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Screening of suppressors of bax-induced cell death identifies glycerophosphate oxidase-1 as a mediator of debcl-induced apoptosis in Drosophila.

Colin J, Garibal J, Clavier A, Szuplewski S, Risler Y, Milet C, Gaumer S, Guénal I, Mignotte B - Genes Cancer (2015)

Bottom Line: We have previously shown that the mammalian pro-apoptotic Bcl-2 family member Bax is very efficient in inducing apoptosis in Drosophila, allowing the study of bax-induced cell death in a genetic animal model.Most of these suppressors also inhibit debcl-induced phenotypes, suggesting that the activities of both proteins can be modulated in part by common signaling or metabolic pathways.Among these suppressors, Glycerophosphate oxidase-1 is found to participate in debcl-induced apoptosis by increasing mitochondrial reactive oxygen species accumulation.

View Article: PubMed Central - PubMed

Affiliation: Université Versailles St-Quentin, Laboratoire de Génétique et Biologie Cellulaire, Montigny-le-Bretonneux, France ; Ecole Pratique des Hautes Etudes, Laboratoire de Génétique Moléculaire et Physiologique, Montigny-le-Bretonneux, France.

ABSTRACT
Members of the Bcl-2 family are key elements of the apoptotic machinery. In mammals, this multigenic family contains about twenty members, which either promote or inhibit apoptosis. We have previously shown that the mammalian pro-apoptotic Bcl-2 family member Bax is very efficient in inducing apoptosis in Drosophila, allowing the study of bax-induced cell death in a genetic animal model. We report here the results of the screening of a P[UAS]-element insertion library performed to identify gene products that modify the phenotypes induced by the expression of bax in Drosophila melanogaster. We isolated 17 putative modifiers involved in various function or process: the ubiquitin/proteasome pathway; cell growth, proliferation and death; pathfinding and cell adhesion; secretion and extracellular signaling; metabolism and oxidative stress. Most of these suppressors also inhibit debcl-induced phenotypes, suggesting that the activities of both proteins can be modulated in part by common signaling or metabolic pathways. Among these suppressors, Glycerophosphate oxidase-1 is found to participate in debcl-induced apoptosis by increasing mitochondrial reactive oxygen species accumulation.

No MeSH data available.


Related in: MedlinePlus

Gpo-1 loss of function suppresses debcl-induced apoptosis by limiting mitochondrial ROS accumulation(A)ptc expression domain visualized by GFP fluorescence in a wing imaginal disc. (B-D) TUNEL staining of wing imaginal discs from ptc>+; ptc>debcl2; and ptc>debcl2,Gpo1n322 wing imaginal discs. (E) Quantification of TUNEL positive cells of wing imaginal discs. (F) Quantification by flow cytometry of MitoSOX staining in larval wing imaginal discs. All these experiments were performed at 18°C. Error bars are the S.E.M. **: Student's t test α<0.01.
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Figure 3: Gpo-1 loss of function suppresses debcl-induced apoptosis by limiting mitochondrial ROS accumulation(A)ptc expression domain visualized by GFP fluorescence in a wing imaginal disc. (B-D) TUNEL staining of wing imaginal discs from ptc>+; ptc>debcl2; and ptc>debcl2,Gpo1n322 wing imaginal discs. (E) Quantification of TUNEL positive cells of wing imaginal discs. (F) Quantification by flow cytometry of MitoSOX staining in larval wing imaginal discs. All these experiments were performed at 18°C. Error bars are the S.E.M. **: Student's t test α<0.01.

Mentions: The line UY1039 being lost we assumed that the suppressor effect of UY1039 was due to a LOF mutation in the Glycerophosphate oxidase-1 gene. Given that this gene is involved in the mitochondrial metabolism, and that numerous Bcl-2 family members act at the mitochondrial level, Gpo-1 seemed of high interest and we decided to focus on its study. To confirm our hypothesis, we tested whether RNAi against Gpo-1 or heterozygosity for a Gpo-1 hypomorph (Gpo-1291) or an amorph (Gpo-1n322) allele, could suppress Debcl overexpression-induced wing phenotype. Both RNAi and both Gpo-1 LOF heterozygous alleles suppressed debcl-induced phenotypes but the most complete and fully penetrant suppression was observed in flies heterozygous for the Gpo-1n322 mutation (Figure 2). Therefore, we decided to assess the apoptosis level in wing imaginal discs of flies heterozygous for Gpo-1n322. The number of TUNEL positive cells in wing discs overexpressing debcl was dramatically reduced by Gpo-1n322 heterozygosity when compared to discs that are not mutated in Gpo-1 (Figure 3A-E), thus confirming that a reduction of Gpo-1 dosage suppresses debcl-induced apoptosis.


