InteractiveFly: GeneBrief

Buffy: Biological Overview | Regulation | Developmental Biology | Effects of RNAi, Overexpression and Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Bcl-2 family proteins are key regulators of apoptosis. Both pro-apoptotic and anti-apoptotic members of this family are found in mammalian cells, but only the pro-apoptotic protein Debcl has been characterized in Drosophila. Buffy, the second Drosophila Bcl-2-like protein, is a pro-survival protein. Ablation of Buffy by RNA interference leads to ectopic apoptosis, whereas overexpression of buffy results in the inhibition of developmental programmed cell death and gamma irradiation-induced apoptosis. Buffy interacts genetically and physically with Debcl to suppress Debcl-induced cell death. Genetic interactions suggest that Buffy acts downstream of Reaper, Grim and Head involution defective, and upstream of the apical caspase Dronc. Furthermore, overexpression of buffy inhibits ectopic cell death in diap1 (th⁵) mutants. Taken together these data suggest that Buffy can act downstream of Rpr, Grim and Hid to block caspase-dependent cell death. Overexpression of Buffy in the embryo results in inhibition of the cell cycle, consistent with a G₁/early-S phase arrest. These data suggest that Buffy is functionally similar to the mammalian pro-survival Bcl-2 family of proteins (Quinn, 2003).

Accumulated evidence suggests that in mammalian cells, mitochondrial Bcl-2 prevents the release of cytochrome c, required for the formation of the Apaf-1 apoptosome and therefore caspase activation. Conversely, the pro-apoptotic Bcl-2 proteins promote mitochondrial permeability and cytochrome c release. Life or death of the cell is determined by whether the balance is tipped towards the pro-survival or the pro-apoptotic Bcl-2 members. In Drosophila, there are two homologs of the Bcl-2/Ced-9 family of programmed cell death (PCD) proteins, Debcl/dBorg-1/dRob-1 and Buffy/dBorg-2. Although both Debcl and Buffy share the BH1, BH2, BH3 and C-terminal transmembrane domains of the Bcl-2 family of proteins, they appear to lack the N terminal BH4 domain. In mammals, the BH4 domain distinguishes the pro-apoptotic Bcl-2 family members, e.g. Bax and Bok, from the anti-apoptotic members, e.g. Bcl-2, Bcl-xL and Bcl-w. Based upon this, both Debcl and Buffy were expected to be pro-apoptotic. Although both Debcl and Buffy are most closely related to the mammalian pro-apoptotic Bok, only Debcl has been shown to have a pro-apoptotic function in Drosophila, since ectopic overexpression of debcl in transgenic flies results in ectopic PCD and functional knockout of Debcl by RNA interference (RNAi) leads to an inhibition of cell death. Buffy is a pro-survival relative of Bcl-2/Ced-9. Buffy is required for cell survival and can prevent developmental and irradiation-induced cell death. Also, Buffy overexpression prevents cell cycle progression and results in the accumulation of cells in G₁, like its mammalian pro-survival counterpart Bcl-2. Thus, both pro-survival and cell cycle functions of Bcl-2 have been evolutionarily conserved in Buffy, suggesting that Buffy is the Drosophila homolog of the pro-survival Bcl-2 proteins (Quinn, 2003).

It is concluded that Buffy functions in a similar manner to the pro-survival mammalian Bcl-2 proteins since it (1) is required for cell survival, (2) inhibits developmentally regulated apoptosis, (3) inhibits gamma-irradiation-induced apoptosis, (4) binds the Drosophila pro-apoptotic Bcl-2 homolog Debcl, and can suppress Debcl-induced cell death, and (5) when overexpressed has an inhibitory effect on cell cycle progression (Quinn, 2003).

In mammals there are multiple pro-survival Bcl-2 proteins that play a tissue-specific role in protecting cells from apoptosis. Therefore, targeted knockout of individual pro-survival Bcl-2 members does not result in the death of the entire organism, suggesting that other pro-survival Bcl-2 proteins can compensate. For example, knockout studies show that murine Bcl-2 is required for survival of stem cells from kidney and melanocytes and adult lymphocytes, while Bcl-X_L is required for survival of neuronal and erythroid cells. In contrast, RNAi knockdown of buffy results in general embryonic cell death, suggesting that Buffy is a principle cell survival protein at this stage of Drosophila development. Indeed, database analysis shows no other Bcl-2-related proteins, apart from Buffy and Debcl (Quinn, 2003).

