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Necrotic Cells Actively Attract Phagocytes through the Collaborative Action of Two Distinct PS-Exposure Mechanisms.

Li Z, Venegas V, Nagaoka Y, Morino E, Raghavan P, Audhya A, Nakanishi Y, Zhou Z - PLoS Genet. (2015)

Bottom Line: Necrosis, a kind of cell death closely associated with pathogenesis and genetic programs, is distinct from apoptosis in both morphology and mechanism.Phosphatidylserine (PS) is an important apoptotic-cell surface signal that attracts engulfing cells.The extracellular domain of CED-1 associates with PS in vitro.

View Article: PubMed Central - PubMed

Affiliation: Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
Necrosis, a kind of cell death closely associated with pathogenesis and genetic programs, is distinct from apoptosis in both morphology and mechanism. Like apoptotic cells, necrotic cells are swiftly removed from animal bodies to prevent harmful inflammatory and autoimmune responses. In the nematode Caenorhabditis elegans, gain-of-function mutations in certain ion channel subunits result in the excitotoxic necrosis of six touch neurons and their subsequent engulfment and degradation inside engulfing cells. How necrotic cells are recognized by engulfing cells is unclear. Phosphatidylserine (PS) is an important apoptotic-cell surface signal that attracts engulfing cells. Here we observed PS exposure on the surface of necrotic touch neurons. In addition, the phagocytic receptor CED-1 clusters around necrotic cells and promotes their engulfment. The extracellular domain of CED-1 associates with PS in vitro. We further identified a necrotic cell-specific function of CED-7, a member of the ATP-binding cassette (ABC) transporter family, in promoting PS exposure. In addition to CED-7, anoctamin homolog-1 (ANOH-1), the C. elegans homolog of the mammalian Ca(2+)-dependent phospholipid scramblase TMEM16F, plays an independent role in promoting PS exposure on necrotic cells. The combined activities from CED-7 and ANOH-1 ensure efficient exposure of PS on necrotic cells to attract their phagocytes. In addition, CED-8, the C. elegans homolog of mammalian Xk-related protein 8 also makes a contribution to necrotic cell-removal at the first larval stage. Our work indicates that cells killed by different mechanisms (necrosis or apoptosis) expose a common "eat me" signal to attract their phagocytic receptor(s); furthermore, unlike what was previously believed, necrotic cells actively present PS on their outer surfaces through at least two distinct molecular mechanisms rather than leaking out PS passively.

No MeSH data available.


