Buffy: Biological Overview | Regulation | Developmental Biology | Effects of RNAi, Overexpression and Mutation | Evolutionary Homologs | References
Gene name - Buffy
Cytological map position - 48C1
Function - signaling
Symbol - Buffy
FlyBase ID: FBgn0040491
Genetic map position - 2R
Classification - Bcl-2 inhibitors of programmed cell death
Cellular location - cytoplasmic
|Recent literature||Clavier, A., Ruby, V., Rincheval-Arnold, A.,
Mignotte, B and Guénal, I. (2015). The Drosophila retinoblastoma protein, Rbf1, induces a debcl
and drp1-dependent mitochondrial apoptosis. J Cell Sci
[Epub ahead of print]. PubMed ID: 26208635
In accordance with its tumor suppressor role, the Retinoblastoma protein pRb can ensure pro-apoptotic functions. rbf1, the Drosophila homolog of Rb, also displays a pro-apoptotic activity in proliferative cells. It has been previously shown that rbf1 pro-apoptotic activity depends on its ability to decrease the level of anti-apoptotic proteins such as the Bcl-2 family protein Buffy. Buffy often acts opposite to Debcl, the other Drosophila Bcl-2-family protein. Both proteins can localize at the mitochondrion, but the way they control apoptosis still remains unclear. This study demonstrates that debcl and the pro-fission gene drp1 are necessary downstream of buffy to trigger a mitochondrial fragmentation during rbf1-induced apoptosis. Interestingly, rbf1-induced apoptosis leads to a debcl- and drp1-dependent Reactive Oxygen Species production, which in turn activates the Jun Kinase pathway to trigger cell death. Moreover, Debcl and Drp1 can interact and Buffy inhibits this interaction. Notably, Debcl modulates Drp1 mitochondrial localization during apoptosis. These results provide a mechanism by which Drosophila Bcl-2 family proteins can control apoptosis and shed light on a link between Rbf1 and mitochondrial dynamics, in vivo.
|M'Angale, P. G. and Staveley, B. E. (2016). The Bcl-2 homologue Buffy rescues alpha-synuclein-induced Parkinson disease-like phenotypes in Drosophila. BMC Neurosci 17: 24. PubMed ID: 27192974
Only two Bcl-2 family genes have been found in Drosophila melanogaster including the pro-cell survival, human Bok-related orthologue, Buffy. The directed expression of alpha-synuclein, a gene contributing to inherited forms of Parkinson disease (PD), in the dopaminergic neurons (DA) of flies provides a robust model of PD complete with the loss of neurons and accompanying motor defects. This study altered the expression of Buffy in the dopamine producing neurons and in the developing neuron-rich eye, with and without the expression of alpha-synuclein. To alter the expression of Buffy in the dopaminergic neurons of Drosophila. The directed expression of Buffy in the dopamine producing neurons, via aDdc-Gal4 transgene, resulted in flies with increased climbing ability and enhanced survival, while the inhibition of Buffy in the dopaminergic neurons reduced climbing ability over time prematurely, similar to the phenotype observed in the alpha-synuclein-induced Drosophila model of PD. Subsequently, the expression of Buffy was altered in the alpha-synuclein-induced Drosophila model of PD. Analysis revealed that Buffy acted to rescue the associated loss of locomotor ability observed in the alpha-synuclein-induced model of PD, while Buffy RNA interference resulted in an enhanced alpha-synuclein-induced loss of climbing ability. In complementary experiments the overexpression of Buffy in the developing eye suppressed the mild rough eye phenotype that results from Gal4 expression and from alpha-synuclein expression. When Buffy is inhibited the roughened eye phenotype is enhanced. It is concluded that the inhibition of Buffy in DA neurons produces a novel model of PD in Drosophila. The directed expression of Buffy in DA neurons provides protection and counteracts the alpha-synuclein-induced Parkinson disease-like phenotypes. Taken all together this demonstrates a role for Buffy, a Bcl-2 pro-cell survival gene, in neuroprotection.
