Fig 1.
TBK1 activation is membrane damage dependent.
(A) U2OS cells were infected with Ad WT (left panel), TS1 (middle panel) and M1 (right panel) and analyzed by western blot at different times pi (from 30 min to 3 h) using antibodies against S172 phosphorylated TBK1 (pTBK1), total TBK1 (TBK1) and actin as a loading control. (B-C) Quantification of TBK1 (B) or pTBK1 (C) signal from western blots upon Ad WT infection over time normalized to non infected (NI) conditions. (D-E) Quantification of TBK1 (D) or pTBK1 (E) signal from western blots upon infection with Ad WT (black), TS1 (grey) or M1 (red) at 30 min pi. Data shown are normalized to NI conditions. All data of B to E are the mean ± SD of 3 independent experiments. P values are based on Ordinary ONE-WAY ANOVA analysis and Dunnett’s multiple comparison.
Fig 2.
Phosphorylated TBK1 recruitment to the site of membrane damage.
U2OS cells were infected with fluorescently labeled (cyan) Ad WT, TS1 or M1 and analyzed by immunofluorescence at different time pi (from 20 min to 120 min) using anti protein VI (green) and anti pTBK1 (magenta) antibodies. (A) Representative confocal images at 20 min pi are presented. An enlarged inset section is shown on the right indicating VI and pTBK1 double colocalizations or VI, pTBK1 and Ad triple colocalizations (arrows). (B) Quantification of pTBK1 foci from A (n>30). (C) Proportion of VI colocalization with pTBK1 over time of infection (n>30). (D) Same analysis as in C using M1 infected cells. (E-F) Quantification of pTBK1 (E) and VI (F) dots at 40 min pi for Ad WT (black), and M1 (red) (n>30). (G) Proportion of Ad capsid colocalization with both VI and pTBK1 for Ad WT (black), TS1 (grey) and M1(red) at 20 min pi (n>30). P values are based on Ordinary ONE-WAY ANOVA analysis and Dunnett’s multiple comparison test for B, C, D, G and unpaired t-test for E and F.
Fig 3.
TBK1 activation involves Gal8 but not Gal3.
(A) U2OS cells were transfected with a control siRNA (siCTRL) or siRNA targeting either Gal3 (siGal3) (top panel) or Gal8 (siGal8) (bottom panel) and analyzed by western blot using anti Gal3 and Gal8 antibodies, respectively. (B) U2OS control or depleted for Gal3 (green) or Gal8 (orange) cells were infected for 30 min with Ad WT and analyzed by western as in Fig 1A. Levels of TBK1 (C) and pTBK1 (D) were quantified for 3 independent experiments and normalized as in Fig 1B and Fig 1C, respectively. (E) U2OS and gal3 KO cells were analyzed by western blot using anti Gal3 antibody. (F) U2OS and gal3 KO cells were infected with Ad WT for 30 min and analyzed by immunofluorescence using anti pTBK1 antibody (magenta). A representative confocal image is shown. (G) Quantification of pTBK1 foci in U2OS control or gal3 KO cells from F (n>30). P values are based on Ordinary ONE-WAY ANOVA analysis and Dunnett’s multiple comparison test for C and D and unpaired t-test for G.
Fig 4.
TBK1 activation and recruitment requires Gal8.
(A) U2OS control or gal8 KO cells were analyzed by western blot (top panel) and immunofluorescence (bottom panel) using anti Gal8 antibodies. (B) Control U2OS or gal8 KO were infected with 50 pp/cell of Ad WT (black) or M1 (red), both expressing GFP. Percentage of GFP expressing cells was determined 24 h pi by flow cytometry. Data for Ad M1 were normalized to Ad WT. (C) Quantification of VI dots at 30 min pi for Ad WT (black), and M1 (red) in U2OS and gal8 KO cells (n>30). (D) U2OS and gal8 KO cells were infected with Ad WT for 30 min and analyzed by western blot as in Fig 1A. Levels of TBK1 (E) and pTBK1 (F) were quantified for 3 independent experiments and normalized as in Fig 1B and 1C, respectively. (G) U2OS control or gal8 KO cells were infected with fluorescently labeled (cyan) Ad WT and analyzed at 30 min pi by immunofluorescence using anti protein VI (green) and anti pTBK1 (magenta) antibodies. Representative confocal images at 30 min pi are shown. An enlarged inset is shown to the right of each panel. White arrows indicate colocalizations between VI and pTBK1. (H) Quantification of pTBK1 foci in U2OS control or gal8 KO cells (n>30). (I) Proportion of VI colocalization with pTBK1 at 30 min pi (n>30). P values are based on Ordinary ONE-WAY ANOVA analysis and Dunnett’s multiple comparison test for B, C, E, F and unpaired t-test for H and I.
