The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

Bcl-2 family proteins play a central role in regulating apoptosis. We previously reported that human Bcl-rambo, also termed BCL2L13, localized to mitochondria and induced apoptosis when overexpressed in human embryonic kidney 293T cells. However, the physiological function of Bcl-rambo currently remains unclear. In the present study, human Bcl-rambo was ectopically expressed in Drosophila melanogaster. Bcl-rambo mainly localized to the mitochondria of Drosophila Schneider 2 (S2) cells. The overexpression of Bcl-rambo, but not Bcl-rambo lacking a C-terminal transmembrane domain, induced apoptosis in S2 cells. Moreover, the ectopic expression of Bcl-rambo by a GAL4-UAS system induced aberrant morphological changes characterized by atrophied wing, split thorax, and rough eye phenotypes. Bcl-rambo induced the activation of effector caspases in eye imaginal discs. The rough eye phenotype induced by Bcl-rambo was partly rescued by the co-expression of p35, Diap1, and Diap2. By using this Drosophila model, we showed that human Bcl-rambo interacted genetically with Drosophila homologues of adenine nucleotide translocators and the autophagy-related 8 protein. The results of the present study demonstrated that human Bcl-rambo localized to mitochondria and at least regulated an apoptosis signaling pathway in Drosophila.


Introduction
Programmed cell death plays an essential role in the development and maintenance of tissue homeostasis in animals [1]. Apoptosis is one type of programmed cell death that is mainly regulated by a family of cysteinyl aspartate-specific proteinases (caspases) [2]. Caspases have been classified as initiator caspases (e.g., caspase-8, -9, and -10) and effector caspases (e.g., caspase-rambo , and Bcl-rambo  were generated by PCR amplification (Fig 1A and  panel A in S1 Fig). Full-length Bcl-rambo, Bcl-rambo (ΔTM), Bcl-rambo (TM), Bcl-rambo (1-441), Bcl-rambo , and DsRed-monomer (TAKARA BIO, Shiga, Japan) were inserted into pUAST and/or pMT/V5-His A expression vectors. The genes encoding Drob-1/Debcl and Buffy were generated by PCR amplification using a cDNA library prepared from Drosophila early embryos. Full-length Drob-1/Debcl and Buffy were inserted into pCR3-based expression vectors containing N-terminal FLAG or VSV tags. FLAG-tagged Drob-1 was inserted into pMT/V5-His A expression vectors. with pMT-V5-His A, pMT-V5-His A/Bcl-rambo, or pMT-V5-His A/Bcl-rambo (ΔTM) and then incubated in the presence of CuSO 4 (500 μM) and Z-VAD-fmk (20 μM) for 24 h. S2 cells were stained for Bcl-rambo (green) and with DAPI (blue) and MitoTracker 1 Red (red). The stained cells in at least five different fields were observed by confocal laser scanning microscopy. Optical sections containing single transfected cells are shown. Data were representative of two independent experiments. Scale bars indicate 10 μm. (C) S2 cells were transfected with pMT-V5-His A, pMT-V5-His A/Bcl-rambo, or pMT-V5-His A/Bcl-rambo (ΔTM) and incubated in the presence of CuSO 4 (500 μM) for 24 h. S2 cells were homogenized and separated into PNL, P10, P100, and S100 fractions. The fractions were analyzed by Western blotting using anti-Bcl-rambo, anti-HSP60, and anti-α-tubulin antibodies. Data were representative of three independent experiments.

Establishment of transgenic flies
pUAST-Bcl-rambo and pUAST-Bcl-rambo (ΔTM) were injected into embryos to obtain stable transformant lines carrying UAS-Bcl-rambo and UAS-Bcl-rambo (ΔTM). P element-mediated germline transformation was accomplished as described [30]. F1 transformants were selected on the basis of white eye color rescue. Eight and six independent lines were established for UAS-Bcl-rambo and UAS-Bcl-rambo (ΔTM), respectively.

