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Loss of the interferon-γ-inducible regulatory immunity-related GTPase (IRG), Irgm1, causes activation of effector IRG proteins on lysosomes, damaging lysosomal function and predicting the dramatic susceptibility of Irgm1-deficient mice to infection.

Maric-Biresev J, Hunn JP, Krut O, Helms JB, Martens S, Howard JC - BMC Biol. (2016)

Bottom Line: We show that the three regulatory IRG proteins (GMS sub-family), including Irgm1, each of which localizes to distinct sets of endocellular membranes, play an important role during the cellular response to IFN-γ, each protecting specific membranes from off-target activation of effector IRG proteins (GKS sub-family).In the absence of Irgm1, which is localized mainly at lysosomal and Golgi membranes, activated GKS proteins load onto lysosomes, and are associated with reduced lysosomal acidity and failure to process autophagosomes.The membrane targeting properties of the three GMS proteins to specific endocellular membranes prevent accumulation of activated GKS protein effectors on the corresponding membranes and thus enable GKS proteins to distinguish organellar cellular membranes from the membranes of pathogen vacuoles.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, University of Cologne, Cologne, Germany.

ABSTRACT

Background: The interferon-γ (IFN-γ)-inducible immunity-related GTPase (IRG), Irgm1, plays an essential role in restraining activation of the IRG pathogen resistance system. However, the loss of Irgm1 in mice also causes a dramatic but unexplained susceptibility phenotype upon infection with a variety of pathogens, including many not normally controlled by the IRG system. This phenotype is associated with lymphopenia, hemopoietic collapse, and death of the mouse.

Results: We show that the three regulatory IRG proteins (GMS sub-family), including Irgm1, each of which localizes to distinct sets of endocellular membranes, play an important role during the cellular response to IFN-γ, each protecting specific membranes from off-target activation of effector IRG proteins (GKS sub-family). In the absence of Irgm1, which is localized mainly at lysosomal and Golgi membranes, activated GKS proteins load onto lysosomes, and are associated with reduced lysosomal acidity and failure to process autophagosomes. Another GMS protein, Irgm3, is localized to endoplasmic reticulum (ER) membranes; in the Irgm3-deficient mouse, activated GKS proteins are found at the ER. The Irgm3-deficient mouse does not show the drastic phenotype of the Irgm1 mouse. In the Irgm1/Irgm3 double knock-out mouse, activated GKS proteins associate with lipid droplets, but not with lysosomes, and the Irgm1/Irgm3(-/-) does not have the generalized immunodeficiency phenotype expected from its Irgm1 deficiency.

Conclusions: The membrane targeting properties of the three GMS proteins to specific endocellular membranes prevent accumulation of activated GKS protein effectors on the corresponding membranes and thus enable GKS proteins to distinguish organellar cellular membranes from the membranes of pathogen vacuoles. Our data suggest that the generalized lymphomyeloid collapse that occurs in Irgm1(-/-) mice upon infection with a variety of pathogens may be due to lysosomal damage caused by off-target activation of GKS proteins on lysosomal membranes and consequent failure of autophagosomal processing.

No MeSH data available.


Related in: MedlinePlus

Lysosomal processing is impaired in IFN-γ-induced Irgm1−/− mouse embryonic fibroblasts (MEFs). aIrgm1−/− MEFs were transfected with EGFP-Irga6 and simultaneously induced with 200 U/mL IFN-γ. After 24 hours, non-fixed cells were incubated with 50 nM Lysotracker for 15 minutes and pictures of live cells were taken. Representative microscopic images of Irga6 and Lysotracker co-localization are shown. Arrows point at the Irga6 structures magnified at the end of each panel in the following array: upper left: Irga6, upper right: Lysotracker, lower left: merge, lower right: phase contrast. Scale bars represent 10 μM. b Quantification of 7a showing the number of Irga6 structures co-localizing with Lysotracker. 25 cells per sample were quantified and the means of three independent experiments ± standard deviation are shown. c Wild type (WT), Irgm1−/−, Irgm3−/−, and Irgm1/Irgm3−/− MEFs were induced with 200 U/mL IFN-γ for 27 hours, 200 nM μg/mL Bafilomycin A1 for 4 hours, or left untreated. After 24 hours, non-fixed cells were incubated with 10 μg/mL DQ-BSA, incubated for 3 hours, and images of live cells were taken. Representative microscopic images of DQ-BSA staining are shown. Scale bars represent 10 μM. d Quantification of Fig. 7c showing the average numbers of DQ-positive structures per cells; 20 cells per sample were evaluated and results of three independent experiments ± standard deviation are shown. Differences were tested for statistical significance using the Mann-Whitney test (NS P > 0.03)
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Fig7: Lysosomal processing is impaired in IFN-γ-induced Irgm1−/− mouse embryonic fibroblasts (MEFs). aIrgm1−/− MEFs were transfected with EGFP-Irga6 and simultaneously induced with 200 U/mL IFN-γ. After 24 hours, non-fixed cells were incubated with 50 nM Lysotracker for 15 minutes and pictures of live cells were taken. Representative microscopic images of Irga6 and Lysotracker co-localization are shown. Arrows point at the Irga6 structures magnified at the end of each panel in the following array: upper left: Irga6, upper right: Lysotracker, lower left: merge, lower right: phase contrast. Scale bars represent 10 μM. b Quantification of 7a showing the number of Irga6 structures co-localizing with Lysotracker. 25 cells per sample were quantified and the means of three independent experiments ± standard deviation are shown. c Wild type (WT), Irgm1−/−, Irgm3−/−, and Irgm1/Irgm3−/− MEFs were induced with 200 U/mL IFN-γ for 27 hours, 200 nM μg/mL Bafilomycin A1 for 4 hours, or left untreated. After 24 hours, non-fixed cells were incubated with 10 μg/mL DQ-BSA, incubated for 3 hours, and images of live cells were taken. Representative microscopic images of DQ-BSA staining are shown. Scale bars represent 10 μM. d Quantification of Fig. 7c showing the average numbers of DQ-positive structures per cells; 20 cells per sample were evaluated and results of three independent experiments ± standard deviation are shown. Differences were tested for statistical significance using the Mann-Whitney test (NS P > 0.03)

