The mammalian pro-apoptotic Bcl-2 proteins function by binding and sequestering pro-survival Bcl-2 members. Debcl binds most mammalian pro-survival Bcl-2 proteins, including Bcl-2 and Bcl-XL, but not their pro-apoptotic counterparts (Colussi, 2000). In order to determine whether Debcl heterodimerizes with Buffy, co-immunoprecipitation experiments were carried out. FLAG-tagged Buffy was coexpressed with HA-tagged Debcl in 293T cells. Immunoprecipitation was performed with anti-FLAG or anti-HA antibodies. The control immunoblot with anti-FLAG shows that FLAG-Buffy (33 kDa) is precipitated. Immunoblotting of the FLAG immunoprecipitates with anti-HA reveals the HA-Debcl protein, suggesting that the two proteins can co-immunoprecipitate. Therefore, like the pro- and anti-apoptotic members of the mammalian Bcl-2 family, Debcl and Buffy can physically interact (Quinn, 2003).
To examine the expression of buffy mRNA during development, Northern blot and RT-PCR analysis were carried out. Due to the low level of buffy mRNA expression, the 1.2-kb buffy transcript was scarcely detectable upon Northern analysis. By RT-PCR, however, buffy mRNA is detected at all developmental stages, with the strongest expression detected from the late larval/early pupal stages (Quinn, 2003).
The spatial distribution of buffy mRNA was determined using in situ hybridization. The buffy transcript was expressed at very low levels and, as with debcl mRNA, detection required indirect tyramide-amplification. In situ hybridization analysis of Drosophila embryos reveals buffy transcript in non-cellularized, stage 5 embryos. Since zygotic transcription does not occur prior to stage 5, this represents maternally deposited mRNA. General ubiquitous expression was observed in germ band extended, stage 10 embryos. Later in embryogenesis the pattern of buffy mRNA becomes more restricted, with staining in the midgut, the hindgut and a segmental pattern throughout the epidermal tissue. buffy message becomes more restricted at stage 16 of embryogenesis and is prominent in the epidermis of the gut and regions of the head, including the pharynx and clypeolabrum. buffy mRNA is detected in the same pattern as the pro-apoptotic Drosophila Bcl-2-related gene debcl. The similarity between the expression patterns of debcl and buffy is particularly striking in stage 16 embryos. Such similar expression patterns suggest that coordinated expression may be important for regulating cell death. The patterns of buffy and debcl expression correlate with regions of cell death in the developing embryo (Quinn, 2003).
During oogenesis, the nurse cells dump their cytoplasm into the oocyte, a process coordinated with nurse cell apoptosis, and are regulated by apoptotic stimuli. buffy mRNA is abundant in the nurse cell chambers from stage 10a ovaries, which undergo apoptosis at stage 10b. During early pupal stages, most larval tissues are histolysed, an extensive apoptotic process regulated by pulses of the steroid hormone ecdysone. During third instar, buffy mRNA is strongest in larval midgut and salivary glands, tissues destined for histolysis in pupariation. buffy mRNA is also detected (albeit at lower levels) in larval tissues that are remodeled into the adult tissue during pupal development -- a process requiring a balance between apoptosis and cell survival -- including the brain lobes and eye imaginal discs. Therefore buffy is expressed throughout development, in the same pattern as the pro-apoptotic gene debcl and in tissues susceptible to apoptosis (Quinn, 2003).
The subcellular distribution of Buffy protein was determined using a rat polyclonal Buffy antibody. The specificity of this antibody was tested using the en-GAL4 driver to ectopically express the upstream activator sequence (UAS)-buffy transgene and a UAS-GFP transgene to mark cells expressing engrailed. Significantly, increased levels of anti-Buffy antibody staining were observed in the Engrailed (En) stripes compared with the levels of protein in adjacent cells. Leaky expression of the UAS-buffy transgene was suggested by the finding that Buffy antibody staining was consistently higher across the entire embryo when compared with the level of endogenous protein from wild-type. Further evidence for leaky expression was provided by the greater general protection from irradiation-induced cell death. Although mutants were not available to further verify the specificity of these antibodies, a clear reduction was found in the level of staining for buffy double-stranded (ds) RNA ablation embryos. In addition, the pattern of Buffy antibody staining in stage-16 wild-type embryos was similar to that observed for buffy mRNA expression (Quinn, 2003).
