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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

Other GKS proteins co-localize with lysosomes in Irgm1−/− cells. a Wild type (WT), Irgm1−/−, Irgm3−/−, and Irgm1/Irgm3−/− mouse embryonic fibroblasts (MEFs) were induced with 200 U/mL IFN-γ for 24 hours. Cells were fixed and stained with anti-Irgb6 antiserum (141/3) and anti-LAMP1 antibody. Representative microscopic images of Irgb6 and lysosome co-localization are shown. Arrows point at the Irgb6 structures magnified at the end of each panel in the following array: upper left: Irgb6, upper right: LAMP1, lower left: merge, lower right: phase contrast. Scale bars represent 10 μM. b Quantification of 3a, S3A and S3B, showing percent of Irgb6, Irgb10, and Irgd structures co-localizing with LAMP1; 50 cells per sample were quantified and results of two independent experiments are shown. cIrgm1−/− MEFs were induced with 200 U/mL IFN-γ for 24 hours, fixed and stained for Irga6 (10D7), Irgb6 (141/3) and LAMP1; Irgd, Irga6 (10D7) and LAMP1; Irga6 (10D7), Irgb10 and LAMP1; or Irgb6 (A20), Irgb10 and LAMP1. Representative microscopic images of GKS structures and lysosome co-localization are shown. Arrows point at the GKS structures which are magnified at the end of each panel in the following array: upper left: first GKS protein, upper right: second GKS protein, lower left: LAMP1, lower right: merge. Scale bar represents 10 μM
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Fig3: Other GKS proteins co-localize with lysosomes in Irgm1−/− cells. a Wild type (WT), Irgm1−/−, Irgm3−/−, and Irgm1/Irgm3−/− mouse embryonic fibroblasts (MEFs) were induced with 200 U/mL IFN-γ for 24 hours. Cells were fixed and stained with anti-Irgb6 antiserum (141/3) and anti-LAMP1 antibody. Representative microscopic images of Irgb6 and lysosome co-localization are shown. Arrows point at the Irgb6 structures magnified at the end of each panel in the following array: upper left: Irgb6, upper right: LAMP1, lower left: merge, lower right: phase contrast. Scale bars represent 10 μM. b Quantification of 3a, S3A and S3B, showing percent of Irgb6, Irgb10, and Irgd structures co-localizing with LAMP1; 50 cells per sample were quantified and results of two independent experiments are shown. cIrgm1−/− MEFs were induced with 200 U/mL IFN-γ for 24 hours, fixed and stained for Irga6 (10D7), Irgb6 (141/3) and LAMP1; Irgd, Irga6 (10D7) and LAMP1; Irga6 (10D7), Irgb10 and LAMP1; or Irgb6 (A20), Irgb10 and LAMP1. Representative microscopic images of GKS structures and lysosome co-localization are shown. Arrows point at the GKS structures which are magnified at the end of each panel in the following array: upper left: first GKS protein, upper right: second GKS protein, lower left: LAMP1, lower right: merge. Scale bar represents 10 μM

Mentions: To investigate whether other GKS proteins also co-localize to LAMP1 in the absence of Irgm1, WT MEFs, Irgm1−/− MEFs, Irgm3−/− MEFs, and Irgm1/Irgm3−/− MEFs were induced with IFN-γ and stained for the GKS proteins Irgb6, Irgb10, and Irgd and for LAMP1 (Fig. 3a, Additional file 3: Figure S3). More than 90 % of all GKS protein accumulations tested co-localized with LAMP1 in Irgm1−/− cells and less than 5 % did so in Irgm3−/− and Irgm1/Irgm3−/− cells (Fig. 3b).Fig. 3


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)

Other GKS proteins co-localize with lysosomes in Irgm1−/− cells. a Wild type (WT), Irgm1−/−, Irgm3−/−, and Irgm1/Irgm3−/− mouse embryonic fibroblasts (MEFs) were induced with 200 U/mL IFN-γ for 24 hours. Cells were fixed and stained with anti-Irgb6 antiserum (141/3) and anti-LAMP1 antibody. Representative microscopic images of Irgb6 and lysosome co-localization are shown. Arrows point at the Irgb6 structures magnified at the end of each panel in the following array: upper left: Irgb6, upper right: LAMP1, lower left: merge, lower right: phase contrast. Scale bars represent 10 μM. b Quantification of 3a, S3A and S3B, showing percent of Irgb6, Irgb10, and Irgd structures co-localizing with LAMP1; 50 cells per sample were quantified and results of two independent experiments are shown. cIrgm1−/− MEFs were induced with 200 U/mL IFN-γ for 24 hours, fixed and stained for Irga6 (10D7), Irgb6 (141/3) and LAMP1; Irgd, Irga6 (10D7) and LAMP1; Irga6 (10D7), Irgb10 and LAMP1; or Irgb6 (A20), Irgb10 and LAMP1. Representative microscopic images of GKS structures and lysosome co-localization are shown. Arrows point at the GKS structures which are magnified at the end of each panel in the following array: upper left: first GKS protein, upper right: second GKS protein, lower left: LAMP1, lower right: merge. Scale bar represents 10 μM
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Fig3: Other GKS proteins co-localize with lysosomes in Irgm1−/− cells. a Wild type (WT), Irgm1−/−, Irgm3−/−, and Irgm1/Irgm3−/− mouse embryonic fibroblasts (MEFs) were induced with 200 U/mL IFN-γ for 24 hours. Cells were fixed and stained with anti-Irgb6 antiserum (141/3) and anti-LAMP1 antibody. Representative microscopic images of Irgb6 and lysosome co-localization are shown. Arrows point at the Irgb6 structures magnified at the end of each panel in the following array: upper left: Irgb6, upper right: LAMP1, lower left: merge, lower right: phase contrast. Scale bars represent 10 μM. b Quantification of 3a, S3A and S3B, showing percent of Irgb6, Irgb10, and Irgd structures co-localizing with LAMP1; 50 cells per sample were quantified and results of two independent experiments are shown. cIrgm1−/− MEFs were induced with 200 U/mL IFN-γ for 24 hours, fixed and stained for Irga6 (10D7), Irgb6 (141/3) and LAMP1; Irgd, Irga6 (10D7) and LAMP1; Irga6 (10D7), Irgb10 and LAMP1; or Irgb6 (A20), Irgb10 and LAMP1. Representative microscopic images of GKS structures and lysosome co-localization are shown. Arrows point at the GKS structures which are magnified at the end of each panel in the following array: upper left: first GKS protein, upper right: second GKS protein, lower left: LAMP1, lower right: merge. Scale bar represents 10 μM
Mentions: To investigate whether other GKS proteins also co-localize to LAMP1 in the absence of Irgm1, WT MEFs, Irgm1−/− MEFs, Irgm3−/− MEFs, and Irgm1/Irgm3−/− MEFs were induced with IFN-γ and stained for the GKS proteins Irgb6, Irgb10, and Irgd and for LAMP1 (Fig. 3a, Additional file 3: Figure S3). More than 90 % of all GKS protein accumulations tested co-localized with LAMP1 in Irgm1−/− cells and less than 5 % did so in Irgm3−/− and Irgm1/Irgm3−/− cells (Fig. 3b).Fig. 3

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