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The Drosophila TRPP cation channel, PKD2 and Dmel/Ced-12 act in genetically distinct pathways during apoptotic cell clearance.

Van Goethem E, Silva EA, Xiao H, Franc NC - PLoS ONE (2012)

Bottom Line: As anticipated, we have found that Dmel\ced-12 is required for apoptotic cell clearance, as for its C. elegans and mammalian homologues, ced-12 and elmo, respectively.However, the loss of Dmel\ced-12 did not solely account for the phenotypes of all three deficiencies, as zygotic mutations and germ line clones of Dmel\ced-12 exhibited weaker phenotypes.However, we have not found any genetic interaction between Dmel\ced-12 and simu.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Cell Biology Unit, MRC Laboratory for Molecular Cell Biology and Anatomy and Developmental Biology Department, University College London, London, United Kingdom.

ABSTRACT
Apoptosis, a genetically programmed cell death, allows for homeostasis and tissue remodelling during development of all multi-cellular organisms. Phagocytes swiftly recognize, engulf and digest apoptotic cells. Yet, to date the molecular mechanisms underlying this phagocytic process are still poorly understood. To delineate the molecular mechanisms of apoptotic cell clearance in Drosophila, we have carried out a deficiency screen and have identified three overlapping phagocytosis-defective mutants, which all delete the fly homologue of the ced-12 gene, known as Dmel\ced12. As anticipated, we have found that Dmel\ced-12 is required for apoptotic cell clearance, as for its C. elegans and mammalian homologues, ced-12 and elmo, respectively. However, the loss of Dmel\ced-12 did not solely account for the phenotypes of all three deficiencies, as zygotic mutations and germ line clones of Dmel\ced-12 exhibited weaker phenotypes. Using a nearby genetically interacting deficiency, we have found that the polycystic kidney disease 2 gene, pkd2, which encodes a member of the TRPP channel family, is also required for phagocytosis of apoptotic cells, thereby demonstrating a novel role for PKD2 in this process. We have also observed genetic interactions between pkd2, simu, drpr, rya-r44F, and retinophilin (rtp), also known as undertaker (uta), a gene encoding a MORN-repeat containing molecule, which we have recently found to be implicated in calcium homeostasis during phagocytosis. However, we have not found any genetic interaction between Dmel\ced-12 and simu. Based on these genetic interactions and recent reports demonstrating a role for the mammalian pkd-2 gene product in ER calcium release during store-operated calcium entry, we propose that PKD2 functions in the DRPR/RTP pathway to regulate calcium homeostasis during this process. Similarly to its C. elegans homologue, Dmel\Ced-12 appears to function in a genetically distinct pathway.

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Genetic interactions between pkd2, uta and drpr  alleles reveal a role for pkd2 in phagocytosis of apoptotic cells.In A–F are projected confocal images of macrophages in stage 13 y,w (wild-type) embryos (A), amo1 homozygous embryos (B), amo1/rya-r44Fk04913 heterozygous embryos (C), double heterozygous amo1/+; Df(3R)3-4/+ (D) and amo1/+; drprrecΔ5/+ (E) embryos, and amo1/Df(2L)b87e25 heterozygous embryos (F). All embryos were stained with the CRQ Ab (green) and 7-AAD (red). Embryos in B–F had macrophages that were smaller and less efficient at engulfing apoptotic cells than that of wild-type embryos (A). In G is a graph summarizing the PIs of wild-type (y,w), amo1, rya-r44Fk04913, rya-r44F16, Df(3R)3-, drprrecΔ5 and Df(2L)b87e25 homozygous macrophages, that of amo1/rya-r44F16, amo1/rya-r44FK04913, amo1/Df(2L)b87e25 and ced-1219F3/simu2 trans-heterozygous macrophages, as well as that of amo1/+;Df(3R)3-4/+ and amo1/+; drprrecΔ5/+ double heterozygous macrophages. Each bar represents the mean value ± SEM of the relative PIs for each genotype. Scale bars in panels A–F are 5 µm. In A–F, dotted white circles are indicative of individual macrophage cell body based on 7-AAD staining of their regular nuclei and CRQ staining.
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pone-0031488-g006: Genetic interactions between pkd2, uta and drpr alleles reveal a role for pkd2 in phagocytosis of apoptotic cells.In A–F are projected confocal images of macrophages in stage 13 y,w (wild-type) embryos (A), amo1 homozygous embryos (B), amo1/rya-r44Fk04913 heterozygous embryos (C), double heterozygous amo1/+; Df(3R)3-4/+ (D) and amo1/+; drprrecΔ5/+ (E) embryos, and amo1/Df(2L)b87e25 heterozygous embryos (F). All embryos were stained with the CRQ Ab (green) and 7-AAD (red). Embryos in B–F had macrophages that were smaller and less efficient at engulfing apoptotic cells than that of wild-type embryos (A). In G is a graph summarizing the PIs of wild-type (y,w), amo1, rya-r44Fk04913, rya-r44F16, Df(3R)3-, drprrecΔ5 and Df(2L)b87e25 homozygous macrophages, that of amo1/rya-r44F16, amo1/rya-r44FK04913, amo1/Df(2L)b87e25 and ced-1219F3/simu2 trans-heterozygous macrophages, as well as that of amo1/+;Df(3R)3-4/+ and amo1/+; drprrecΔ5/+ double heterozygous macrophages. Each bar represents the mean value ± SEM of the relative PIs for each genotype. Scale bars in panels A–F are 5 µm. In A–F, dotted white circles are indicative of individual macrophage cell body based on 7-AAD staining of their regular nuclei and CRQ staining.