Mentions: Since lysosomes of IFN-γ-induced Irgm1−/− cells were apparently not able to process autophagosomes, we asked whether the general processing activity of these lysosomes is impaired. We first analyzed the pH status of Irga6-coated lysosomes with the pH sensitive lysosomal dye Lysotracker on live, non-fixed cells. Irgm1−/− MEFs were transfected with EGFP-Irga6 and simultaneously induced with IFN-γ (Fig. 7a). EGFP-Irga6 structures that co-localized with Lysotracker were quantified (Fig. 7b). Out of 11.7 Irga6 structures per cell, only 3.1 were Lysotracker-positive. We show above that EGFP-Irga6 structures co-localize completely with LAMP1 (Fig. 2e). Therefore, the data show that the pH of the majority of Irga6-coated lysosomes in IFN-γ-induced Irgm1−/− cells is raised, resulting in an impairment of autophagosome processing.Fig. 7


Loss of the interferon-γ-inducible regulatory immunity-related GTPase (IRG), Irgm1, causes activation of effector IRG proteins on lysosomes, damaging lysosomal function and predicting the dramatic susceptibility of Irgm1-deficient mice to infection.

Maric-Biresev J, Hunn JP, Krut O, Helms JB, Martens S, Howard JC - BMC Biol. (2016)