Overexpression of Bcl-2 impairs the apoptotic response to DNA damaging agents such as ionizing radiation. One of the reasons many tumors are resistant to chemotherapy and radiation therapy is that they mis-express Bcl-2. In the fly, increased levels of Buffy are sufficient to inhibit the Drosophila apoptotic pathway that normally responds to DNA damaging agents (Quinn, 2003).

Overexpression of mammalian Bcl-2 in Drosophila tissues has been shown to inhibit apoptosis, induced by either irradiation or Rpr overexpression (Gaumer, 2000; Brun, 2002). As has been found for Buffy, genetic analysis places the anti-apoptotic activity of Bcl-2 downstream of Rpr when expressed in flies (Brun, 2002). This is most likely a consequence of Bcl-2 protein binding to and sequestering Debcl (Colussi, 2000). In mammals, Bcl-2-mediated inhibition of apoptosis requires an alpha-helical domain within the N-terminal BH4 domain (Hunter, 1996). In contrast, Buffy’s N-terminus, and the putative alpha-helices therein, were not required for either inhibition of irradiation-induced apoptosis or suppression of Rpr-, Grim- and Hid-induced apoptosis. Thus, the C-terminal region containing the BH1, BH2, BH3 and membrane anchor is sufficient for Buffy’s cell survival function (Quinn, 2003).

Certain factors controlling cell cycle progression are also sensitive to apoptotic stimuli. Indeed, cell cycle factors may promote apoptosis under conditions unfavorable for proliferation, thus rendering cycling cells more vulnerable to apoptosis. Evidence that Bcl-2 plays a role in controlling cell cycle progression has been accumulating steadily, and in this study the first in vivo evidence is provided that a member of the Bcl-2 family can result in cell cycle inhibition. However, in contrast to the serum-deprived G0 cells that have been used previously, Drosophila embryonic cells cycle normally prior to overexpression of Buffy (Quinn, 2003 and references therein).

The cell cycle delay that occurs as a consequence of Buffy overexpression is dose dependent. Expression of two copies of UAS-buffy via en-GAL4 is embryonic lethal, presumably as a consequence of the G₁-S cell cycle arrest, which results in less cells in the En-stripes and insufficient cells to complete embryonic development. Two copies of en-GAL4,UAS-buffy (i.e., high level of Buffy protein) induce a cell cycle arrest and are embryonic lethal; however, flies expressing one copy (lower level of expression) are viable. Indeed, a low level of ubiquitous Buffy expression does not cause a cell cycle arrest, but results in the production of additional neural cells. Thus, the effect of Buffy overexpression is dose dependent; high levels of Buffy overexpression induce cell cycle arrest, which will ultimately result in fewer cells, while lower levels can inhibit developmental cell death and are associated with increased cell numbers (Quinn, 2003).

Consistent with cell cycle arrest, Bcl-2 overexpression in mammalian cells correlates with increased levels of the CycE/Cdk2 inhibitor p27 (Brady, 1996; Linette, 1996), hyperphosphorylated and inactive retinoblastoma (RB) tumor suppressor (Mazel, 1996), and increased levels of RB-related protein p130 (Lind, 1999; Vairo, 2000). The inhibitory effect of Bcl-2 on the cell cycle is independent of p53, the cdk4/6 inhibitor p16 and RB, but requires p130 and p27 (O’Reilly, 1996; Vairo, 1996, 2000). The cell cycle inhibitory function of Bcl-2 can be separated from its cell survival function since the tyrosine residue, Y28, in the N-terminal BH4 domain is important for Bcl-2 inhibition of cell cycle re-entry, but is not required for cell survival (Huang, 1997). Although Buffy does not have an obvious BH4 domain, the cell cycle inhibitory function has been conserved between Buffy and Bcl-2. Thus, it will now be important to determine whether Buffy uses a mechanism similar to Bcl-2 to inhibit cell cycle progression (Quinn, 2003 and references therein).