Mentions: We asked whether pkd2 might be required for phagocytosis of apoptotic cells in vivo by assessing the phagocytosis phenotype of a previously generated knock-out allele of pkd2, amo1[43]. We found that homozygous mutant macrophages for amo1 were less efficient at engulfing apoptotic cells than wild-type macrophages (compare figures 6A and B) with a relative PI of 51.7±2.6% (p<0.001)(figure 6G), thereby confirming a role for pkd2 in phagocytosis of apoptotic cells. We also asked whether pkd2, rya-r44F and rtp might genetically interact in phagocytosis. As anticipated, we found an interaction between pkd2 and rya-r44F, as heterozygous mutant macrophages for the pkd2 knockout allele amo1 and the rya-r44F16 hypomorph allele were small and poorly engulfed apoptotic cells (figures 6C). These results are consistent with previous genetic interactions found between pkd2 and rya-r44F in cell body wall contraction, and argue that pkd2 acts in phagocytosis similarly to its role in body cell wall contraction by regulating Ca2+ homeostasis. We also observed a similar phenotype in double heterozygous mutant embryos for amo1 and the rtp deficiency Df(3R)3-4, with smaller macrophages gathering around apoptotic corpses but engulfing poorly (figure 6D) with a relative PI of 55.4±2.4% similar to that of amo1 homozygous macrophages (p>0.05)(figure 6G).


The Drosophila TRPP cation channel, PKD2 and Dmel/Ced-12 act in genetically distinct pathways during apoptotic cell clearance.

Van Goethem E, Silva EA, Xiao H, Franc NC - PLoS ONE (2012)

Genetic interactions between pkd2, uta and drpr  alleles reveal a role for pkd2 in phagocytosis of apoptotic cells.In A–F are projected confocal images of macrophages in stage 13 y,w (wild-type) embryos (A), amo1 homozygous embryos (B), amo1/rya-r44Fk04913 heterozygous embryos (C), double heterozygous amo1/+; Df(3R)3-4/+ (D) and amo1/+; drprrecΔ5/+ (E) embryos, and amo1/Df(2L)b87e25 heterozygous embryos (F). All embryos were stained with the CRQ Ab (green) and 7-AAD (red). Embryos in B–F had macrophages that were smaller and less efficient at engulfing apoptotic cells than that of wild-type embryos (A). In G is a graph summarizing the PIs of wild-type (y,w), amo1, rya-r44Fk04913, rya-r44F16, Df(3R)3-, drprrecΔ5 and Df(2L)b87e25 homozygous macrophages, that of amo1/rya-r44F16, amo1/rya-r44FK04913, amo1/Df(2L)b87e25 and ced-1219F3/simu2 trans-heterozygous macrophages, as well as that of amo1/+;Df(3R)3-4/+ and amo1/+; drprrecΔ5/+ double heterozygous macrophages. Each bar represents the mean value ± SEM of the relative PIs for each genotype. Scale bars in panels A–F are 5 µm. In A–F, dotted white circles are indicative of individual macrophage cell body based on 7-AAD staining of their regular nuclei and CRQ staining.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3275576&req=5