Western blotting
Subcellular fractions were prepared as described above. Salivary glands were taken from third instar larva and heated at 95°C for 2 min in 0.11 mM Tris-HCl (pH 7.5) and Complete™. They were homogenized with a dounce homogenizer in the presence of SDS sample buffer. After centrifugation (10, 000 x g, 10 min), supernatants were collected. Protein samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Wako Pure Chemical Industries, Osaka, Japan). The membranes were incubated with primary antibodies and then HRP-conjugated secondary antibodies. Protein bands were visualized using ECL Western Blotting Detection Reagents (GE Healthcare, Piscataway, NJ, USA) and analyzed by ImageQuant LAS 4000 mini (GE Healthcare).

Apoptosis assay
S2 cells were stained with Hoechst 33342 (7.5 μM). Nuclear morphology was observed under a fluorescence light microscope (Axiovert 200M, Carl Zeiss, Jena, Germany). Apoptotic cells (%) were calculated as (condensed and/or fragmented nuclei / total nuclei) x 100. In parallel with the apoptosis assay, transfection efficiency was evaluated by transfection with expression vectors encoding DsRed-monomer.

Light microscopy and scanning electron microscopy (SEM)
Adult wings were dehydrated with isopropanol and mounted in Hoyer's medium. Samples were observed under a light microscope SZX12 (Olympus) or SEM VE-7800 (Keyence, Osaka, Japan) in the low vacuum mode. The phenotypes of the eye and thorax from at least four adult female flies (3 to 5 d old) were observed. The phenotypes of the wings from 4-5 adult female flies (3 to 5 d old) were observed. In these experiments, no significant variations were observed in eye, thorax, and wing phenotypes among the individuals. The L3-L4 area of the wings was quantified by Image J software. The mean of the L3-L4 area was calculated from 4-5 flies and normalized by the mean of the control L3-L4 area. The L3-L4 area (%) was calculated as the mean ± S.E. of three independent experiments. The brightness and contrast of all images taken by the light microscope were adjusted uniformly using Microsoft Office Picture Manager, which was applied equally to all images in each figure.

Caspase assay
Third instar larvae were dissected in PBS, and imaginal discs were suspended in Grace's insect medium in the presence of Cellevent™ Caspase-3/7 Green Detection Reagent (Life Technologies) at the final concentration of 5 μM for 1 h at 37°C. Eye imaginal discs were fixed with 4% PFA-PBS and permeabilized with 0.5% Triton X-100-PBS, followed by staining with Hoechst 33342 (5 μM). Samples were analyzed by confocal laser scanning microscopy.

Immunoprecipitation
HEK293T cells were transfected with expression vectors by the calcium phosphate method and incubated for in the presence of Z-VAD-fmk (20 μM) for 16 h. Cells were solubilized in Nonidet P-40 lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% Nonidet P-40, 10% glycerol, and 2 mM sodium vanadate, Complete™). Postnuclear lysates were precleared with Sepharose 6B for 1 h and then immunoprecipitated with anti-FLAG M2 affinity gels (Sigma-Aldrich) for 3 h. The immunoprecipitates were washed several times with Nonidet P-40 lysis buffer and analyzed by Western blotting.

Statistical analysis
Statistical analyses were performed using an analysis of variance followed by the Tukey's test for multiple comparisons.