Related in: MedlinePlus

anoh-1 is required for the efficient removal of necrotic touch cells but not apoptotic cells.(A) Gene structure of two isoforms of anoh-1. The blue open boxes indicate the region deleted in the anoh-1(tm4762) allele. (B) Domain structure of ANOH-1b protein. Dark grey bars indicate 8 predicted transmembrane domains. Red color labels amino acids 1–18, which exists in ANOH-1b but missing from ANOH-1a. (C) The anoh-1(tm4762) mutant embryos are normal in the removal of somatic apoptotic cells. (D) The anoh-1(tm4762) mutant adults are normal in the removal of apoptotic germ cells. (E) The engulfment and degradation processes of embryonic apoptotic cells C1, C2, and C3 are normal in anoh-1(tm4762) mutant embryos, measured by time-lapse microscopy. (F) The anoh-1(tm4762) mutation perturbs the removal of necrotic cells, and the anoh-1b but not the anoh-1a form rescues the necrotic cell-removal defect when expressed in touch cells under the control of Pmec-7. For each sample, 6 groups of 10 worms at indicated stages were scored and displayed as mean. Error bars indicate standard deviations. *, 0.01<p<0.05 (student t-test). ***, p<0.001 (student t-test). ns, no significant difference, p>0.05 (student t-test). (G) The expression of anoh-1b in the necrotic cells, not engulfing cells, rescues anoh-1(tm4762) mutant phenotype. In anoh-1(tm4762); mec-4(e1611dm) mutant animals expressing indicated transgenes, the number of necrotic cells in the tail of each of the 6 groups of 10 newly hatched L1 larvae were scored. Data are presented as mean ± sd. (H) The GFP fusion forms of ANOH-1b but not ANOH-1a are localized to the plasma membrane. GFP (a, c, e, g) and DIC (b, d, f, h) images of the tails of mec-4(e1611dm) L1-stage larvae. Arrowheads in (a, b, e, f) label necrotic cells on which ANOH-1b is observed on the cell surface. Arrows in (c, d, g, h) label necrotic cells in which ANOH-1a is observed on the nuclear surface. All GFP reporters are expressed under the Pmec-7 promoter control. (I) The anoh-1b promoter is expressed in touch neurons. Shown here are epifluorescence (b-c) and the corresponding DIC (a) images of the tail region of a wild-type L2 larva co-expressing Pmec-7 mCherry (b), which is a touch neuron-specific reporter, and Panoh-1b NLS::GFP (c). White arrowheads mark a touch neuron. Dorsal is up. Scale bars are 5μm.
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pgen.1005285.g005: anoh-1 is required for the efficient removal of necrotic touch cells but not apoptotic cells.(A) Gene structure of two isoforms of anoh-1. The blue open boxes indicate the region deleted in the anoh-1(tm4762) allele. (B) Domain structure of ANOH-1b protein. Dark grey bars indicate 8 predicted transmembrane domains. Red color labels amino acids 1–18, which exists in ANOH-1b but missing from ANOH-1a. (C) The anoh-1(tm4762) mutant embryos are normal in the removal of somatic apoptotic cells. (D) The anoh-1(tm4762) mutant adults are normal in the removal of apoptotic germ cells. (E) The engulfment and degradation processes of embryonic apoptotic cells C1, C2, and C3 are normal in anoh-1(tm4762) mutant embryos, measured by time-lapse microscopy. (F) The anoh-1(tm4762) mutation perturbs the removal of necrotic cells, and the anoh-1b but not the anoh-1a form rescues the necrotic cell-removal defect when expressed in touch cells under the control of Pmec-7. For each sample, 6 groups of 10 worms at indicated stages were scored and displayed as mean. Error bars indicate standard deviations. *, 0.01<p<0.05 (student t-test). ***, p<0.001 (student t-test). ns, no significant difference, p>0.05 (student t-test). (G) The expression of anoh-1b in the necrotic cells, not engulfing cells, rescues anoh-1(tm4762) mutant phenotype. In anoh-1(tm4762); mec-4(e1611dm) mutant animals expressing indicated transgenes, the number of necrotic cells in the tail of each of the 6 groups of 10 newly hatched L1 larvae were scored. Data are presented as mean ± sd. (H) The GFP fusion forms of ANOH-1b but not ANOH-1a are localized to the plasma membrane. GFP (a, c, e, g) and DIC (b, d, f, h) images of the tails of mec-4(e1611dm) L1-stage larvae. Arrowheads in (a, b, e, f) label necrotic cells on which ANOH-1b is observed on the cell surface. Arrows in (c, d, g, h) label necrotic cells in which ANOH-1a is observed on the nuclear surface. All GFP reporters are expressed under the Pmec-7 promoter control. (I) The anoh-1b promoter is expressed in touch neurons. Shown here are epifluorescence (b-c) and the corresponding DIC (a) images of the tail region of a wild-type L2 larva co-expressing Pmec-7 mCherry (b), which is a touch neuron-specific reporter, and Panoh-1b NLS::GFP (c). White arrowheads mark a touch neuron. Dorsal is up. Scale bars are 5μm.

Mentions: Mammalian TMEM16F, a multispan transmembrane domain protein, is a Ca2+-activated phospholipid scramblase that triggers PS exposure in response to Ca2+-influx [36]. Given that the mec-4(dm)-induced touch neuron necrosis is mediated by Ca2+ influx [22,24], we examined whether ANOH-1, a close C. elegans homolog of TMEM16F (Figs 5B and S4), mediated PS exposure when necrosis occurred. We analyzed an anoh-1(tm4762) deletion allele (www.wormbase.org) for the removal of dying cells. The tm4762 allele carries a 202-bp deletion that results in a frameshift and a premature stop codon after amino acid 17 of the predicted ANOH-1b open reading frame and removes the start codon of the alternatively-spliced ANOH-1a open reading frame (S4 and S5B(b) Figs) (also see the next section), presumably generating a allele. In anoh-1(tm4762) mutant embryos, the numbers of apoptotic cells are the same as that displayed in wild-type embryos at 5 different embryonic developmental stages (Fig 5C). Furthermore, the dynamics of the engulfment and degradation processes of three individual apoptotic cells, C1, C2, and C3, are normal comparing to wild-type embryos (Fig 5E), using previously established protocols [48,55]. Similarly, the number of apoptotic germ cells are virtually the same in wild-type and anoh-1(tm4762) mutant adult hermaphrodites at four time points (Fig 5D). These results indicate that anoh-1, unlike ced-7, is not involved in the removal of apoptotic cells. In contrast, in anoh-1(tm4762); mec-4(e1611dm) double mutant animals, the removal of necrotic touch neurons is siginificantly delayed: at L1 and L2 stages, the mean numbers of persistent necrotic PLML/R in anoh-1(tm4762) background are approximately 1.6-fold and 1.5-fold of that in wild-type larvae, respectively, whereas at L3 and L4 stages, the mean numbers are not significantly different from wild-type animals (Fig 5F). These results strongly suggest that anoh-1 specifically contributes to efficient removal of necrotic but not apoptotic cells.