Lysosomal processing is impaired in IFN-γ-induced Irgm1−/− mouse embryonic fibroblasts (MEFs). aIrgm1−/− MEFs were transfected with EGFP-Irga6 and simultaneously induced with 200 U/mL IFN-γ. After 24 hours, non-fixed cells were incubated with 50 nM Lysotracker for 15 minutes and pictures of live cells were taken. Representative microscopic images of Irga6 and Lysotracker co-localization are shown. Arrows point at the Irga6 structures magnified at the end of each panel in the following array: upper left: Irga6, upper right: Lysotracker, lower left: merge, lower right: phase contrast. Scale bars represent 10 μM. b Quantification of 7a showing the number of Irga6 structures co-localizing with Lysotracker. 25 cells per sample were quantified and the means of three independent experiments ± standard deviation are shown. c Wild type (WT), Irgm1−/−, Irgm3−/−, and Irgm1/Irgm3−/− MEFs were induced with 200 U/mL IFN-γ for 27 hours, 200 nM μg/mL Bafilomycin A1 for 4 hours, or left untreated. After 24 hours, non-fixed cells were incubated with 10 μg/mL DQ-BSA, incubated for 3 hours, and images of live cells were taken. Representative microscopic images of DQ-BSA staining are shown. Scale bars represent 10 μM. d Quantification of Fig. 7c showing the average numbers of DQ-positive structures per cells; 20 cells per sample were evaluated and results of three independent experiments ± standard deviation are shown. Differences were tested for statistical significance using the Mann-Whitney test (NS P > 0.03)
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Fig7: Lysosomal processing is impaired in IFN-γ-induced Irgm1−/− mouse embryonic fibroblasts (MEFs). aIrgm1−/− MEFs were transfected with EGFP-Irga6 and simultaneously induced with 200 U/mL IFN-γ. After 24 hours, non-fixed cells were incubated with 50 nM Lysotracker for 15 minutes and pictures of live cells were taken. Representative microscopic images of Irga6 and Lysotracker co-localization are shown. Arrows point at the Irga6 structures magnified at the end of each panel in the following array: upper left: Irga6, upper right: Lysotracker, lower left: merge, lower right: phase contrast. Scale bars represent 10 μM. b Quantification of 7a showing the number of Irga6 structures co-localizing with Lysotracker. 25 cells per sample were quantified and the means of three independent experiments ± standard deviation are shown. c Wild type (WT), Irgm1−/−, Irgm3−/−, and Irgm1/Irgm3−/− MEFs were induced with 200 U/mL IFN-γ for 27 hours, 200 nM μg/mL Bafilomycin A1 for 4 hours, or left untreated. After 24 hours, non-fixed cells were incubated with 10 μg/mL DQ-BSA, incubated for 3 hours, and images of live cells were taken. Representative microscopic images of DQ-BSA staining are shown. Scale bars represent 10 μM. d Quantification of Fig. 7c showing the average numbers of DQ-positive structures per cells; 20 cells per sample were evaluated and results of three independent experiments ± standard deviation are shown. Differences were tested for statistical significance using the Mann-Whitney test (NS P > 0.03)
Mentions: Since lysosomes of IFN-γ-induced Irgm1−/− cells were apparently not able to process autophagosomes, we asked whether the general processing activity of these lysosomes is impaired. We first analyzed the pH status of Irga6-coated lysosomes with the pH sensitive lysosomal dye Lysotracker on live, non-fixed cells. Irgm1−/− MEFs were transfected with EGFP-Irga6 and simultaneously induced with IFN-γ (Fig. 7a). EGFP-Irga6 structures that co-localized with Lysotracker were quantified (Fig. 7b). Out of 11.7 Irga6 structures per cell, only 3.1 were Lysotracker-positive. We show above that EGFP-Irga6 structures co-localize completely with LAMP1 (Fig. 2e). Therefore, the data show that the pH of the majority of Irga6-coated lysosomes in IFN-γ-induced Irgm1−/− cells is raised, resulting in an impairment of autophagosome processing.Fig. 7

Bottom Line: We show that the three regulatory IRG proteins (GMS sub-family), including Irgm1, each of which localizes to distinct sets of endocellular membranes, play an important role during the cellular response to IFN-γ, each protecting specific membranes from off-target activation of effector IRG proteins (GKS sub-family).In the absence of Irgm1, which is localized mainly at lysosomal and Golgi membranes, activated GKS proteins load onto lysosomes, and are associated with reduced lysosomal acidity and failure to process autophagosomes.The membrane targeting properties of the three GMS proteins to specific endocellular membranes prevent accumulation of activated GKS protein effectors on the corresponding membranes and thus enable GKS proteins to distinguish organellar cellular membranes from the membranes of pathogen vacuoles.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, University of Cologne, Cologne, Germany.

ABSTRACT

Background: The interferon-γ (IFN-γ)-inducible immunity-related GTPase (IRG), Irgm1, plays an essential role in restraining activation of the IRG pathogen resistance system. However, the loss of Irgm1 in mice also causes a dramatic but unexplained susceptibility phenotype upon infection with a variety of pathogens, including many not normally controlled by the IRG system. This phenotype is associated with lymphopenia, hemopoietic collapse, and death of the mouse.

Results: We show that the three regulatory IRG proteins (GMS sub-family), including Irgm1, each of which localizes to distinct sets of endocellular membranes, play an important role during the cellular response to IFN-γ, each protecting specific membranes from off-target activation of effector IRG proteins (GKS sub-family). In the absence of Irgm1, which is localized mainly at lysosomal and Golgi membranes, activated GKS proteins load onto lysosomes, and are associated with reduced lysosomal acidity and failure to process autophagosomes. Another GMS protein, Irgm3, is localized to endoplasmic reticulum (ER) membranes; in the Irgm3-deficient mouse, activated GKS proteins are found at the ER. The Irgm3-deficient mouse does not show the drastic phenotype of the Irgm1 mouse. In the Irgm1/Irgm3 double knock-out mouse, activated GKS proteins associate with lipid droplets, but not with lysosomes, and the Irgm1/Irgm3(-/-) does not have the generalized immunodeficiency phenotype expected from its Irgm1 deficiency.

Conclusions: The membrane targeting properties of the three GMS proteins to specific endocellular membranes prevent accumulation of activated GKS protein effectors on the corresponding membranes and thus enable GKS proteins to distinguish organellar cellular membranes from the membranes of pathogen vacuoles. Our data suggest that the generalized lymphomyeloid collapse that occurs in Irgm1(-/-) mice upon infection with a variety of pathogens may be due to lysosomal damage caused by off-target activation of GKS proteins on lysosomal membranes and consequent failure of autophagosomal processing.

No MeSH data available.


Related in: MedlinePlus