|M'Angale, P. G. and Staveley, B. E. (2016). Loss of porin function in dopaminergic neurons of Drosophila is suppressed by Buffy. J Biomed Sci 23(1): 84. PubMed ID: 27881168
Mitochondrial porin, also known as the voltage-dependent anion channel (VDAC), is a multi-functional channel protein that shuttles metabolites between the mitochondria and the cytosol and implicated in cellular life and death decisions. The inhibition of porin under the control of neuronal Ddc-Gal4 result in short lifespan and in an age-dependent loss in locomotor function, phenotypes that are strongly associated with Drosophila models of Parkinson disease. Loss of porin function was achieved through exploitation of RNAi while derivative lines were generated by homologous recombination and tested by PCR. The expression of human α-synuclein transgene in neuronal populations that include dopamine producing neurons under the control of Ddc-Gal4 produces a robust Parkinson disease model, and results in severely reduced lifespan and locomotor dysfunction. In addition, the porin-induced phenotypes are greatly suppressed when the pro-survival Bcl-2 homologue Buffy is overexpressed in these neurons and in the developing eye adding to the cellular advantages of altered expression of this anti-apoptotic gene. When α-synuclein was co-expressed along with porin, it results in a decrease in lifespan and impaired climbing ability. This enhancement of the α-synuclein-induced phenotypes observed in neurons was demonstrated in the neuron rich eye, where the simultaneous co-expression of porin-RNAi and α-synuclein resulted in an enhanced eye phenotype, marked by reduced number of ommatidia and increased disarray of the ommatidia. It is concluded that the inhibition of porin in dopaminergic neurons among others results in reduced lifespan and age-dependent loss in climbing ability, phenotypes that are suppressed by the overexpression of the sole pro-survival Bcl-2 homologue Buffy. The inhibition of porin phenocopies Parkinson disease phenotypes in Drosophila, while the overexpression of Buffy can counteract these phenotypes to improve the overall "healthspan" of the organism.
|M'Angale, P. G. and Staveley, B. E. (2016). The HtrA2 Drosophila model of Parkinson's disease is suppressed by the pro-survival Bcl-2 Buffy. Genome: 1-7. PubMed ID: 27848260
Mutations in High temperature requirement A2 (HtrA2), also designated PARK13, which lead to the loss of its protease activity, have been associated with Parkinson's disease (PD). HtrA2 is a mitochondrial protease that translocates to the cytosol upon the initiation of apoptosis where it participates in the abrogation of inhibitors of apoptosis (IAP) inhibition of caspases. This study has demonstrated that the loss of the HtrA2 function in the dopaminergic neurons of Drosophila melanogaster results in PD-like phenotypes, and attempts were made to restore the age-dependent loss in locomotor ability by co-expressing the sole pro-survival Bcl-2 homologue Buffy. The inhibition of HtrA2 in the dopaminergic neurons of Drosophila resulted in shortened lifespan and impaired climbing ability, and the overexpression of Buffy rescued the reduction in lifespan and the age-dependent loss of locomotor ability. In supportive experiments, the inhibition of HtrA2 in the Drosophila eye results in eye defects, marked by reduction in ommatidia number and increased disruption of the ommatidial array; phenotypes that are suppressed by the overexpression of Buffy.
|M'Angale, P. G. and Staveley, B. E. (2016). Overexpression of Buffy enhances the loss of parkin and suppresses the loss of Pink1 phenotypes in Drosophila. Genome: 1-7 [Epub ahead of print]. PubMed ID: 28106473
Mutations in parkin (PARK2) and Pink1 (PARK6) are responsible for autosomal recessive forms of early onset Parkinson's disease (PD). Attributed to the failure of neurons to clear dysfunctional mitochondria, loss of gene expression leads to loss of nigrostriatal neurons. The Pink1/parkin pathway plays a role in the quality control mechanism aimed at eliminating defective mitochondria, and the failure of this mechanism results in a reduced lifespan and impaired locomotor ability, among other phenotypes. Inhibition of parkin or Pink1 through the induction of stable RNAi transgene in the Ddc-Gal4-expressing neurons results in such phenotypes to model PD. To further evaluate the effects of the overexpression of the Bcl-2 homologue Buffy, this study analysed lifespan and climbing ability in both parkin-RNAi- and Pink1-RNAi-expressing flies. In addition, the effect of Buffy overexpression upon parkin-induced developmental eye defects was examined through GMR-Gal4-dependent expression. Curiously, Buffy overexpression produced very different effects: the parkin-induced phenotypes were enhanced, whereas the Pink1-enhanced phenotypes were suppressed. Interestingly, the overexpression of Buffy along with the inhibition of parkin in the neuron-rich eye results in the suppression of the developmental eye defects.