Necrotic Cells Actively Attract Phagocytes through the Collaborative Action of Two Distinct PS-Exposure Mechanisms.

Li Z, Venegas V, Nagaoka Y, Morino E, Raghavan P, Audhya A, Nakanishi Y, Zhou Z - PLoS Genet. (2015)

anoh-1 is required for the efficient removal of necrotic touch cells but not apoptotic cells.(A) Gene structure of two isoforms of anoh-1. The blue open boxes indicate the region deleted in the anoh-1(tm4762) allele. (B) Domain structure of ANOH-1b protein. Dark grey bars indicate 8 predicted transmembrane domains. Red color labels amino acids 1–18, which exists in ANOH-1b but missing from ANOH-1a. (C) The anoh-1(tm4762) mutant embryos are normal in the removal of somatic apoptotic cells. (D) The anoh-1(tm4762) mutant adults are normal in the removal of apoptotic germ cells. (E) The engulfment and degradation processes of embryonic apoptotic cells C1, C2, and C3 are normal in anoh-1(tm4762) mutant embryos, measured by time-lapse microscopy. (F) The anoh-1(tm4762) mutation perturbs the removal of necrotic cells, and the anoh-1b but not the anoh-1a form rescues the necrotic cell-removal defect when expressed in touch cells under the control of Pmec-7. For each sample, 6 groups of 10 worms at indicated stages were scored and displayed as mean. Error bars indicate standard deviations. *, 0.01<p<0.05 (student t-test). ***, p<0.001 (student t-test). ns, no significant difference, p>0.05 (student t-test). (G) The expression of anoh-1b in the necrotic cells, not engulfing cells, rescues anoh-1(tm4762) mutant phenotype. In anoh-1(tm4762); mec-4(e1611dm) mutant animals expressing indicated transgenes, the number of necrotic cells in the tail of each of the 6 groups of 10 newly hatched L1 larvae were scored. Data are presented as mean ± sd. (H) The GFP fusion forms of ANOH-1b but not ANOH-1a are localized to the plasma membrane. GFP (a, c, e, g) and DIC (b, d, f, h) images of the tails of mec-4(e1611dm) L1-stage larvae. Arrowheads in (a, b, e, f) label necrotic cells on which ANOH-1b is observed on the cell surface. Arrows in (c, d, g, h) label necrotic cells in which ANOH-1a is observed on the nuclear surface. All GFP reporters are expressed under the Pmec-7 promoter control. (I) The anoh-1b promoter is expressed in touch neurons. Shown here are epifluorescence (b-c) and the corresponding DIC (a) images of the tail region of a wild-type L2 larva co-expressing Pmec-7 mCherry (b), which is a touch neuron-specific reporter, and Panoh-1b NLS::GFP (c). White arrowheads mark a touch neuron. Dorsal is up. Scale bars are 5μm.
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pgen.1005285.g005: anoh-1 is required for the efficient removal of necrotic touch cells but not apoptotic cells.(A) Gene structure of two isoforms of anoh-1. The blue open boxes indicate the region deleted in the anoh-1(tm4762) allele. (B) Domain structure of ANOH-1b protein. Dark grey bars indicate 8 predicted transmembrane domains. Red color labels amino acids 1–18, which exists in ANOH-1b but missing from ANOH-1a. (C) The anoh-1(tm4762) mutant embryos are normal in the removal of somatic apoptotic cells. (D) The anoh-1(tm4762) mutant adults are normal in the removal of apoptotic germ cells. (E) The engulfment and degradation processes of embryonic apoptotic cells C1, C2, and C3 are normal in anoh-1(tm4762) mutant embryos, measured by time-lapse microscopy. (F) The anoh-1(tm4762) mutation perturbs the removal of necrotic cells, and the anoh-1b but not the anoh-1a form rescues the necrotic cell-removal defect when expressed in touch cells under the control of Pmec-7. For each sample, 6 groups of 10 worms at indicated stages were scored and displayed as mean. Error bars indicate standard deviations. *, 0.01<p<0.05 (student t-test). ***, p<0.001 (student t-test). ns, no significant difference, p>0.05 (student t-test). (G) The expression of anoh-1b in the necrotic cells, not engulfing cells, rescues anoh-1(tm4762) mutant phenotype. In anoh-1(tm4762); mec-4(e1611dm) mutant animals expressing indicated transgenes, the number of necrotic cells in the tail of each of the 6 groups of 10 newly hatched L1 larvae were scored. Data are presented as mean ± sd. (H) The GFP fusion forms of ANOH-1b but not ANOH-1a are localized to the plasma membrane. GFP (a, c, e, g) and DIC (b, d, f, h) images of the tails of mec-4(e1611dm) L1-stage larvae. Arrowheads in (a, b, e, f) label necrotic cells on which ANOH-1b is observed on the cell surface. Arrows in (c, d, g, h) label necrotic cells in which ANOH-1a is observed on the nuclear surface. All GFP reporters are expressed under the Pmec-7 promoter control. (I) The anoh-1b promoter is expressed in touch neurons. Shown here are epifluorescence (b-c) and the corresponding DIC (a) images of the tail region of a wild-type L2 larva co-expressing Pmec-7 mCherry (b), which is a touch neuron-specific reporter, and Panoh-1b NLS::GFP (c). White arrowheads mark a touch neuron. Dorsal is up. Scale bars are 5μm.
Mentions: Mammalian TMEM16F, a multispan transmembrane domain protein, is a Ca2+-activated phospholipid scramblase that triggers PS exposure in response to Ca2+-influx [36]. Given that the mec-4(dm)-induced touch neuron necrosis is mediated by Ca2+ influx [22,24], we examined whether ANOH-1, a close C. elegans homolog of TMEM16F (Figs 5B and S4), mediated PS exposure when necrosis occurred. We analyzed an anoh-1(tm4762) deletion allele (www.wormbase.org) for the removal of dying cells. The tm4762 allele carries a 202-bp deletion that results in a frameshift and a premature stop codon after amino acid 17 of the predicted ANOH-1b open reading frame and removes the start codon of the alternatively-spliced ANOH-1a open reading frame (S4 and S5B(b) Figs) (also see the next section), presumably generating a allele. In anoh-1(tm4762) mutant embryos, the numbers of apoptotic cells are the same as that displayed in wild-type embryos at 5 different embryonic developmental stages (Fig 5C). Furthermore, the dynamics of the engulfment and degradation processes of three individual apoptotic cells, C1, C2, and C3, are normal comparing to wild-type embryos (Fig 5E), using previously established protocols [48,55]. Similarly, the number of apoptotic germ cells are virtually the same in wild-type and anoh-1(tm4762) mutant adult hermaphrodites at four time points (Fig 5D). These results indicate that anoh-1, unlike ced-7, is not involved in the removal of apoptotic cells. In contrast, in anoh-1(tm4762); mec-4(e1611dm) double mutant animals, the removal of necrotic touch neurons is siginificantly delayed: at L1 and L2 stages, the mean numbers of persistent necrotic PLML/R in anoh-1(tm4762) background are approximately 1.6-fold and 1.5-fold of that in wild-type larvae, respectively, whereas at L3 and L4 stages, the mean numbers are not significantly different from wild-type animals (Fig 5F). These results strongly suggest that anoh-1 specifically contributes to efficient removal of necrotic but not apoptotic cells.