Drosophila larvae lacking the bcl-2 gene, buffy, are sensitive to nutrient stress, maintain increased basal target of rapamycin (Tor) signaling and exhibit characteristics of altered basal energy metabolism

B cell lymphoma 2 (Bcl-2) proteins are the central regulators of apoptosis. The two bcl-2 genes in Drosophila modulate the response to stress-induced cell death, but not developmental cell death. Because null mutants are viable, Drosophila provides an optimum model system to investigate alternate functions of Bcl-2 proteins. This report explores the role of one bcl-2 gene in nutrient stress responses. Starvation of Drosophila larvae lacking the bcl-2 gene buffy decreases survival rate by more than twofold relative to wild-type larvae. The buffy null mutant reacted to starvation with the expected responses such as inhibition of target of rapamycin (Tor) signaling, autophagy initiation and mobilization of stored lipids. However, the autophagic response to starvation initiated faster in larvae lacking buffy and was inhibited by ectopic buffy. Unusually high basal Tor signaling, indicated by more phosphorylated S6K, was detected in the buffy mutant, and removal of a genomic copy of S6K, but not inactivation of Tor by rapamycin, reverted the precocious autophagy phenotype. Instead, Tor inactivation also required loss of a positive nutrient signal to trigger autophagy and loss of both was sufficient to activate autophagy in the buffy mutant even in the presence of enforced phosphoinositide 3-kinase (PI3K) signaling. Prior to starvation, the fed buffy mutant stored less lipid and glycogen, had high lactate levels and maintained a reduced pool of cellular ATP. These observations, together with the inability of buffy mutant larvae to adapt to nutrient restriction, indicate altered energy metabolism in the absence of buffy. All animals in their natural habitats are faced with periods of reduced nutrient availability. This study demonstrates that buffy is required for adaptation to both starvation and nutrient restriction. Thus, Buffy is a Bcl-2 protein that plays an important non-apoptotic role to promote survival of the whole organism in a stressful situation (Monserrate, 2012).

This study has demonstrated that buffy is required for normal larval responses to nutrient stress. This could not be attributed to a role for buffy in sensing nutrient starvation and activating normal starvation responses. Instead, larvae lacking buffy displayed characteristics of altered energy metabolism and increased growth signaling through Tor, as demonstrated by increased phosphorylated S6K. This study did not address whether the increased Tor signaling is a cause or result of the energy metabolism of the buffy mutant. It is conceivable that upregulation of Tor signaling results in increased energy consumption to promote growth. However, it was not observed that the increased phosphorylated S6K was correlated with increased growth in the buffy mutant, suggesting that the Tor signaling was balanced by the altered energy metabolism in the mutant. Taking into account the current understanding of Bcl-2 proteins, it is postulated that Buffy is required to maintain energy homeostasis at a set point that is optimal for both growth and starvation responses. Loss of buffy results in a change of this homeostatic set point that may directly or indirectly upregulate growth signaling and that places the animal closer to a metabolic cliff in terms of its ability to survive nutrient stress (Monserrate, 2012).

In investigating starvation responses in the buffy mutant, it was observed that fat body autophagy was initiated faster in buffy mutant larvae. Although Tor signaling normally inhibits autophagy, the high level of phosphorylated S6K maintained by the mutant was required for precocious starvation-induced autophagy. Since autophagy is a mechanism to recycle essential building blocks when nutrients in the environment are scarce, whether reduced energy storage was correlated with precocious autophagy was investigated. Wild-type animals, with normal Tor signaling, provided with 20% of the normal nutrients (20% CY, 1.8% sucrose) were autophagic after 2 h of starvation. But this nutrient-restriction diet resulted in a much greater reduction in stored nutrients in the fat body than the 15% reduction in lipid storage observed in the buffy mutant. In addition, excess growth signaling by ectopic activation of Tor signaling in wild-type larvae, was not sufficient to induce precocious autophagy. It is proposed that the unique combination of an altered metabolism and increased Tor signaling in larvae lacking buffy that render the animal more sensitive to nutrient stress and results in precocious autophagy (Monserrate, 2012).