The pro-survival Bcl-2 proteins are localized to intracellular membranes, including mitochondria, endoplasmic reticulum and nuclear envelope. The Buffy C-terminus contains a putative hydrophobic membrane anchor, similar to the sequence found in many Bcl-2 family proteins. To determine whether Buffy localizes to mitochondria, Drosophila tissues were co-stained with the mitochondrial marker mitotracker and anti-Buffy antibody. Mitotracker has been used previously to show that the mitochondria of Drosophila larval brain are scattered throughout the entire cytoplasm and surround the nucleus. This pattern of mitotracker staining was reproduced in larval neuroblast cells; co-localization was found with Buffy protein predominantly in mitotracker-positive regions. Ionizing radiation has been used to induce apoptosis in Drosophila tissues. Similar Buffy staining was seen in gamma-irradiated tissues compared with untreated ones, including eye discs, wing discs, salivary glands and midgut. Therefore, like Bcl-2, Buffy localizes to mitochondria in both normal and irradiated cells, unlike the pro-apoptotic Bax proteins that only become localized to the mitochondrial membrane following stress signals (Quinn, 2003).
Since no specific buffy mutants are currently available, RNAi was used to knock down Buffy expression. buffy dsRNA was injected into pre-blastoderm embryos, which were then allowed to develop for 6-7 h before TUNEL and staining with anti-Buffy antibody, to measure the efficiency of Buffy protein ablation. On average, a 7-fold increase in TUNEL cells was observed in buffy dsRNA-injected embryos. Wild-type, stage-11 embryos have small populations of apoptotic cells in the amnioserosa, brain lobes and developing central nervous system (CNS), and ubiquitous staining for Buffy protein. Control embryos injected with GFP dsRNA and aged to stage 11 have a similar low level of apoptosis and ubiquitous staining for Buffy protein. Reduction of Buffy protein, shown using the Buffy antibody, correlates with increased levels of ectopic apoptosis. In a separate experiment, embryos were aged to between stages 14 and 16, following injection with either buffy dsRNA or buffer only. Antibody staining revealed that Buffy protein was barely detectable by stage 14-16 in embryos injected with buffy dsRNA, compared with control embryos at stage 14, where epidermal and neural staining is observed, and at stage 16. The older buffy RNAi embryos were fragile, with many disintegrating during collection; those remaining had three times the number of TUNEL-positive cells compared with control embryos at stages 14 or 16. Reduced numbers of cells and very few surviving neural cells were also observed in similarly injected and aged embryos. Thus, ablation of Buffy function results in cell death, indicating that Buffy is necessary for embryonic cell survival (Quinn, 2003).
Apoptosis commences during stage 11 of Drosophila embryogenesis, and as development proceeds, TUNEL-labeled cells are observed throughout the embryo, particularly in cells of the nervous system. In order to determine whether buffy overexpression inhibits developmental PCD, buffy was overexpressed in Drosophila embryos using the UAS-GAL4 system. Buffy protein was expressed using the En-GAL4 driver, which drives expression in the pair-rule striped pattern of the embryo. En stripes from En-GAL4,UAS-GFP,UAS-Buffy embryos contain approximately half the number of TUNEL-positive cells when compared with control embryos. Therefore, ectopic expression of Buffy can inhibit developmentally regulated PCD during Drosophila embryogenesis. Furthermore, ubiquitous expression of buffy using the Armadillo-GAL4 driver results in additional neural cells, suggesting that Buffy overexpression can block the normal pattern of PCD in the developing peripheral nervous system (Quinn, 2003).