Results
Bcl-rambo localized to mitochondria in Drosophila S2 cells Bcl-2 family proteins regulate the intrinsic apoptosis pathway via mitochondria. In HEK293T cells, full-length Bcl-rambo (1-485), but not Bcl-rambo lacking the C-terminal TM (1-459), localized to mitochondria [12]. Bcl-rambo, Bcl-rambo (ΔTM), Bcl-rambo (1-441), and Bclrambo (1-459) were transiently expressed in Drosophila S2 cells in order to examine whether the C-terminal TM of Bcl-rambo was necessary for localization to mitochondria in Drosophila. Confocal microscopy observations revealed that Bcl-rambo primarily co-localized with Mito-Tracker ( Fig 1B). In contrast, Bcl-rambo (ΔTM) was uniformly distributed over the cytoplasm (Fig 1B). Consistent with this, Bcl-rambo (1-441) and Bcl-rambo (1-459) did not localize to the mitochondria and exhibited a cytoplasmic distribution (panel B in S1 Fig). The localization of Bcl-rambo and Bcl-rambo (ΔTM) was further analyzed by subcellular fractionation. The mitochondrial chaperone HSP60 was present in the P10 fraction and, to a lesser extent, in the S100 fraction, while α-tubulin was only present in the S100 fractions (Fig 1C), indicating that the P10 fraction was enriched with mitochondria. Similar to HSP60, Bcl-rambo was present mainly in the P10 fraction and marginally in the S100 fraction, whereas Bcl-rambo (ΔTM) was only present in the S100 fraction ( Fig 1C). These results indicated that Bcl-rambo mainly localized to mitochondria in Drosophila S2 cells.
We further investigated the kinetics of apoptosis induced by human Bcl-rambo and the Drosophila pro-apoptotic Bcl-2 family member Drob-1/Debcl/dBorg-1/DBok [32][33][34][35]. In S2 cells transiently transfected with expression vectors driven by the metallothionein promoter, the expression of Bcl-rambo and Drob-1 was induced within 4 h of the CuSO 4 treatment, and their expression levels were maintained at least up to 16 h (Fig 2B). The ectopic expression of Bclrambo or Drob-1 initiated the induction of apoptosis during 4-8 h, and the number of apoptotic cells steadily increased up to 16 h (Fig 2C). Cytochrome c was detected in the cytosolic fraction within 8 h of the CuSO 4 treatment in Bcl-rambo-or Drob-1-transfected S2 cells, but not control S2 cells (Fig 2D). These results suggest that Bcl-rambo induces the mitochondrial pathway of apoptosis in Drosophila.

Ectopic expression of Bcl-rambo, but not Bcl-rambo (ΔTM), induced morphological aberrations in Drosophila tissues
In order to gain an insight into the function of Bcl-rambo, it was ectopically overexpressed in several tissues of Drosophila by the GAL4-UAS expression system. GAL4 is expressed along the anterior-posterior compartment boundary under the control of the dpp (blk) promoter in the dpp-GAL4 driver line [36,37]. The ectopic expression of Bcl-rambo using the dpp-GAL4 driver fly lines induced morphological changes in a wing vein ( Fig 3A). In particular, the L3-L4 area of Bcl-rambo flies became smaller than that of control ( Fig 3B). In contrast, the overexpression of Bcl-rambo (ΔTM) did not cause obviously different phenotypes from those of control ( Fig 3A and 3B). In the pnr-GAL4 driver line, GAL4 is expressed in the dorsal mesothorax [38,39]. The expression of Bcl-rambo using the pnr-GAL4 driver fly lines induced a split-thorax phenotype, while no significant differences were observed between control and Bcl-rambo (ΔTM) (Fig 3C). The en gene is expressed in the posterior compartment of each segment of the embryo and plays an essential role in the development stages of Drosophila. The overexpression of Bcl-rambo using the en-GAL4 driver fly lines induced embryonic lethality, and no adult flies expressing Bcl-rambo were observed (Table 1). In contrast, Bcl-rambo (ΔTM) did not affect the number of adult flies (Table 1). In Drosophila, salivary gland is suitable for preparing cell lysates without the contamination of other tissues. The amount of Bcl-rambo and Bclrambo (ΔTM) at the protein level was almost equivalent in the sg-GAL4 driver fly lines ( Fig 3D  and 3E). Thus, these results indicated that the ectopic expression of Bcl-rambo interfered with the proper differentiation of the wing and thorax as well as embryonic development.