Energy sensing has been linked to autophagy initiation in mammals. ULK1 (mammalian ATG1) function is regulated by both Tor and AMPK. In the simplest current thinking, nutrient deprivation both inactivates Tor and activates AMPK to phosphorylate and activate ULK1 to initiate autophagy. In Drosophila, the complex of ATG1/ATG13 is regulated by Tor and AMPK is required for starvation-induced autophagy, suggesting that regulation of autophagy initiation by phosphorylation is similar in fruit flies. In larvae lacking buffy, decreased cellular energy might more efficiently activate ATG1/ATG13, possibly mediated through AMPK. This model does not take into account that precocious autophagy in the buffy mutant required phosphorylated S6K. There is conflicting data as to the role of S6K in autophagy. Because inhibition of Tor induces autophagy, phosphorylation of S6K is inversely correlated with autophagy. However, S6K has been shown to be required for starvation-induced autophagy in Drosophila, and plays a positive role in autophagic induction in mammals. Faster autophagy in the buffy mutant may reflect a positive signaling role for S6K in autophagy initiation that contributes to this phenotype. Indeed it is intriguing to postulate that a metabolic signal from loss of the positive nutrient signal is transmitted through phosphorylated S6K in all animals, and that augmented phosphorylated S6K merely potentiates this signal in the buffy mutant (Monserrate, 2012).

The metabolism phenotypes observed in the buffy mutant larvae (smaller energy stores in the fat body, increased glucose utilization inferred from less glycogen storage, a reduced pool of ATP and increased lactate) are most simply explained by a shift in the balance of glycolysis to oxidative phosphorylation toward glycolysis. Glycolysis is less efficient at generating ATP and increased glycolysis generates excessive pyruvate that is converted to lactate. To maintain glycolysis at a higher rate, a higher percentage of ingested glucose and lipids must be shuttled into glycolysis at the expense of storage in the fat body. Animals that rely more on glycolysis for energy generation would certainly be more sensitive to nutrient restriction. This hypothesis is supported by recent evidence that oxygen consumption and cellular ATP levels were reduced, while glycolysis was increased, in Bcl-2-associated X protein (BAX)-deficient cells. Two recent studies on Bcl-x_L also support direct regulation of oxidative phosphorylation: one demonstrated that Bcl-x_L controls the levels of the metabolite acetyl coenzyme A (acetyl-CoA) and the other proposed that neuronal Bcl-x_L directly regulates the efficiency of ATP synthesis by the F₁F₀ ATP synthase complex. Consistent with less efficient oxidative phosphorylation, buffy mutant larvae are sensitive to the reactive oxygen species (ROS) generator, paraquat, and have a twofold increase in ROS. Increased ROS has also been reported to result from enforced Tor signaling in Drosophila . Intriguingly, ROS has been proposed to affect S6K phosphorylation (Monserrate, 2012).

Bcl-2 proteins govern permeabilization of the mitochondrial outer membrane that leads to loss of mitochondrial energy production and release of apoptogenic factors such as cytochrome c. Buried within the vast quantity of publications investigating Bcl-2 proteins are studies that support a role for some of the Bcl-2 proteins in mitochondrial energetics, generally with a focus on ectopic expression of Bcl-2 proteins and effects on metabolism with regard to apoptosis. Many studies have shown an interaction between Bcl-2 proteins and the voltage-dependent anion channel (VDAC) that regulates movement of metabolites between the mitochondria and the cytosol. Although this interaction is not required for mitochondrial-dependent cell death, it may be that Bcl-2 proteins modulate mitochondrial energetics through VDAC. One of the metabolites whose uptake is facilitated by VDAC is Ca²⁺. Intracellular Ca²⁺ signaling is regulated by the ER and Bcl-2 proteins influence ER calcium content through modulation of the inositol triphosphate receptor (IP3R) and the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Uptake of Ca²⁺ released by the ER can stimulate mitochondrial energy metabolism through several targets. Ectopic Buffy decorates both the mitochondria and the ER in various cell types, leaving open the possibility that Buffy has a functional role in ER-mitochondria Ca²⁺ signaling. Additionally, Bcl-2 proteins play a role in mitochondrial morphogenesis, both in the fragmentation observed upon apoptosis induction. Mitochondria in Drosophila also fragment prior to cell death. This study observed that buffy mutant fat body had a higher density of mitochondria that were in general smaller and less 'snake like'. However, buffy mutant animals did not have more mitochondria since no increase in mitochondrial genomes was observed in larval or fat body extracts (Monserrate, 2012).