pone-0031488-g006: Genetic interactions between pkd2, uta and drpr alleles reveal a role for pkd2 in phagocytosis of apoptotic cells.In A–F are projected confocal images of macrophages in stage 13 y,w (wild-type) embryos (A), amo1 homozygous embryos (B), amo1/rya-r44Fk04913 heterozygous embryos (C), double heterozygous amo1/+; Df(3R)3-4/+ (D) and amo1/+; drprrecΔ5/+ (E) embryos, and amo1/Df(2L)b87e25 heterozygous embryos (F). All embryos were stained with the CRQ Ab (green) and 7-AAD (red). Embryos in B–F had macrophages that were smaller and less efficient at engulfing apoptotic cells than that of wild-type embryos (A). In G is a graph summarizing the PIs of wild-type (y,w), amo1, rya-r44Fk04913, rya-r44F16, Df(3R)3-, drprrecΔ5 and Df(2L)b87e25 homozygous macrophages, that of amo1/rya-r44F16, amo1/rya-r44FK04913, amo1/Df(2L)b87e25 and ced-1219F3/simu2 trans-heterozygous macrophages, as well as that of amo1/+;Df(3R)3-4/+ and amo1/+; drprrecΔ5/+ double heterozygous macrophages. Each bar represents the mean value ± SEM of the relative PIs for each genotype. Scale bars in panels A–F are 5 µm. In A–F, dotted white circles are indicative of individual macrophage cell body based on 7-AAD staining of their regular nuclei and CRQ staining.
Mentions: We asked whether pkd2 might be required for phagocytosis of apoptotic cells in vivo by assessing the phagocytosis phenotype of a previously generated knock-out allele of pkd2, amo1[43]. We found that homozygous mutant macrophages for amo1 were less efficient at engulfing apoptotic cells than wild-type macrophages (compare figures 6A and B) with a relative PI of 51.7±2.6% (p<0.001)(figure 6G), thereby confirming a role for pkd2 in phagocytosis of apoptotic cells. We also asked whether pkd2, rya-r44F and rtp might genetically interact in phagocytosis. As anticipated, we found an interaction between pkd2 and rya-r44F, as heterozygous mutant macrophages for the pkd2 knockout allele amo1 and the rya-r44F16 hypomorph allele were small and poorly engulfed apoptotic cells (figures 6C). These results are consistent with previous genetic interactions found between pkd2 and rya-r44F in cell body wall contraction, and argue that pkd2 acts in phagocytosis similarly to its role in body cell wall contraction by regulating Ca2+ homeostasis. We also observed a similar phenotype in double heterozygous mutant embryos for amo1 and the rtp deficiency Df(3R)3-4, with smaller macrophages gathering around apoptotic corpses but engulfing poorly (figure 6D) with a relative PI of 55.4±2.4% similar to that of amo1 homozygous macrophages (p>0.05)(figure 6G).

Bottom Line: As anticipated, we have found that Dmel\ced-12 is required for apoptotic cell clearance, as for its C. elegans and mammalian homologues, ced-12 and elmo, respectively.However, the loss of Dmel\ced-12 did not solely account for the phenotypes of all three deficiencies, as zygotic mutations and germ line clones of Dmel\ced-12 exhibited weaker phenotypes.However, we have not found any genetic interaction between Dmel\ced-12 and simu.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Cell Biology Unit, MRC Laboratory for Molecular Cell Biology and Anatomy and Developmental Biology Department, University College London, London, United Kingdom.

ABSTRACT
Apoptosis, a genetically programmed cell death, allows for homeostasis and tissue remodelling during development of all multi-cellular organisms. Phagocytes swiftly recognize, engulf and digest apoptotic cells. Yet, to date the molecular mechanisms underlying this phagocytic process are still poorly understood. To delineate the molecular mechanisms of apoptotic cell clearance in Drosophila, we have carried out a deficiency screen and have identified three overlapping phagocytosis-defective mutants, which all delete the fly homologue of the ced-12 gene, known as Dmel\ced12. As anticipated, we have found that Dmel\ced-12 is required for apoptotic cell clearance, as for its C. elegans and mammalian homologues, ced-12 and elmo, respectively. However, the loss of Dmel\ced-12 did not solely account for the phenotypes of all three deficiencies, as zygotic mutations and germ line clones of Dmel\ced-12 exhibited weaker phenotypes. Using a nearby genetically interacting deficiency, we have found that the polycystic kidney disease 2 gene, pkd2, which encodes a member of the TRPP channel family, is also required for phagocytosis of apoptotic cells, thereby demonstrating a novel role for PKD2 in this process. We have also observed genetic interactions between pkd2, simu, drpr, rya-r44F, and retinophilin (rtp), also known as undertaker (uta), a gene encoding a MORN-repeat containing molecule, which we have recently found to be implicated in calcium homeostasis during phagocytosis. However, we have not found any genetic interaction between Dmel\ced-12 and simu. Based on these genetic interactions and recent reports demonstrating a role for the mammalian pkd-2 gene product in ER calcium release during store-operated calcium entry, we propose that PKD2 functions in the DRPR/RTP pathway to regulate calcium homeostasis during this process. Similarly to its C. elegans homologue, Dmel\Ced-12 appears to function in a genetically distinct pathway.

Show MeSH
Related in: MedlinePlus