RETRACTED ARTICLE: Molecular characterization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins (original) (raw)

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Acknowledgements

We thank the veterinary pathology core lab (VPCL) at St Jude for their work in processing of H&E and Gomori slides. We also thank C. Guy in the Department of Immunology for his help with confocal microscopy. We thank M. Yang for assistance with maintenance of the mouse colony. This work was supported by research grants from the National Institutes of Health, the Lupus Research Institute, and ALSAC.

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Author notes

  1. Jennifer Martinez
    Present address: Present address: Immunity, Inflammation, and Disease Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27703, USA.,

Authors and Affiliations

  1. Department of Immunology, St Jude Children’s Research Hospital, Memphis, Tennessee 38105, USA
    Jennifer Martinez, R. K. Subbarao Malireddi, Larissa Dias Cunha, Stephane Pelletier, Sebastien Gingras, Thirumala-Devi Kanneganti & Douglas R. Green
  2. Department of Pathology and Immunology, Washington University School of Medicine, St Louis, Missouri 63110, USA
    Qun Lu, Robert Orchard & Herbert W. Virgin
  3. Embryonic Stem Cell Laboratory, St Jude Children’s Research Hospital, Memphis, Tennessee 38105, USA
    Stephane Pelletier & Sebastien Gingras
  4. Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA
    Jun-Lin Guan
  5. St Jude Proteomics Facility, St Jude Children’s Research Hospital, Memphis, Tennessee 38105, USA
    Haiyan Tan & Junmin Peng
  6. Departments of Structural Biology and Developmental Neurobiology, St Jude Children’s Research Hospital, Memphis, Tennessee 38105, USA
    Junmin Peng

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  1. Jennifer Martinez
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  2. R. K. Subbarao Malireddi
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Contributions

J.M., T.-D.K., H.W.V. and D.R.G. designed the experiments; J.M. performed and analysed the experiments; R.K.S.M., Q.L., L.D.C., R.O., H.T. and J.P. performed and analysed specific experiments; S.P. and S.G. performed CRISPR/Cas9 technology; J.-L.G. contributed materials; and J.M., H.W.V. and D.R.G. wrote the manuscript.

Corresponding author

Correspondence toDouglas R. Green.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 5 LAP is a pathway distinct from canonical autophagy.

(a) RAW cells were fed inert beads or Pam3csk4–beads for 1 h. Immunofluorescent staining was performed for LC3B and analyzed by microscopy. Representative images are shown (above), and per cent of LC3 + phagosomes is calculated (below). Data are presented as mean ± s.d. (n = 50 cells per genotype pooled from two independent experiments, ∗∗p,0.001 using Student’s t_-test). (b) RAW cells were allowed to phagocytose inert beads or Pam3csk4–beads for 1 h. Phagosomes were purified using sucrose gradient as described in experimental procedures. Phagosome proteins were solubilized in SDS-PAGE and blotted with the indicated antibodies. The results presented are representative of three independent experiments. (c) RAW-GFP-LC3 cells were fed inert beads or Alexa Fluor 594-zymosan, and internalization and GFP-LC3 translocation were followed at 4-min intervals for 6 h. Representative images from three independent experiments are shown (top). Time (in minutes) is indicated above each panel. Time course of GFP-LC3 translocation to the inert bead- or zymosan-containing phagosome. Data are presented as mean ± s.d. (n = 75 cells per genotype pooled from three independent experiments, ∗∗_p, 0.001 using Student’s _t_-test). (d) RAW-GFP-LC3 cells were fed inert beads or Alexa Fluor 594-zymosan for 1 h. Whole cell lysates from pre-digitonin treated samples (right), and digitonin-treated samples (left) were solubilized in SDS-PAGE and blotted for LC3B. The results presented are representative of two independent experiments.

Supplementary Figure 6 Generation of the Rubicon-deficient mouse model using the CRISP/Cas9 method.