This study has demonstrated that Drosophila larvae lacking buffy are sensitive to nutrient restriction and starvation. buffy mutant larvae have unusual basal characteristics: increased Tor signaling, reduced energy source storage, reduced ATP levels and increased lactate levels. The data provides evidence that, in the normal animal, Buffy maintains basal energy homeostasis to enable appropriate responses to nutrient stress. Future studies will determine how Buffy influences basal energy metabolism and clarify the relationship between energy metabolism and S6K regulation. The recent reports demonstrating that Bcl-x_L regulates metabolic efficiency in neurons and that Bax promotes bioenergetics in HCT-116 cells and primary hepatocytes support the hypothesis that some Bcl-2 proteins have a non-apoptotic role to produce resistance to stressors by maintaining mitochondrial energetics. The current data adds to these reports, and is unique because it investigates the effect on organismal health of loss of a bcl-2 gene and provides evidence for crosstalk with Tor signaling. It is important to note that the Drosophila Bcl-2 proteins are bona fide Bcl-2 proteins containing BH1-4 domains and a C-terminal transmembrane domain, have the ability to bind other Bcl-2 proteins and can substitute for their mammalian counterparts (Monserrate, 2012).

Apoptosis is most often considered at the cellular level: cells that are unnecessary, damaged or diseased are removed by cell suicide. But it is essential to keep in mind that apoptosis promotes survival of the entire organism. It is certainly plausible that the same proteins that function as a rheostat for apoptosis also perform a similar function for survival, through energy modulation, in stressful life situations that are normally encountered by the organism (Monserrate, 2012).

Knockdown of the putative Lifeguard homologue CG3814 in neurons of Drosophila melanogaster

Lifeguard is an integral transmembrane protein that modulates FasL-mediated apoptosis by interfering with the activation of caspase 8. It is evolutionarily conserved, with homologues present in plants, nematodes, zebra fish, frog, chicken, mouse, monkey, and human. The Lifeguard homologue in Drosophila, CG3814, contains the Bax inhibitor-1 family motif of unknown function. Downregulation of Lifeguard disrupts cellular homeostasis and disease by sensitizing neurons to FasL-mediated apoptosis. Bioinformatic analyses was used to identify CG3814, a putative homologue of Lifeguard, and knocked down CG3814/LFG expression under the control of the Dopa decarboxylase (Ddc-Gal4) transgene in Drosophila melanogaster neurons to investigate whether it possesses neuroprotective activity. Knockdown of CG3814/LFG in Ddc-Gal4-expressing neurons resulted in a shortened lifespan and impaired locomotor ability, phenotypes that are strongly associated with the degeneration and loss of dopaminergic neurons. Lifeguard interacts with anti-apoptotic Bcl-2 proteins and possibly pro-apoptotic proteins to exert its neuroprotective function. The co-expression of Buffy, the sole anti-apoptotic Bcl-2 gene family member in Drosophila, and CG3814/LFG by stable inducible RNA interference, suppresses the shortened lifespan and the premature age-dependent loss in climbing ability. Suppression of CG3814/LFG in the Drosophila eye reduces the number of ommatidia and increases disruption of the ommatidial array. Overexpression of Buffy, along with the knockdown of CG3814/LFG, counteracts the eye phenotypes. Knockdown of CG3814/LFG in Ddc-Gal4-expressing neurons in Drosophila diminishes its neuroprotective ability and results in a shortened lifespan and loss of climbing ability, phenotypes that are improved upon overexpression of the pro-survival Buffy (M'Angale, 2016).

Lifeguard (LFG) or Fas apoptotic inhibitory molecule 2, also known as Transmembrane Bcl-2 associated protein X (Bax) inhibitor motif 2, belongs to a diverse membrane-spanning protein family and inhibits apoptosis mediated by the Fas/CD95/Apo-1 receptor but not the closely related TNFR . LFG was first identified as neuronal membrane protein 35 when it was found to be differentially upregulated during rat postnatal development and predominantly localized to the endoplasmic reticulum (ER). A different nomenclature categorizes this protein into a family referred to as LFG , which is adopted from Lifeguard, and classifies LFG as LFG2. This conserved protein consists of seven transmembrane domains and is found in plants, insects, amphibians, fish, and mammals. LFG regulates cell death by interfering with caspase 8 activation, but not its recruitment to the death-inducing signaling complex, a role that is essential for the survival of neurons during development. Expression of LFG has been shown to be dependent on PI3K/ Akt and its knockdown sensitizes neurons to FasL-induced apoptosis. This appears to occur through its regulation by PI3K. Another mechanism through which LFG regulates apoptosis is via interaction with Bcl-X L and Bcl-2 at the ER to inhibit calcium release. This interaction with Bcl-X L is contrary to previous findings where LFG was shown to interact with Bax. Dysregulation of LFG has been implicated in the disruption of cellular homeostasis involved in many cancers and neuronal diseases. Nevertheless, the antiapoptotic role of LFG has been demonstrated in addition to its interaction with the Bcl-2 family of proteins (M'Angale, 2016).