Screening of suppressors of bax-induced cell death identifies glycerophosphate oxidase-1 as a mediator of debcl-induced apoptosis in Drosophila.

Colin J, Garibal J, Clavier A, Szuplewski S, Risler Y, Milet C, Gaumer S, Guénal I, Mignotte B - Genes Cancer (2015)

Gpo-1 loss of function suppresses debcl-induced apoptosis by limiting mitochondrial ROS accumulation(A)ptc expression domain visualized by GFP fluorescence in a wing imaginal disc. (B-D) TUNEL staining of wing imaginal discs from ptc>+; ptc>debcl2; and ptc>debcl2,Gpo1n322 wing imaginal discs. (E) Quantification of TUNEL positive cells of wing imaginal discs. (F) Quantification by flow cytometry of MitoSOX staining in larval wing imaginal discs. All these experiments were performed at 18°C. Error bars are the S.E.M. **: Student's t test α<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482245&req=5

Figure 3: Gpo-1 loss of function suppresses debcl-induced apoptosis by limiting mitochondrial ROS accumulation(A)ptc expression domain visualized by GFP fluorescence in a wing imaginal disc. (B-D) TUNEL staining of wing imaginal discs from ptc>+; ptc>debcl2; and ptc>debcl2,Gpo1n322 wing imaginal discs. (E) Quantification of TUNEL positive cells of wing imaginal discs. (F) Quantification by flow cytometry of MitoSOX staining in larval wing imaginal discs. All these experiments were performed at 18°C. Error bars are the S.E.M. **: Student's t test α<0.01.
Mentions: The line UY1039 being lost we assumed that the suppressor effect of UY1039 was due to a LOF mutation in the Glycerophosphate oxidase-1 gene. Given that this gene is involved in the mitochondrial metabolism, and that numerous Bcl-2 family members act at the mitochondrial level, Gpo-1 seemed of high interest and we decided to focus on its study. To confirm our hypothesis, we tested whether RNAi against Gpo-1 or heterozygosity for a Gpo-1 hypomorph (Gpo-1291) or an amorph (Gpo-1n322) allele, could suppress Debcl overexpression-induced wing phenotype. Both RNAi and both Gpo-1 LOF heterozygous alleles suppressed debcl-induced phenotypes but the most complete and fully penetrant suppression was observed in flies heterozygous for the Gpo-1n322 mutation (Figure 2). Therefore, we decided to assess the apoptosis level in wing imaginal discs of flies heterozygous for Gpo-1n322. The number of TUNEL positive cells in wing discs overexpressing debcl was dramatically reduced by Gpo-1n322 heterozygosity when compared to discs that are not mutated in Gpo-1 (Figure 3A-E), thus confirming that a reduction of Gpo-1 dosage suppresses debcl-induced apoptosis.

Bottom Line: We have previously shown that the mammalian pro-apoptotic Bcl-2 family member Bax is very efficient in inducing apoptosis in Drosophila, allowing the study of bax-induced cell death in a genetic animal model.Most of these suppressors also inhibit debcl-induced phenotypes, suggesting that the activities of both proteins can be modulated in part by common signaling or metabolic pathways.Among these suppressors, Glycerophosphate oxidase-1 is found to participate in debcl-induced apoptosis by increasing mitochondrial reactive oxygen species accumulation.

View Article: PubMed Central - PubMed

Affiliation: Université Versailles St-Quentin, Laboratoire de Génétique et Biologie Cellulaire, Montigny-le-Bretonneux, France ; Ecole Pratique des Hautes Etudes, Laboratoire de Génétique Moléculaire et Physiologique, Montigny-le-Bretonneux, France.

ABSTRACT
Members of the Bcl-2 family are key elements of the apoptotic machinery. In mammals, this multigenic family contains about twenty members, which either promote or inhibit apoptosis. We have previously shown that the mammalian pro-apoptotic Bcl-2 family member Bax is very efficient in inducing apoptosis in Drosophila, allowing the study of bax-induced cell death in a genetic animal model. We report here the results of the screening of a P[UAS]-element insertion library performed to identify gene products that modify the phenotypes induced by the expression of bax in Drosophila melanogaster. We isolated 17 putative modifiers involved in various function or process: the ubiquitin/proteasome pathway; cell growth, proliferation and death; pathfinding and cell adhesion; secretion and extracellular signaling; metabolism and oxidative stress. Most of these suppressors also inhibit debcl-induced phenotypes, suggesting that the activities of both proteins can be modulated in part by common signaling or metabolic pathways. Among these suppressors, Glycerophosphate oxidase-1 is found to participate in debcl-induced apoptosis by increasing mitochondrial reactive oxygen species accumulation.

No MeSH data available.


Related in: MedlinePlus