Bottom Line: Necrosis, a kind of cell death closely associated with pathogenesis and genetic programs, is distinct from apoptosis in both morphology and mechanism.Phosphatidylserine (PS) is an important apoptotic-cell surface signal that attracts engulfing cells.The extracellular domain of CED-1 associates with PS in vitro.

View Article: PubMed Central - PubMed

Affiliation: Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
Necrosis, a kind of cell death closely associated with pathogenesis and genetic programs, is distinct from apoptosis in both morphology and mechanism. Like apoptotic cells, necrotic cells are swiftly removed from animal bodies to prevent harmful inflammatory and autoimmune responses. In the nematode Caenorhabditis elegans, gain-of-function mutations in certain ion channel subunits result in the excitotoxic necrosis of six touch neurons and their subsequent engulfment and degradation inside engulfing cells. How necrotic cells are recognized by engulfing cells is unclear. Phosphatidylserine (PS) is an important apoptotic-cell surface signal that attracts engulfing cells. Here we observed PS exposure on the surface of necrotic touch neurons. In addition, the phagocytic receptor CED-1 clusters around necrotic cells and promotes their engulfment. The extracellular domain of CED-1 associates with PS in vitro. We further identified a necrotic cell-specific function of CED-7, a member of the ATP-binding cassette (ABC) transporter family, in promoting PS exposure. In addition to CED-7, anoctamin homolog-1 (ANOH-1), the C. elegans homolog of the mammalian Ca(2+)-dependent phospholipid scramblase TMEM16F, plays an independent role in promoting PS exposure on necrotic cells. The combined activities from CED-7 and ANOH-1 ensure efficient exposure of PS on necrotic cells to attract their phagocytes. In addition, CED-8, the C. elegans homolog of mammalian Xk-related protein 8 also makes a contribution to necrotic cell-removal at the first larval stage. Our work indicates that cells killed by different mechanisms (necrosis or apoptosis) expose a common "eat me" signal to attract their phagocytic receptor(s); furthermore, unlike what was previously believed, necrotic cells actively present PS on their outer surfaces through at least two distinct molecular mechanisms rather than leaking out PS passively.

No MeSH data available.


Related in: MedlinePlus