(a) Western blot analysis of organs from Rubicon+/+ and _Rubicon_−/− mice, aged 8 weeks. Representative images from three independent experiments are shown. (b) Relative mRNA level of Rubicon of organs from Rubicon+/+ and Rubicon_−/− mice (aged 8 weeks) was quantified by real-time PCR. Data normalized to actin. Primers were designed to cover a region upstream of the introduced stop codon (N terminus) and downstream of the introduced stop codon (C terminus). Data are presented as mean ± s.d. (n = 3 independent experiments, ∗∗_p < 0.001 using Student’s _t_-test; in each experiment, technical triplicate samples of 3 mice per genotype were assessed). (c) Expected and observed frequency of offspring from Rubicon +/− × Rubicon +/− crosses of mice. All genotypes were observed at Mendelian ratios. (d–f) FACS analysis of immune cell composition in the spleen (d), lymph node (e), and thymus (f) of Rubicon+/+, Rubicon +/−, and _Rubicon_−/− mice, aged 12 weeks. Data are presented as mean ± s.d. (n = 3 independent experiments; in each experiment, technical triplicate samples of 100,000 cells each per genotype were assessed). (g) Rubicon+/+ GFP-LC3+ and _Rubicon_−/− GFP-LC3+ bone marrow-derived macrophages were fed Alexa Fluor 594-zymosan, and the per cent of phagocytosis (% Phagocytosis, left) and the extent of phagocytosis (MFI of Phagocytosed Zymosan, right) was quantified by flow cytometry. Data are presented as mean ± s.d. (n = 3 independent experiments; in each experiment, technical triplicate samples of 50,000 cells each per genotype were assessed). (h) RAW cells were transfected with Scrambled (Scr.) or RAB7 siRNA oligonucleotides. After 48 h of transfection, cells were fed Pam3csk4–beads for 1 h. Phagosomes were purified using sucrose gradient as described in experimental procedures. Phagosome proteins were solubilized in SDS-PAGE and blotted with the indicated antibodies. Representative images from two independent experiments are shown.

Supplementary Figure 7 LAP occurs independently of the pre-initiation complex.

(a,b) Bone marrow-derived macrophages from LysM-Cre−FIP200 flox/flox GFP-LC3+ and LysM-Cre+ FIP200 flox/flox GFP-LC3+ mice were left untreated (NS) or were cultured with 200 nM rapamycin (Rapa., a), starvation conditions (S, b), Inert beads (I, b), or Alexa Fluor 594-zymosan (Zymosan or Z, a,b). (c) Bone marrow-derived macrophages from LysM-Cre−FIP200 flox/flox GFP-LC3+ and LysM-Cre+ FIP200 flox/flox GFP-LC3+ mice were allowed to phagocytose latex beads coated with Pam3csk4 for 1 h. Phagosomes were purified using sucrose gradient as described in experimental procedures. Phagosome proteins were solubilized in SDS-PAGE and blotted with the indicated antibodies. The results presented are representative of three independent experiments. (d) Bone marrow-derived macrophages from ULK1+/+ and ULK1_−/− mice were allowed to phagocytose latex beads coated with Pam3csk4 for 1 h. Phagosomes were purified using sucrose gradient as described in experimental procedures. Phagosome proteins were solubilized in SDS-PAGE and blotted with the indicated antibodies. The results presented are representative of three independent experiments. (e–h) RAW-GFP-LC3 cells were transfected with Scrambled, Ambra1, or WIPI2 siRNA oligonucleotides. After 48 h of transfection, cells were left untreated (NS) or were cultured with 200 nM rapamycin (Rapa., e), starvation conditions (S, f), Inert beads (I, f), or Alexa Fluor 594-zymosan (Zymosan or Z, e,f). GFP-LC3 puncta was assessed at 18 h, and translocation of GFP-LC3 to the LAPosome was assessed at 1 h by confocal microscopy (e) and flow cytometry (f). Whole cell lysates were analyzed for deletion efficiency of Ambra1- (g) or WIPI2-siRNA-treated (h) RAW-GFP-LC3 cells. For a,c,d, and e, representative images from three independent experiments are shown. For b and f, Data are presented as mean ± s.d. (n = 3 independent experiments, ∗_p < 0.05,∗∗p < 0.001 using Student’s _t_-test; for each experiment, technical triplicate samples of 50,000 cells each per genotype were assessed by FACS).

Supplementary Figure 8 LAP uses a UVRAG-containing Class III PI3K Complex.