Ectopic expression of Bcl-rambo, but not Bcl-rambo (ΔTM), induced a rough eye phenotype in Drosophila
The expression of Bcl-rambo using the GMR-GAL4 driver fly lines induced a rough eye phenotype, such as a reduction in eye size and the loss of ommatidia, bristles, and pigmentation ( Fig  4A). In contrast, Bcl-rambo (ΔTM) did not exert any phenotypic changes from control and green fluorescent protein (GFP) (Fig 4A). Bcl-rambo and Bcl-rambo (ΔTM) were similarly expressed in eye imaginal discs (Fig 4B), excluding the possibility that Bcl-rambo (ΔTM) was not or insufficiently expressed. A rough eye phenotype was also observed in other independent transgenic lines for Bcl-rambo, but not Bcl-rambo (ΔTM) ( Table 1 and

Ectopic expression of Bcl-rambo induced activation of effector caspases in Drosophila
Bcl-rambo was found to induce apoptosis in S2 cells (Fig 2). Drosophila effector caspases have been shown to exhibit DEVD-cleaving activity in a manner similar to mammalian effector caspases [40,41]. In order to determine whether the ectopic expression of Bcl-rambo caused caspase activation, a fluorogenic substrate for caspase-3/7 was used to visualize activated effector caspases. When Bcl-rambo was expressed in eye imaginal discs, the activated effector caspases greatly increased from the posterior region to the morphogenetic furrow (Fig 5A and 5B). However, no obvious signals of activated caspases were detected when Bcl-rambo (ΔTM) was expressed (Fig 5A and 5B). These results indicated that the ectopic expression of Bcl-rambo, but not Bcl-rambo (ΔTM), significantly induced the activation of effector caspases.
Reduced pigmentation induced by Bcl-rambo was rescued by p35, Diap-1, and Diap-2 The baculovirus p35 and Drosophila Diap1 and Diap2 have been shown to inhibit caspase activity and regulate caspase-dependent apoptosis [28,42]. We further investigated whether the  rough eye phenotype induced by Bcl-rambo was rescued by p35, Diap1, and Diap2. The reduction in eye pigments induced by Bcl-rambo was rescued by the co-expression of p35, Diap1, and Diap2 (Fig 6A). In contrast, the co-expression of p35, Diap1, and Diap2 only weakly suppressed the abnormal formation of ommatidia and bristles as well as the reduction in eye size induced by Bcl-rambo (Fig 6A). Bcl-rambo (ΔTM) did not cause any changes with the coexpression of p35, Diap1, and Diap2 ( Fig 6B). Moreover, additional UAS repeats in the UAS-GFP insertion did not rescue the rough eye phenotype induced by Bcl-rambo (S4 Fig), excluding the effect of diluting GMR-GAL4.

Aberrant morphology of ommatidia induced by Bcl-rambo was partly rescued by p35
The Drosophila compound eye is composed of eight photoreceptor cells, four cone cells, two primary pigment cells, six secondary pigment cells, three tertiary pigment cells, and three mechanosensory bristles. The differentiation of these cells was completed~42 h after pupal formation at 28°C. Since the loss-of-pigment phenotype induced by Bcl-rambo was effectively rescued by the co-expression of caspase inhibitors, we examined the morphology of the Drosophila pupal retina 42 h after pupal formation. When Bcl-rambo was expressed using the GMR-GAL4 driver fly lines, the shapes of the cone cells and pigment cells were markedly altered because four cone cells per ommatidium and the surrounding pigment cells were hardly distinguishable (Fig 7A and 7B). In contrast, the morphology of the pupal retina expressing Bcl-rambo (ΔTM) was not obviously changed, but showed a slight defect in the ommatidia rotation (Fig 7A). The aberrant morphology of ommatidia induced by Bcl-rambo was partly rescued by p35 (Fig 7A and 7B). We further investigated whether Bcl-rambo induced apoptosis in the pupal retinae at the same period. The signals for activated caspase-3/7 were rarely detected in the control pupal retinae, and they were not increased in the pupal retinae when Bcl-rambo or Bcl-rambo (ΔTM) was expressed (data not shown). These results suggest that Bcl-rambo does not induce apoptosis in the pupal retinae at this period.
In order to determine whether Bcl-rambo affected eye development, eye imaginal discs were stained with Elav for a marker of all photoreceptor cells, Prospero for a marker of R7 cells, and Cut for a maker of cone cell precursors. Elav, Prospero, and Cut were expressed in eye imaginal discs even when Bcl-rambo or Bcl-rambo (ΔTM) was expressed (Fig 8A and 8B). These results suggested that Bcl-rambo did not affect the differentiation of photoreceptor cells in the eye discs of third instar larvae. Although the formation of Cut-positive cone cell precursors was not affected by Bcl-rambo (Fig 8B), the differentiation of cone cells and pigment cells were clearly affected by the expression of Bcl-rambo in pupal retinae (Fig 7A). The spacing of Elav clusters appeared to be abnormal (Fig 8A and 8B), and this may have been due to the death of interommatidial cells. Therefore, the primary effect of Bcl-rambo appears to be the induction of apoptosis, while defects in the differentiation of cone cells and pigment cells may be secondary effects during the later stage of eye development.