|M'Angale, P. G. and Staveley, B. E. (2017). Bax-inhibitor-1 knockdown phenotypes are suppressed by Buffy and exacerbate degeneration in a Drosophila model of Parkinson disease. PeerJ 5: e2974. PubMed ID: 28243526
Bax inhibitor-1 (BI-1) is an evolutionarily conserved cytoprotective transmembrane protein that acts as a suppressor of Bax-induced apoptosis by regulation of endoplasmic reticulum stress-induced cell death. BI-1 was knocked down in the sensitive dopa decarboxylase (Ddc) expressing neurons of Drosophila to investigate its neuroprotective functions. BI-1-induced phenotypes were rescied by co-expression with the pro-survival Buffy, and the effect of BI-1 knockdown on the neurodegenerative alpha-synuclein-induced Parkinson disease (PD) model was determined. Knockdown of BI-1 was achieved under the direction of the Ddc-Gal4 transgene and resulted in shortened lifespan and precocious loss of locomotor ability. Co-expression of Buffy with BI-1-RNAi resulted in suppression of the reduced lifespan and impaired climbing ability. Expression of human alpha-synuclein in Drosophila dopaminergic neurons results in neuronal degeneration, accompanied by the age-dependent loss in climbing ability. It is concluded that knockdown of BI-1 in the dopaminergic neurons of Drosophila results in a shortened lifespan and premature loss in climbing ability, phenotypes that appear to be strongly associated with models of PD in Drosophila, and which are suppressed upon overexpression of Buffy and worsened by co-expression with alpha-synuclein. This suggests that BI-1 is neuroprotective and its knockdown can be counteracted by the overexpression of the pro-survival Bcl-2 homologue.
|M'Angale, P. G. and Staveley, B. E. (2017). A loss of Pdxk model of Parkinson disease in Drosophila can be suppressed by Buffy. BMC Res Notes 10(1): 205. PubMed ID: 28606139
The identification of a DNA variant in pyridoxal kinase (Pdxk) associated with increased risk to Parkinson disease (PD) gene has led to a study the inhibition of this gene in the Dopa decarboxylase (Ddc)-expressing neurons of Drosophila. The multitude of biological functions attributable to the vitamers of vitamin B6 catalysed by this kinase reveal an overabundance of possible links to PD, that include dopamine synthesis, antioxidant activity and mitochondrial function. Drosophila possesses a single homologue of Pdxk, and this study used RNAi to inhibit the activity of this kinase in the Ddc-Gal4-expressing neurons. Any association was further investigated between this enhanced disease risk gene with the established PD model induced by expression of alpha-synuclein in the same neurons. The pro-survival functions of Buffy, an anti-apoptotic Bcl-2 homologue, were relied on to rescue the Pdxk-induced phenotypes. Ddc-Gal4, which drives expression in both dopaminergic and serotonergic neurons, was used to drive the expression of Pdxk RNA interference in DA neurons of Drosophila. The inhibition of Pdxk in the alpha-synuclein-induced Drosophila model of PD did not alter longevity and climbing ability of these flies. It has been previously shown that deficiency in vitamers lead to mitochondrial dysfunction and neuronal decay, therefore, co-expression of Pdxk-RNAi with the sole pro-survival Bcl-2 homologue Buffy in the Ddc-Gal4-expressing neurons, resulted in increased survival and a restored climbing ability. In a similar manner, when Pdxk was inhibited in the developing eye using GMR-Gal4, it was found that there was a decrease in the number of ommatidia and the disruption of the ommatidial array was more pronounced. When Pdxk was inhibited with the alpha-synuclein-induced developmental eye defects, the eye phenotypes were unaltered. Interestingly co-expression with Buffy restored ommatidia number and decreased the severity of disruption of the ommatidial array. It is concluded that though Pdxk is not a confirmed Parkinson disease gene, the inhibition of this kinase recapitulated the PD-like symptoms of decreased lifespan and loss of locomotor function, possibly producing a new model of PD.
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-XL 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, Buffys 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 Buffys 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 (OReilly, 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).
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-xL also support direct regulation of oxidative phosphorylation: one demonstrated that Bcl-xL controls the levels of the metabolite acetyl coenzyme A (acetyl-CoA) and the other proposed that neuronal Bcl-xL 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-xL 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).
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).
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).
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).