(a,b) Whole cell lysates were analyzed for deletion efficiency of LysM-Cre−Beclin1 flox/flox and LysM-Cre+ Beclin1 flox/flox macrophages (a). Macrophages were fed Alexa Fluor 594-zymosan, and the per cent of phagocytosis (% Phagocytosis, left) and the extent of phagocytosis (MFI of Phagocytosed Zymosan, right) were quantified by flow cytometry (b). (c,d) Whole cell lysates were analyzed for deletion efficiency of LysM-Cre−VPS34 flox/flox and LysM-Cre+ VPS34 flox/flox macrophages (c). Macrophages were fed Alexa Fluor 594-zymosan, and the per cent of phagocytosis (% Phagocytosis, left) and the extent of phagocytosis (MFI of Phagocytosed Zymosan, right) were quantified by flow cytometry (d). (e,f) Whole cell lysates were analyzed for deletion efficiency of LysM-Cre−ATG14 flox/flox and LysM-Cre+ ATG14 flox/flox macrophages (e). Macrophages were fed Alexa Fluor 594-zymosan, and the per cent of phagocytosis (% Phagocytosis, left) and the extent of phagocytosis (MFI of Phagocytosed Zymosan, right) were quantified by flow cytometry (f). (g,h) Whole cell lysates were analyzed for deletion efficiency of Scrambled siRNA- and UVRAG siRNA-treated RAW-GFP-LC3 cells (g). Macrophages were fed Alexa Fluor 594-zymosan, and the per cent of phagocytosis (% Phagocytosis, left) and the extent of phagocytosis (MFI of Phagocytosed Zymosan, right) were quantified by flow cytometry (h). (i,j) Bone marrow-derived macrophages from wild-type, _Rubicon_−/−, or _NOX2_−/− mice were fed inert beads or Pam3csk4–beads for 1 h. Immunofluorescent staining was performed for the proteins indicated above each panel and analyzed by microscopy. Representative images (i) and signal intensity profiles (j) for Beclin1, UVRAG, and VPS34 across phagocytosed beads are quantified. Data (intensity measurements across beads) are presented as mean ± s.d. (n = 50 cells per genotype per stain pooled from two independent experiments). For b,d,f and h, data are presented as mean ± s.d. (n = 3 independent experiments; technical triplicate samples of 50,000 cells each per genotype per experiment were assessed).

Supplementary Figure 9 NOX2 is downstream of the Class III PI3K Complex and required for LAP.

(a) NOX2+/+ GFP-LC3+ and _NOX2_−/− GFP-LC3+ bone marrow-derived macrophages were fed Alexa Fluor 594-zymosan, and the per cent of phagocytosis (% Phagocytosis, left) and the extent of phagocytosis (MFI of Phagocytosed Zymosan, right) were quantified by flow cytometry. (b) Bone marrow-derived macrophages from genetic knockout strains were fed inert beads or Alexa Fluor 594-zymosan and analyzed for ROS production at 1 h by flow cytometry using dihydroethidium (DHE). Filled grey histogram represents inert bead. Representative plots from three independent experiments are shown. (c) NOX2+/+ and _NOX2_−/− bone marrow-derived macrophages were fed Pam3csk4–beads (30 min). mVPS34 was immunoprecipitated from the purified LAPosomes and used in the Class III PI3K Activity assay. Data are presented as pM of PI(3)P,. (d) Bone marrow-derived macrophages from WT, LysM-Cre+ Beclin1 flox/flox, and _ULK1_−/− mice were allowed to phagocytose latex beads coated with Pam3csk4 for 1 h. Phagosomes were purified using sucrose gradient as described in experimental procedures. Phagosome proteins were solubilized in SDS-PAGE and blotted with the indicated antibodies. The results presented are representative of three independent experiments. For a and c, data are presented as mean ± s.d. (n = 3 independent experiments; for each experiment, technical triplicate samples of 50,000 cells each per genotype were assessed by FACS).

Supplementary Figure 10 The activity of Rubicon and NOX2 are required for the translocation of downstream conjugation systems to the LAPosome.

(a) Bone marrow-derived macrophages from wild-type, _Rubicon_−/−, or _NOX2_−/− mice were fed inert beads or Pam3csk4–beads for 1 h. Immunofluorescent staining was performed for the proteins indicated above each panel and analyzed by microscopy. Representative images from two independent experiments are shown. (b) Model of proposed activity of Rubicon in the crosstalk of Class III PI3K and NOX2 complexes. Recruitment of the Rubicon- and UVRAG-containing Class III PI3K complex allows for sustained VPS34 activity at the LAPosome, resulting in significant PI(3)P deposition on the LAPosome membrane. This PI(3)P allows for the recruitment of autophagic downstream conjugation systems to the LAPosome and stabilizes the NOX2 complex via its binding to p-p40PHOX. Rubicon itself also stabilizes the NOX2 complex, promoting optimal ROS production. Rubicon mediates the crosstalk between the Class III PI3K and NOX2 complexes, resulting in lipid oxidation and PI(3)P generation, both required for conjugation of LC3 to the lipids of the LAPosome.