The Bcl-2 family of genes are key regulators of cell death and survival in animals and contain anti-apoptotic and proapoptotic members. These genes regulate life and death decisions at the cellular level by maintaining a delicate balance between pro- apoptotic and anti-apoptotic mediators. Homologues of Bcl-2 family member in Drosophila melanogaster are limited to the anti-apoptotic Buffy and the pro-apoptotic Debcl. In previous studies, overexpression of Buffy has been shown to confer survival advantages in response to external stimuli and under conditions of stress. A role for this gene in the mitochondrial pathway has been described during Drosophila oogenesis. This suggests that this protein has an important role in aspects of cell death (M'Angale, 2016).

The Drosophila homologue was initially reported to be NMDARA1, although a previous study reported this transcript to be NMDARA1 and a homologue of the rat glutamate-binding protein. It was originally listed as CG3798 in FlyBase and is currently listed as Nmda1, and is also known as NMDA receptor-associated protein. The protein sequence used for bioinformatic comparisons in the present study was Nmda1 polypeptide C. The accession number of the earlier protein sequence, has since been updated in both FlyBase and National Center for Biotechnology Information the (NCBI) and currently represents the CG3814 isoform A (Polypeptide Dmel\CG3814-PA). Interestingly, CG3814 and Nmda1 are adjacent to each other on chromosome 2 and are transcribed in the same direction. Recent bioinformatic studies have made comparisons between Drosophila CG3814 and/or CG9722, and human LFG. The existence of homologues of this protein family that are implicated in the regulation of FasL-mediated apoptosis may underpin their evolutionary importance in cytoprotection. Drosophila is used as a model organism to study changes in gene expression and to model human diseases. Previous studies have used Ddc-Gal4 -expressing neurons since they are sensitive to subtle disruptions in gene expression and degenerate in an age-dependent manner, which manifests as deficiency in locomotor function. Since these neurons are highly sensitive, measurements of survival and climbing ability can be used as rapid assays to assess the organismal effects of altered gene expression and to identify genetic interactions. The present study investigated the effect of CG3814/LFG knockdown, which has a wider expression pattern than the testes- specific CG9722, under the control of the Dopa decarboxylase transgene in neurons of Drosophila. It was further determined whether there is interaction with Bcl-2 proteins by overexpressing the pro-survival Bcl-2 homologue Buffy (M'Angale, 2016).

Bioinformatic analysis of protein sequences showed CG3814 to be the strongest candidate for Drosophila LFG, with a sequence identity of 45% and similarity of 65%. However, this does not exempt CG9722; CG3814 was widely expressed when compared to CG9722, which is predominantly expressed in the testis. Therefore, it is proposed that CG3814 is the putative Drosophila homologue of LFG (M'Angale, 2016).

The conditional knockdown of CG3814 by stable inducible RNA interference in Drosophila neurons under the control of the Ddc-Gal4 transgene resulted in decreased median survival and severely impaired climbing ability, phenotypes that were consistently present in both RNAi lines tested. The 'healthspan' of these flies was highly compromised as determined by their shortened lifespan and precocious loss in locomotor function. LFG is able to block FasL-induced cell death and has been demonstrated to be neuroprotective, is highly expressed in neurons, and its loss-of-function induces cell death. The profound loss in climbing ability that results from knockdown of CG3814/LFG in Drosophila neurons appears to be the result of neuronal loss, since compared to control flies at around the same age and at the same time point, there is a significant difference in climbing abilities. In this study, no cell death assays were performed and as such the conclusions are mostly based on behavioral phenotypes that are comparable to the controls. This does not negate the evidence obtained following the knockdown of CG3814/LFG . In addition, RNAi does not exclude off-target regions that share homology with other BI-1 containing motifs, although data from the Vienna Drosophila Resource Centre show there are no off targets for these RNAi lines, which target the various isoforms present. Taken together, these results suggest a strong neuroprotective role for CG3814 in Drosophila Ddc-Gal4 -expressing neurons (M'Angale, 2016).