Overexpression of Bcl-2 impairs the stress-induced apoptotic response of cells. In order to determine whether buffy overexpression inhibits stress-induced apoptosis, TUNEL from gamma-irradiated en-GAL4,UAS-GFP,UAS-Buffy embryos was compared with control embryos. Two UAS-buffy constructs were generated: a wild-type construct predicted to generate full-length protein and an N-terminal deletion construct (buffyDeltaN). The deletion eliminates 128 amino acids from the N-terminus, removing two putative alpha-helices that may be ancestral to the amphipathic alpha-helix from the BH4 domain of pro-survival Bcl-2 proteins. Since the BH4 domain is required for anti-apoptotic function of Bcl-2, by comparing the properties of buffyDeltaN with full-length buffy it was determine whether the extended N-terminal region of Buffy is important for cell survival (Quinn, 2003).
Embryos expressing either full-length buffy or buffyDeltaN in the En pattern are protected from gamma-irradiation-induced apoptosis. TUNEL labeling within the En stripe was reduced 7-fold for full-length buffy and 6-fold for buffyDeltaN, compared with wild type. There was also a reduced level of TUNEL in the inter-stripe region for full-length buffy and buffyDeltaN compared with wild type. This general reduction of TUNEL labeling, which was reproducible over three experiments, may be due to leaky expression of the UAS-buffy transgene. To determine whether Buffy could inhibit stress-induced apoptosis in other tissues, Buffy was expressed using en-GAL4, which is also expressed in the posterior of third instar larval wing discs. Expression of two copies of UAS-buffy with en-GAL4 is embryonic lethal, therefore wing discs expressing only one copy of full-length buffy were examined. Cells in the posterior compartment of the wing disc were protected from gamma-irradiation-induced apoptosis, compared with the high level of cell death observed in the anterior compartment, or with the extensive TUNEL observed in irradiated wild-type discs. The observation that protection from apoptosis does not occur in the anterior compartment of the wing disc suggests that either (1) leaky expression of the UAS-buffy transgene does not occur to the same degree in the wing discs as in the embryo, or (2) that when only one copy of UAS-buffy is present, leaky expression does not occur at a level that provides protection from irradiation-induced cell death (Quinn, 2003).
Increased levels of Buffy are therefore sufficient to inhibit the Drosophila apoptotic pathway that normally responds to DNA damaging agents such as ionizing radiation. Furthermore, since both full-length Buffy and BuffyDeltaN protect embryos from gamma-irradiation-induced apoptosis, the region of the protein encompassing the three BH domains, and C-terminal membrane anchor, is sufficient for the anti-apoptotic function of Buffy (Quinn, 2003).
To examine genetic interactions between Buffy and other apoptotic pathway genes, Glass multimer reporter (GMR)-GAL4 was used to drive the UAS-buffy transgene in the posterior region of the third instar eye imaginal disc. Recombinants of the UAS-buffy transgene with GMR-GAL4 on the second chromosome, when heterozygous (GMR-GAL4:UAS-buffy/+), produce flies with eyes of wild-type appearance. Similarly, ectopic expression of the Drosophila inhibitor of apoptosis, DIAP1, using the GMR driver results in normal-appearing adult eyes. However, expression of diap1 can inhibit apoptotic phenotypes generated by overexpression of caspases, rpr and hid. GMR-diap1 also suppresses the GMR-GAL4/+;UAS-debcl/+ ablated eye phenotype, consistent with the notion that Debcl induces apoptosis by functioning upstream of DIAP1-dependent caspase inhibition (Quinn, 2003).
The strong ablated eye phenotype from GMR-GAL4/+;UAS-debcl/+ can be partially suppressed by coexpression of buffy. The extreme nature of the GMR-GAL4;UAS-Debcl/+ phenotype suggests a high level of Debcl protein expression, and thus the slight suppression by Buffy suggests that this UAS-buffy line is not expressed at high enough levels to sequester the excess Debcl protein. However, the en-GAL4-UAS-debcl ablated wing phenotype was suppressed by coexpression of UAS-buffy. TUNEL labeling of third instar wing imaginal discs reveals that this suppression is due to Buffy inhibiting Debcl-induced apoptosis in the posterior compartment (Quinn, 2003).