Bcl-rambo interacted genetically with adenine nucleotide translocators and the autophagy-related 8a protein
Recent studies reported that Bcl-rambo bound to adenine nucleotide translocators [21], ceramide synthases 2 (CerS2) and 6 (CerS6) [17], and LC3, a mammalian homologue of the autophagy-related 8 (Atg8) protein [23]. We herein investigated whether Bcl-rambo interacted genetically with stress-sensitive B (SesB) and Ant2 (a major Drosophila homologue of adenine nucleotide translocators and the second isogene, respectively), Schlank (a Drosophila homologue of ceramide synthases), and Drosophila Atg8a. The loss-of-pigment phenotype induced by Bcl-rambo was effectively rescued by the sesB org or Ant2 G0247 sesB G0247 mutation (Fig 11A), while Bcl-rambo (ΔTM) did not exhibit any phenotypic changes in sesB org or Ant2 G0247 sesB G0247 flies (Fig 11B). The schlank G0061 mutation did not rescue the loss-of-pigment phenotype induced by Bcl-rambo (Fig 11C). The Atg8a EP362 mutation rescued the loss-of-pigmentation induced by Bcl-rambo, and partly suppressed the loss of bristles induced by Bcl-rambo, while Bcl-rambo (ΔTM) flies did not exhibit the clear effects observed in Atg8a EP362 flies ( Fig  11D). Due to the insertion of UAS repeats in Atg8a EP362 flies, Atg8a may be overexpressed in the presence of the GMR-GAL4 driver.