The CG3814 -induced phenotypes may occur through a mechanism that does not involve interaction with pro-survival Bcl-2 proteins at the ER membrane, to regulate the release of calcium from the ER. Therefore, knockdown of CG3814/ LFG in the neurons appears to result in neuronal degeneration and death. The only known pro-survival Bcl-2 homologue in Drosophila is Buffy. Overexpression of Buffy is known to confer a survival advantage to cells under normal and stress conditions. Overexpression of Buffy, along with the knockdown of CG3814, suppressed the CG3814 -induced phenotypes markedly, by significantly improving survival and locomotor function. This action of Buffy on 'healthspan' may be specific to an interaction with CG3814, or it may be attributed to a general pro-survival signaling pathway that is initiated by Buffy in response to stress mediated by the loss of CG3814 function. This indicates a strong pro-survival role for CG3814/LFG since the phenotypes that result from its knockdown are rescued by the pro-survival Buffy (M'Angale, 2016).

In supportive experiments, the directed knockdown of CG3814 in the neuron-rich developing Drosophila eye under the direction of the GMR response elements resulted in a decreased number of ommatidia. The reduced ommatidium number was attributed to the high degree of fusion of the ommatidium and consequently resulted in ommatidium disarray. Knockdown of CG3814 in the Drosophila eye seems to exacerbate the Gal4 -induced apoptosis that manifests as the roughened eye phenotype. The overexpression of Buffy, along with the knockdown of CG3814, results in the suppression of the phenotype, with the number of ommatidia and the degree of roughened eye being restored to control levels. Buffy seems to ameliorate this phenotype possibly via a general action on survival signals or through a concerted function that rescues CG3814-induced apoptosis (M'Angale, 2016).

In conclusion, knockdown of CG3814/LFG in the Ddc-Gal4 -expressing neurons of Drosophila results in a severely shortened lifespan and a marked age-dependent loss in climbing ability, phenotypes that are strongly associated with the degeneration and loss of DA neurons. Overexpression of the pro-cell survival mediator Buffy along with the knockdown of CG3814/LFG , rescues the observed phenotypes, which suggests strong pro-survival and neuroprotective roles for CG3814/LFG in Drosophila neurons (M'Angale, 2016).

REGULATION

The pro-apoptotic activity of Drosophila Rbf1 involves dE2F2-dependent downregulation of diap1 and buffy mRNA

The retinoblastoma gene, rb, ensures at least its tumor suppressor function by inhibiting cell proliferation. Its role in apoptosis is more complex and less described than its role in cell cycle regulation. Rbf1, the Drosophila homolog of Rb, has been found to be pro-apoptotic in proliferative tissue. However, the way it induces apoptosis at the molecular level is still unknown. To decipher this mechanism, rbf1 expression was induced in wing proliferative tissue. It was found that Rbf1-induced apoptosis depends on dE2F2/dDP heterodimer, whereas dE2F1 transcriptional activity is not required. Furthermore, Rbf1 and dE2F2 downregulate two major anti-apoptotic genes in Drosophila: buffy, an anti-apoptotic member of Bcl-2 family and diap1, a gene encoding a caspase inhibitor. On the one hand, Rbf1/dE2F2 repress buffy at the transcriptional level, which contributes to cell death. On the other hand, Rbf1 and dE2F2 upregulate how expression. How is a RNA binding protein involved in diap1 mRNA degradation. By this way, Rbf1 downregulates diap1 at a post-transcriptional level. Moreover, the dREAM complex (see Rbf) has a part in these transcriptional regulations. Taken together, these data show that Rbf1, in cooperation with dE2F2 and some members of the dREAM complex, can downregulate the anti-apoptotic genes buffy and diap1, and thus promote cell death in a proliferative tissue (Clavier, 2014).

Protein Interactions

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-X_L, 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).

DEVELOPMENTAL BIOLOGY

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).

EVOLUTIONARY HOMOLOGS

Information about anti-apoptotic BCL2 homologs can be found at death executioner Bcl-2 homologue (debcl), Evolutionary Homologs section.