The Inhibitor of apoptosis (IAP) antagonists Reaper (Rpr), Grim and Hid are central regulators of developmental apoptosis in Drosophila. Ectopic expression of each is sufficient to trigger apoptosis, and hid and rpr have been shown to be important for programmed cell death (PCD). To investigate the role for grim in PCD, a grim null mutant was generated. grim was not a key proapoptotic gene for embryonic PCD, confirming that grim cooperates with rpr and hid in embryogenesis. In contrast, PCD of glial cells in the microchaete lineage required grim, identifying a death process dependent upon endogenous grim. Grim associates with mitochondria and has been shown to activate a mitochondrial death pathway distinct from IAP antagonization; therefore, the Drosophila bcl-2 genes buffy and debcl were investigated for genetic interaction withgrim. Loss of buffy led to microchaete glial cell survival and suppressed death in the eye induced by ectopic Grim. This is the first example of a developmental PCD process influenced by buffy, and places buffy in a proapoptotic role. PCD of microchaete glial cells represents an exceptional opportunity to study the mitochondrial proapoptotic process induced by Grim (Wu, 2010).
Shaping of the Drosophila embryo involves a series of morphogenetic movements, all of which are accompanied by cell death. Embryos homozygous for the H99 deletion lack developmental apoptosis. The three known proapoptotic genes in the H99 region, rpr, hid and grim are expressed during embryogenesis. Data using overlapping deficiencies, mutants and ectopic expression studies suggest that the combined activities of rpr, hid and grim are required for initiation of PCD during embryogenesis. This study of the first grim null mutant confirms that grim works cooperatively with the other RHG genes for embryonic PCD (Wu, 2010).
In development of the mechanosensory bristles of the Drosophila notum, an SOP gives rise to the four cells of the sensory organ and a glial cell that is doomed to die. Evaluation of the grim null mutant, knockdown of grim, and a large deletion that removes genomic sequences upstream of grim all demonstrated that PCD of this glial cell is dependent upon grim. Additionally, a small deletion within the glutamine-rich domain of Grim was identified that reduced the proapoptotic activity of Grim. Future investigations that focus on the role of the glutamine-rich domain in Grim function will first require separation of the grimΔ52-57 mutation from the f04656 P-element insertion (Wu, 2010).
An increase was observed in glial cell survival in the absence of buffy. Death of microchaete glial cells is the first developmental PCD identified that is modified by loss of buffy. buffy played a proapoptotic role in this process as well as in grim-dependent cell death in the eye. Expression studies in the eye have led to the conclusion that over-expressed Debcl is a proapoptotic Bcl-2 protein, whereas over-expressed Buffy moderately blocks the killing activity of Debcl and other pro-death proteins such as Rpr, Grim and Hid. In stress-induced apoptosis this study also found that Buffy inhibits apoptosis. Why then, does Buffy appear to be proapoptotic in the current study? To begin, although over-expressed Buffy suppresses cell death due to ectopic Grim in the eye, this study found that a high level of Buffy was unable to block PCD of the microchaete glial cell. This suggests that there is an important difference in the killing function of over-expressed Grim relative to a physiological amount of Grim. It is not simply that over-expressed Grim induces Debcl-dependent apoptosis that would be enhanced by loss of Buffy and suppressed by ectopic Buffy. If this were the case, then Grim-induced death should have been suppressed by loss of debcl, which it was not. How ectopic Buffy inhibits over-expressed Grim-dependent cell death (as well as other proapoptotic proteins when over-expressed may instead relate to its ability to suppress mitochondrial dysfunction. But in cells that respond to endogenous levels of Grim, Buffy is not antiapoptotic. This establishes that physiological levels of Grim do not utilize a killing mechanism that can be suppressed by Buffy. Furthermore, ectopic Buffy is not generally inhibitory to PCD in the animal. Secondly, the Drosophila Bcl-2 proteins can function as both pro- and anti-death proteins: Debcl protects cells from CED-3 and serum-deprivation induced cell death and protects neurons from expanded polyglutamine-mediated neurodegeneration whereas Buffy can promote cell death. The finding that Bcl-2 proteins have dual functions is not novel. Certain mammalian antiapoptotic Bcl-2 proteins can be converted into proapoptotic proteins that induce Cytochrome c release from mitochondria. CED-9, the C. elegans Bcl-2 protein, also has both pro- and antiapoptotic activity. Conversely, Bax, Bak and Bad are mammalian proapoptotic Bcl-2 proteins that can promote cell survival. In Drosophila, as in mammals, the ability of Bcl-2 proteins to promote or inhibit cell death likely depends on the specific cellular context (Wu, 2010).