Discussion
We previously identified human Bcl-rambo and showed that its overexpression induced apoptosis in HEK293T cells [12]. Consistent with these findings, previous studies reported that Bcl-rambo induced apoptosis in other cell lines [20][21][22]. However, Bcl-rambo has been also shown to inhibit pro-apoptotic ceramide synthases in glioblastoma [17]. Bcl-rambo is known to be strongly expressed in several cancer cells [13][14][15][16][17], implying that Bcl-rambo may inhibit apoptosis or promote growth. Thus, it currently remains unclear whether Bcl-rambo plays either a pro-apoptotic role or anti-apoptotic role or even both roles under physiological conditions. In order to gain an insight into this issue, we investigated the biological activity of human Bcl-rambo in Drosophila. The ectopic expression of Bcl-rambo induced apoptosis in Drosophila S2 cells. Bcl-rambo also induced apoptosis and morphological aberrations representative of the rough eye phenotype in transgenic flies.
Bcl-rambo, but not Bcl-rambo (ΔTM), mainly localized to mitochondria in Drosophila S2 cells. This result was consistent with our previous findings obtained using HEK293T cells [12], and indicated that the C-terminal TM was essential for the mitochondrial localization of Bclrambo. A previous study showed that the mitochondrial targeting of Bcl-x L required the C-terminal TM domain to be flanked at both ends by at least two basic amino acids [43]. This consensus sequence proposed for mitochondrial localization was found to be conserved in human Bcl-rambo [12]. Drosophila has two Bcl-2 family proteins Drob-1/Debcl and Buffy [32][33][34][35]. Drob-1/Debcl has been shown to predominantly localize to mitochondria, whereas Buffy localizes to the endoplasmic reticulum in Drosophila and mammalian cell lines [44]. Drob-1/Debcl, but not Buffy, has C-terminal positively-charged residues homologous with Bcl-x L [44]. Thus, Bcl-rambo localizes to mitochondria in a manner similar to Drob-1/Debcl in Drosophila.
In Drosophila, Bcl-2 family members were found to not play a central role in the regulation of apoptosis [9,26]. Drob-1/Debcl and Buffy are not required for normal development, but participate in stress-induced apoptosis [45]. The ectopic expression of Drob-1/Debcl induced apoptosis in cultured cells and transgenic flies [32][33][34][35], indicating that Drob-1/Debcl exhibits proapoptotic activity. In contrast, Buffy has an anti-apoptotic function and has been shown to interact genetically and physically with Drob-1/Debcl to suppress Drob-1/Debcl-induced cell death [46]. A previous study reported that Drob-1/Debcl protected neurons from polyglutamine-induced toxicity, while Buffy enhanced it [47]. These findings suggested that Drob-1/ Debcl and Buffy possess both pro-apoptotic and anti-apoptotic activities. The ectopic expression of Bcl-rambo, but not Bcl-rambo (ΔTM), induced apoptosis in Drosophila S2 cells and caspase activation in eye imaginal discs. This is consistent with our previous findings in which Bcl-rambo induced caspase activation in HEK293T cells [12]. In S2 cells, Bcl-rambo and Drob-1/Debcl induced the release of cytochrome c into the cytosol. These results suggest that Bclrambo regulates the mitochondrial signaling pathway of apoptosis.
Human Bcl-2 and murine Bax have been shown to play roles in the regulation of apoptosis in the Drosophila eye and wing [48]. Human Bcl-2 is known to suppress murine Bax-and reaper-induced mitochondrial defects [49]. Furthermore, Drob-1/Debcl is required for cell death by murine Bax in the Drosophila eye [50]. These findings collectively suggest that the apoptosis pathway regulated by Bcl-2 family proteins is highly conserved between mammals and Drosophila. We showed that Bcl-rambo did not interact with anti-apoptotic or pro-apoptotic Bcl-2 family members in HEK293T cells [12]. Consistent with this result, Bcl-rambo interacted genetically with Drob-1/Debcl or Buffy only weakly in Drosophila. In addition, Bclrambo did not bind to Drob-1/Debcl or Buffy in HEK293T cells. Since the BHNo domain of Bcl-rambo was previously shown to induce caspase activation in HEK293T cells [12], the BHNo domain may be essential for the induction of apoptosis in Drosophila.