REFERENCES

Search PubMed for articles about Drosophila Buffy

Brady, H. J. M., Gil-Gümez, Kirberg, J. and Berns, A. J. M. (1996). Baxalpha perturbs T cell development and affects cell cycle entry of T cells. EMBO J. 15: 6991-7001. 9003775

Brun, S., Rincheval, V., Gaumer, S., Mignotte, B. and Guenal, I. (2002). reaper and bax initiate two different apoptotic pathways affecting mitochondria and antagonized by bcl-2 in Drosophila. Oncogene 21: 6458-6470. 12226749

Clavier, A., Baillet, A., Rincheval-Arnold, A., Coleno-Costes, A., Lasbleiz, C., Mignotte, B. and Guenal, I. (2014). The pro-apoptotic activity of Drosophila Rbf1 involves dE2F2-dependent downregulation of diap1 and buffy mRNA. Cell Death Dis 5: e1405. PubMed ID: 25188515

Colussi, P. A., Quinn, L. M., Huang, D. C. S., Coombe, M., Read, S. H., Richardson, H. and Kumar, S. (2000). Debcl a proapoptotic Bcl-2 homologue, is a component of the Drosophila melanogaster cell death machinery. J. Cell Biol. 148: 703-710. 10684252

Huang, D. C. S., O’Reilly, L. A., Strasser, A. and Cory, S. (1997). The anti-apoptosis function of Bcl-2 can be genetically separated from its inhibitory effect on cell cycle entry. EMBO J. 16: 4628-4638. 9303307

Gaumer, S., Guénal,I., Brun, S., Théodore, L. and Mignotte, B. (2000). Bcl-2 and Bax mammalian regulators of apoptosis are functional in Drosophila. Cell Death Differ. 7: 804-814. 11042675

Hunter, J. J., Bond, B. L. and Parslow, T. G. (1996). Functional dissection of the human Bcl-2 protein: sequence requirements for inhibition of apoptosis. Mol. Cell. Biol. 16: 877-883. 8622689

Lind, E. F., Wayne, J., Wang, Q.-Z., Staeva, T., Stolzer, A. and Petrie, H. T. (1999). Bcl-2 induced changes in E2F regulatory complexes reveal the potential for integrated cell cycle and cell death functions. J. Immunol. 162: 5374-5379. 10228014

Linette, G. P., Li, Y., Roth, K. and Korsmeyer, S. J. (1996). Cross talk between cell death and cell cycle progression: BCL-2 regulates NFAT-mediated activation. Proc. Natl Acad. Sci. 93: 9545-9552. 8790367

M'Angale, P. G. and Staveley, B. E. (2016). Knockdown of the putative Lifeguard homologue CG3814 in neurons of Drosophila melanogaster. Genet Mol Res 15(4). PubMed ID: 28002605

Mazel, S., Burtrum, D. and Petrie, H. T. (1996). Regulation of cell division cycle progression by bcl-2 expression: a potential mechanism for inhibition of programmed cell death. J. Exp. Med. 183: 2219-2226. 8642331

Monserrate, J. P., Chen, M. Y. and Brachmann, C. B. (2012). Drosophila larvae lacking the bcl-2 gene, buffy, are sensitive to nutrient stress, maintain increased basal target of rapamycin (Tor) signaling and exhibit characteristics of altered basal energy metabolism. BMC Biol. 10: 63. PubMed Citation: 22824239

O’Reilly, L. A., Huang, D. C. S. and Strasser, A. (1996). The cell death inhibitor Bcl-2 and its homologues influence control of cell cycle entry. EMBO J. 15: 6979-6990. 9003774

Quinn, L., et al. (2003). Buffy, a Drosophila Bcl-2 protein, has anti-apoptotic and cell cycle inhibitory functions. EMBO J. 22: 3568-3579. 12853472

Vairo, G., Innes, K. M. and Adams, J. M. (1996). Bcl-2 has a cell cycle inhibitory function separable from its enhancement of cell survival. Oncogene 13: 1511-1519. 8875989

Vairo, G., Soos, T. J., Upton, T. M., Zalvide, J., DeCaprio, J. A., Ewen, M. E., Koff, A. and Adams, J. M. (2000). Bcl-2 retards cell cycle entry through p27Kip1, pRB relative p130 and altered E2F reulation. Mol. Cell. Biol. 20: 4745-4753. 10848600

Wu, J. N., et al. (2010). grim promotes programmed cell death of Drosophila microchaete glial cells. Mech. Dev. 127: 407-417. PubMed Citation: 20558283

date revised: 20 September 2012

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