ULEMENTARY INORMATION - static-content.springer.com10.1038/ncb3192... · of the introduced stop codon (C terminus). Data are presented as mean ± SD (n=3 independent experiments,
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S U P P L E M E N TA RY I N F O R M AT I O N
WWW.NATURE.COM/NATURECELLBIOLOGY 1
DOI: 10.1038/ncb3192
Beclin1
VPS34
UVRAG
ATG14
Rubicon
p22 PHOX
p-p40PHOX
ATG7
UNC93B
LC3-II
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Supplementary Figure 1: LAP is a pathway distinct from canonical autophagy.
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Supplementary Figure 1 LAP is a pathway distinct from canonical autophagy. (a) RAW cells were fed inert beads or Pam3csk4-beads for 1 hour. Immunofluorescent staining was performed for LC3B and analyzed by microscopy. Representative images are shown (above), and percent of LC3+ phagosomes is calculated (below). Data are presented as mean ± SD (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 hr. 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 ± SD (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 hour. 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 2 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 ± SD (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+/- x 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 ± SD (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 percent of phagocytosis (% Phagocytosis, left) and the extent of phagocytosis (MFI of Phagocytosed Zymosan, right) was quantified by flow cytometry. Data are presented as mean ± SD (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 hours of transfection, cells were fed Pam3csk4-beads for 1 hr. 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 3: LAP occurs independently of the pre-initiation complex.
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Supplementary Figure 3 LAP occurs independently of the pre-initiation complex. (a-b) Bone marrow-derived macrophages from LysM-Cre- FIP200flox/flox GFP-LC3+ and LysM-Cre+ FIP200flox/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- FIP200flox/flox GFP-LC3+ and LysM-Cre+ FIP200flox/flox GFP-LC3+ mice were allowed to phagocytose latex beads coated with Pam3csk4 for 1 hr. 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 hr. 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 hours 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 ± SD (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 4 LAP utilizes a UVRAG-containing Class III PI3K Complex. (a-b) Whole cell lysates were analyzed for deletion efficiency of LysM-Cre- Beclin1flox/flox and LysM-Cre+ Beclin1flox/flox macrophages (a). Macrophages were fed Alexa Fluor 594-zymosan, and the percent 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- VPS34flox/flox and LysM-Cre+ VPS34flox/flox macrophages (c). Macrophages were fed Alexa Fluor 594-zymosan, and the percent 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- ATG14flox/flox and LysM-Cre+ ATG14flox/flox macrophages (e). Macrophages were fed Alexa Fluor 594-zymosan, and the percent 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 percent 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 hour. 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 ± SD (n=50 cells per genotype per stain pooled from two independent experiments). For (b), (d), (f) and (h), data are presented as mean ± SD (n=3 independent experiments; technical triplicate samples of 50,000 cells each per genotype per experiment were assessed).
Supplementary Figure 5: NOX2 is downstream of the Class III PI3K complex and required for LAP
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Supplementary Figure 5 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 percent 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 minutes). 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+ Beclin1flox/flox, and ULK1-
/- mice were allowed to phagocytose latex beads coated with Pam3csk4 for 1 hr. 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 ± SD (n=3 independent experiments; for each experiment, technical triplicate samples of 50,000 cells each per genotype were assessed by FACS).
Supplementary Figure 6: The activity of Rubicon and NOX2 are required for translocation of the downstream conjugation systems to the LAPosome.
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Supplementary Figure 6 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 hour. 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 7 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 minutes. 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 ± SD (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 hour 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 ± SD (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 8 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 percent 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 ± SD (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 Figure 9 Western blot scans. Phagosome purification samples were prepared as described in Methods. The entire phagosome purification was run on 1 – 2 SDS-PAGE gels due to the relatively lower protein yield compared to whole-cell lysate samples. Membranes were sectioned according to the molecular weight marker, and proteins residing within that range of molecular weights were probed with antibodies listed above. When necessary, membranes were stripped with Restore PLUS
Western Blot Stripping Buffer (Life Technologies), re-blocked in 1X TBST with 5% w/v nonfat dry milk, and probed with fresh antibodies. Images were captured with an Amersham Imager 600 and saved as tiff files. The western images shown are the specific sections that we probed to allow for the analysis of multiple proteins from a limited sample volume. All the original unedited images from key experiments are included in this supplementary figure and are representative of reproducible, independent experiments.
*MM, Number of mismatches in the 20mer target sequence; Chr, chromosome; Start and End, begin and end of the coordinates of the sites (NCBI37/mm9 mouse assembly).
Supplementary Table 1 Proteins uniquely associated with the LAPosome. Proteins associated with the LAPosome with a log2 SILAC ratio greater than 0.7 to the inert bead phagosome. The protein name, description, Log2 ratios for both IgG-bead/inert bead and Pam3csk4-bead/inert beads, and the average Log2 ratio are shown.
*MM, Number of mismatches in the 20mer target sequence; Chr, chromosome; Start and End, begin and end of the coordinates of the sites (NCBI37/mm9 mouse assembly).
Supplementary Table 2 Off-target sequences of Rubicon-E5F2 target site