Bcl-rambo has been reported to regulate apoptosis by interacting with adenine nucleotide translocators [21], ceramide synthases 2 (CerS2) and 6 (CerS6) [17] and the Legionella protein SidF [20]. Adenine nucleotide translocators are components of the permeability transition pore complex in mitochondria, and play a key role in cell death [51]. In Drosophila, adenine nucleotide translocators are encoded by sesB as a major isoform and the second isogene Ant2 [52]. Ceramide synthases have been shown to regulate sphingolipid metabolism and also play a role in regulating cell death [53,54]. In subcellular localization, ceramide synthase activities have been detected in mitochondrial and microsomal fractions [54]. Schlank is the Drosophila member of the ceramide synthase family and controls growth and body fat [55]. Due to its localization to mitochondria, it was possible to postulate that Bcl-rambo may regulate the mitochondrial apoptosis pathway via adenine nucleotide translocators or ceramide synthases. The genetic interaction between Bcl-rambo and SesB, but not Schlank suggests that Bcl-rambo regulates the mitochondrial apoptosis pathway primarily via adenine nucleotide transporters in Drosophila.
The baculovirus p35 and Drosophila Diap1 and Diap2 have been shown to inhibit caspases in order to prevent apoptosis [28,42]. Bcl-rambo induced caspase activity in the eye imaginal discs of third instar larvae, but not the pupal retinae, suggesting that Bcl-rambo has a stronger effect on dividing cells. The ectopic expression of Bcl-rambo induced the rough eye phenotype in Drosophila, accompanied by a reduction in eye size and the loss of eye pigment. Although the reduced eye size caused by Bcl-rambo was only weakly rescued by p35, Diap1, and Diap2, reduced pigmentation was rescued by p35, Diap1 and Diap2. The shapes of cone cells and pigment cells were markedly altered by Bcl-rambo, and their morphologies were also partially rescued by p35 in the pupal retinae. Thus, the overexpression of Bcl-rambo may have multiple effects on cells, and the loss of cells may be secondary to defects rescued by p35. Previous studies reported that the ectopic expression of Drob-1/Debcl resulted in the rough eye phenotype [33,34], while the rough eye phenotype induced by Drob-1/Debcl was inhibited by p35 [33]. Drob-1/Debcl-induced cell death was not antagonized by p35 in Drosophila S2 cells [34], while it was rescued by p35 in CHO cells [32]. These findings suggest that the role of p35 in Drob-1/ Debcl-induced cell death may be influenced by the cellular context. In addition to apoptosis, necrosis and autophagic cell death have been observed during Drosophila development [56,57]. Drob-1/Debcl and Buffy are associated with germinal cell death characterized with the mixed morphologies of apoptosis and necrosis [58]. Thus, the ectopic expression of Bcl-rambo may induce caspase-dependent apoptosis and caspase-independent cell death in Drosophila.
In conclusion, the ectopic expression of Bcl-rambo induced the rough eye phenotype in transgenic flies. Bcl-rambo induced the rough eye phenotype, which suggested that a functional homologue of Bcl-rambo may exist in Drosophila even though amino acid homology is small. A recent study showed that Bcl-rambo had a short WXXL/I motif and was a mammalian homologue of yeast Atg32 that mediated mitophagy and mitochondrial fragmentation [23]. Bcl-rambo has been reported to interact with adenine nucleotide translocators, ceramide synthases, and LC3 (a mammalian homologue of Atg8) [17,21,23], and homologues of these proteins are present in Drosophila. Thus, humans and Drosophila may have a similar pathway regulated by Bcl-rambo. By using Drosophila as a screening system, we revealed that human Bcl-rambo interacted genetically with Drosophila homologues of adenine nucleotide translocators and Atg8, suggesting that Bcl-rambo mediates at least two different pathways for apoptosis and mitophagy in Drosophila. These results indicate that the Drosophila model established in the present study can be a powerful screening tool for investigating genetic interactions. The further identification of gene(s) interacting with Bcl-rambo may help to address its physiological function in humans. Bcl-rambo and Bcl-rambo (ΔTM) were expressed using GMR-GAL4 driver fly lines. (A) (a, e, i) GMR-GAL4/w; UAS-Bcl-rambo/ +; +, (b, f, j) GMR-GAL4/w; UAS-Bcl-rambo/Buffy H37 ; +, (c, g, k) GMR-GAL4/w; UAS-Bclrambo (ΔTM)/+; +, (d, h, l) GMR-GAL4/w; UAS-Bcl-rambo (ΔTM)/ Buffy H37 ; +. The morphology of adult eyes was observed by light microscopy (a-d) and SEM (e-l). Scale bars in e-h and i-l indicate 50 μm and 14.2 μm, respectively. Data were representative of two independent experiments. (TIF)