Supplementary Figure 11 PI(3)P and ROS are both required for LAP.

(a) Bone marrow-derived macrophages from genetic knockout strains were fed Pam3csk4–beads for 30 min. Immunofluorescent staining was performed for oxidized LDL (OxLDL) and PI(3)P and analyzed by confocal microscopy. Representative images (left) and signal intensity profiles for OxLDL (middle) and PI(3)P (left) across phagocytosed beads are quantified. Data (intensity measurements across beads) are presented as mean ± s.d. (n = 60 cells per genotype per stain pooled from two independent experiments). (b) RAW-GFP-LC3 cells were fed inert beads or zymosan, in the presence or absence of tert_-butyl hydroperoxide (TBHP, 100 μM, 50 μM), Tiron (1 mM, 0.5 mM), or 3-MA (25 mM, 5 mM). Cells were analyzed for ROS production at 1 h by flow cytometry using dihydroethidium (DHE). (c) RAW-GFP-LC3 cells were fed inert beads or Pam3csk4–beads, in the presence or absence of Tiron (1 mM, 0.5 mM) or 3-MA (25 mM, 5 mM). After 1 h of phagocytosis, phagosomes were purified using sucrose gradient as described in experimental procedures. Phagosome proteins were solubilized in SDS-PAGE and blotted with the indicated antibodies. The results presented are representative of four independent experiments. (d) RAW cells were fed inert beads, zymosan, or HRP-coupled beads. Cells were analyzed for ROS production at 1 h by flow cytometry using dihydroethidium (DHE). (e) RAW cells were fed inert beads (I), Pam3csk4–beads (P), Catalase-beads (C), or Pam3csk4 + Catalase –beads (PC). Cells were analyzed for ROS production at 1 h by flow cytometry using dihydroethidium (DHE). For b,d, and e, data are presented as mean ± s.d. (n = 3 independent experiments, ∗∗_p < 0.001 using Student’s _t_-test). For each experiment, technical triplicate samples of 50,000 cells each per genotype were assessed by FACS.

Supplementary Figure 12 Clearance of Aspergillus fumigatus requires LAP.

(a,b) Bone marrow-derived macrophages from GFP-LC3 + genetic knockout strains were fed inert beads or A. fumigatus_-dsRed and analyzed by flow cytometry for GFP-LC3 translocation to the LAPosome (a) and phagocytosis (b). The per cent of phagocytosis (% Phagocytosis, left) and the extent of phagocytosis (MFI of Phagocytosed A. fumigatus, right) were quantified by flow cytometry. Data are presented as the average of two independent experiments. (c) Bone marrow-derived macrophages from different genetic knockout strains were fed live A. fumigatus at an MOI of 1, and percentage of killing was calculated as [total cfu at time-point ∖ cfu at infection]. (d,e) Mice of different genetic knockout strains (5 mice/strain) were infected intranasally with live A. fumigatus conidia. Percent weight loss was monitored at days 3 and 7 post-infection (d). Serum was collected at day 7 post-infection and analyzed for cytokines via Luminex technology (e). In c,d, and e, data are presented as mean ± s.d. (n = 3 independent experiments, ∗_p < 0.05,∗∗p < 0.001 using Student’s _t_-test. In c, technical triplicate samples of each genotype per timepoint per experiment were assessed. In d and e, technical triplicate samples from 5 mice per genotype per timepoint per experiment were assessed.

Supplementary Table 1 Proteins uniquely associated with the LAPosome.

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Supplementary Table 2 Off-target sequences of Rubicon-E5F2 target site.

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Supplementary Table 3 Sequencing and targeting primers for Rubicon-E2F2 target site.

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Supplementary Table 4 Real-time PCR primers.

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Martinez, J., Malireddi, R., Lu, Q. et al. RETRACTED ARTICLE: Molecular characterization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins.Nat Cell Biol 17, 893–906 (2015). https://doi.org/10.1038/ncb3192

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