Hay, D Branton, P Fisher, L Pick, H Kramer, G Halder, N Tapon, and the Developmental Studies Hybridoma Bank for antibodies and fly stocks, and D Berry for helpful comments and discussions. This work was supported by NIH NRSA GM067563 to DNM, an HHMI Undergraduate Research Fellowship to SK, and NIH grants CA107988 to BMB, GM073723 to CL, and GM59136 to EHB.

Supplementary Information accompanies the paper on Cell Death and Differentiation website (http://www.nature.com/cdd)