Ectopic expression of Rpr, Hid and Grim causes the Drosophila IAP homolog Diap1/Thread(th) to be sequestered and inactivated, thus resulting in ectopic cell death. Overexpression of buffy with GMR-GAL4 partially suppresses the ablated eye phenotypes of rpr, hid and grim. Furthermore, the ectopic TUNEL observed in homozygous diap1 (th5) mutant embryos is inhibited by overexpression of the UAS-buffy transgene with en-GAL4. Taken together, this suggests that Buffy acts downstream of Rpr, Grim, Hid and DIAP1 to block caspase-dependent cell death (Quinn, 2003).
Expression of the UAS-dronc transgene with GMR-GAL4 results in a small, mottled eye phenotype as a consequence of ectopic cell death, particularly of pigment cells. This phenotype is not modified by coexpression of the UAS-buffy transgene, suggesting that Buffy acts upstream of caspase activation. Similarly, overexpression of the N-terminal deletion construct (UAS-buffyDeltaN) with GMR-GAL4 suppresses the Rpr, Grim, Hid and Debcl-ablated eye phenotypes, but does not alter the Dronc mottled eye phenotype. Therefore, only the C-terminal portion of the Buffy protein is required for suppression (Quinn, 2003).
The mammalian Bcl-2 protein can inhibit cell cycle entry, independent of its anti-apoptotic function. Although the overall growth rate of proliferating cell cultures is not affected by ectopic Bcl-2, increased withdrawal from the cell cycle into G1 phase occurs. Overexpression of the UAS-buffy transgene with the en-GAL4 driver results in inhibition of rapid embryonic cell cycles and an accumulation of cells in G1. Although the cell cycle pattern is dynamic, generally there are comparable numbers of S-phase cells for the same sized region both inside and outside the En stripe in a normal stage-11 embryo. The number of S-phase cells was clearly reduced, although not eliminated, in cells overexpressing two copies of the buffy transgene in the En-stripe compared with the inter-stripe regions and the control embryo. Mitotic cells, visualized using the anti-phosphohistone H3 antibody (PH3), were scattered across the epithelium of stage-11 embryos. Mitotic cells were almost eliminated within the buffy-expressing En stripe, compared with cells between the stripe and control embryos (Quinn, 2003).
That mitotic figures were almost eliminated, while some BrdU incorporation was observed, might suggest a G2 phase arrest, which would result in high levels of the G2-M cyclin, Cyclin B. However, staining with a Cyclin B antibody shows that Cyclin B is low in buffy-overexpressing cells when compared with neighboring regions and control embryos. Therefore, high levels of Buffy do not appear to cause a G2 arrest or delay. Since there are decreased numbers of S-, G2- and M-phase cells, and the nuclei size is smaller, arrest is consistent with a G1/early S phase arrest. Consistent with a cell cycle arrest when Buffy is overexpressed, the En stripe from an equivalent region of the embryo is often thinner and contains fewer cells compared with the control in stage 11/early stage 12 embryos. The variability in the width of the En band is likely to be a consequence of the extremely rapid cycling of stage 11 embryos, combined with the fact the there will be a gradual accumulation of Buffy in the En stripes, because En only starts to be highly expressed at stage 11. Leaky expression of the UAS-buffy transgene does not appear to greatly affect cell cycle progression in the inter-stripe region of en-GAL4,UAS-buffy embryos, possibly because the level of Buffy protein required is higher than that needed to prevent apoptosis. Importantly, these results provide the first evidence within a whole animal that a member of the Bcl-2 family has a cell cycle inhibitory role (Quinn, 2003).
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date revised: 28 December 2003
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