Grim can activate two pathways leading to cell death: the first is dependent upon the IBM to block DIAP1 and release active caspases, and the second requires the GH3 domain for mitochondrial targeting. In the case of Rpr, these two pathways are inter-dependent in that DIAP1 degradation promoted by Rpr is significantly more efficient at the mitochondria. However, this may not be the situation for Grim as there are numerous observations that Grim maintains some killing activity even when unable to bind and antagonize DIAP1 and that the GH3 domain alone targets mitochondria and induces death. These data are suggestive of the existence of a DIAP1-independent killing mechanism for Grim that is mitochondrial. Expression of mutants lacking the ability to engage one or the other of the two mechanisms demonstrated that the two pathways could cooperate. It is not known how these two activities contribute to Grim function in an endogenous PCD setting. This study defines PCD of microchaete glial cells as the first example of grim-dependent PCD, and a very recent report demonstrates that life and death of these cells does not rely upon DIAP1 antagonization. In this report, DIAP1 protein turnover was monitored in live animals. DIAP1 protein was not detectable in cells of the microchaete SOP lineage from the 2-cell stage (PIIa and PIIb) through the 5-cell stage (shaft, socket, neuron, sheath and glial cell). Thus, even from birth, there was no DIAP1 protein in the glial cell. The lack of DIAP1 protein was not because RHG proteins induced DIAP1 protein degradation since there was also no detectable DIAP1 in H99−/− clones. Lastly, Rpr was expressed in cells of the SOP lineage and this did not lead to precocious glial cell death. If IAP protein was present and responsible for maintaining glial cell viability, then Rpr expression should have caused premature cell death. These data demonstrate that Grim does not induce PCD of microchaete glial cells solely through DIAP1 antagonization, and suggest that another mechanism utilized by Grim contributes to glial cell death. The previously described mitochondrial pathway for Grim-dependent death is the most likely candidate and is supported by the genetic and physical interaction of Grim with Buffy. Verification of the dependence of microchaete glial cell death on the GH3 domain of Grim awaits mutational analysis of grim at its endogenous locus (Wu, 2010).
A mitochondrial pathway activated by Grim could involve mitochondrial permeabilization or alternatively mitochondrial fragmentation and dysfunction, but must ultimately lead to cell death by activating caspases (microchaete glial cells have active caspase activity prior to death and p35 expression forces their survival. When expressed in vertebrate cells, Grim induced release of Cytochrome c, through a process that required the GH3 domain but was independent of IAP antagonization or Bcl-2 proteins. Although Cytochrome c is not released, mitochondrial permeabilization may play a role in Drosophila cell death (mitochondria are clearly affected as Cytochrome c changes confirmation early in death). Mitochondrial fragmentation has been observed in Drosophila cells undergoing PCD and occurs prior to caspase activation, suggesting that fragmentation may be causative. A large body of work has demonstrated that mitochondrial fission in mammalian cells accompanies apoptosis (Wu, 2010).
In this study, although it is possible that Grim and Buffy promote cell death through separate mechanisms, it is proposed that the current results are most consistent with Buffy enhancing or amplifying a mitochondrial death process activated by Grim. The physical interaction between the two proteins supports this. Such a model would explain why loss of buffy has only a partial effect on ectopic Grim-dependent death (likely mostly DIAP1-dependent) and why microchaete cell death was affected less by loss of buffy than by loss of grim (because Grim is augmented by Buffy). Endogenous Buffy must be sufficient for grim-dependent cell death because excess Buffy did not further increase microchaete cell death. Since buffy does not modify ectopic Rpr or Hid in the fly eye, and since there are situations in which Grim induces cell death in which neither Rpr nor Hid can, the interaction between Grim and Buffy may be unique to Grim (Wu, 2010).
There is significant evidence that mammalian Bcl-2 family members can influence the dynamics of mitochondrial fission and fusion in both healthy and apoptotic cells, possibly through direct interaction with core components of the mitochondrial fission/fusion machinery. Although a role for Buffy in mitochondrial morphogenesis has not been carefully investigated, Buffy expression suppresses mitochondrial phenotypes of PINK1 and Prel mutant animals. In the simplest scenario, either Grim induces mitochondrial dysfunction more efficiently through interaction with Buffy (perhaps leading to more mitochondrial fission) or mitochondrial dysfunction is actively amplified by Buffy (perhaps through release of a proapoptotic factor) (Wu, 2010).
PCD of microchaete glial cells is the first example of an in vivo death that utilizes buffy and requires grim and provides an unparalleled opportunity to investigate a cell death mechanism that is likely to elucidate a role for mitochondria in Drosophila PCD (Wu, 2010).
Information about anti-apoptotic BCL2 homologs can be found at death executioner Bcl-2 homologue (debcl), Evolutionary Homologs section.
Search PubMed for articles about Drosophila Buffy
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date revised: 20 September 2012
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