Fig 5.
Conventional autophagic receptors are not involved in TBK1 recruitment or activation upon Ad infection.
(A-B) U2OS control or gal8 KO cells were infected with Ad WT and analyzed at 30 min pi by immunofluorescence using anti protein VI and anti NDP52 antibodies. Quantifications of NDP52 dots (A) and proportion of VI colocalization with NDP52 (B) are presented (n>30). (C) U2OS control and ndp52 KO cells are analyzed by western blot (top panel) and immunofluorescence (bottom panel) using anti NDP52 antibody. (D-E) U2OS control or ndp52 KO cells were infected with Ad WT and analyzed at 30 min pi by immunofluorescence using anti protein VI and anti pTBK1 antibodies. Quantification of pTBK1 foci (D) and proportion of VI colocalization with pTBK1(E) are shown (n>30). (F) U2OS ndp52 KO cells were transfected with siCTRL or siRNA targeting Tax1BP1 (siTax1BP1) and analyzed by western blot using antibodies against Tax1BP1 and actin. (G and H) U2OS ndp52 KO control or Tax1BP1 depleted cells were infected with Ad WT and analyzed at 30 min pi by immunofluorescence using anti protein VI and anti pTBK1 antibodies. Quantification of pTBK1 foci (G) and proportion of VI colocalization with pTBK1 (H) are shown (n>30). (I) U2OS control and ndp52 KO cells were transfected with siCTRL or sip62, and then infected with Ad WT for 30 min. Cells were analyzed by western blot as in Fig 1A (left panel) and with anti NDP52, p62 and actin antibodies (right panel). P values are based on unpaired t-test.
Fig 6.
TBK1 kinase activity promotes autophagic degradation of Ad M1.
(A) U2OS cells were pre-treated with vehicle (top panel) or with 5 μM of MRT67-307 (bottom panel) for 3 h and transfected with poly(I:C) for 3 h (right panel). Cells were analyzed by immunofluorescence using antibodies against IRF3 (yellow). Representative confocal images are shown and the nucleus is marked with a white line. (B) U2OS cells were pre-treated for 3 h with vehicle (control) or different concentrations of MRT67-307 and infected with 50 pp/cell of Ad WT (black) or M1 (red), both expressing GFP. Percentage of GFP expressing cells was determined 24 h pi by flow cytometry and normalized to Ad WT. (C) U2OS control or tbk1 KO cells were analyzed by western blot using anti TBK1 antibody. (D) tbk1 KO cells were transfected with plasmid coding for GFP-tagged TBK1 WT or mutant (TBK1K38M or TBK1S172A) and analyzed by western blot using antibodies against TBK1. (E) tbk1 KO cells were transfected by GFP-tagged TBK1 plasmids followed by poly(I:C) transfection as in D and analyzed by immunofluorescence using IRF3 antibody (yellow). The nucleus is marked with a white line. (F) U2OS control or tbk1 KO cells were infected with 50 pp/cell of WT (black) or M1 (red), both expressing GFP. The percentage of GFP expressing cells was determined 24 h pi by flow cytometry and normalized to Ad WT. (G) tbk1 KO cells were transfected with control or GFP-tagged TBK1 plasmids and infected with 10 pp/cell of mCherry Ad WT or M1. The percentage of GFP and mCherry expressing cells was determined by flow cytometry and normalized to Ad WT. P values are based on Ordinary ONE-WAY ANOVA analysis and Dunnett’s multiple comparison test.
Fig 7.
TBK1 is not involved in autophagic receptor recruitment to Ad penetration site.
(A) U2OS control or tbk1 KO cells were infected with fluorescently labeled Ad WT (cyan) and analyzed by immunofluorescence at 30 min pi using anti protein VI (green) and anti NDP52 (red) antibodies. Representative confocal images are shown. An enlarged inset is shown to the right of each panel. White arrows indicate VI and NDP52 colocalizations. (B) U2OS control or tbk1 KO cells were infected with fluorescently labeled Ad WT (cyan) and analyzed by immunofluorescence at 30 min pi using anti protein VI (green) and anti p62 (red) antibodies. Representative confocal images are shown with enlarged insets to the right of each panel. White arrows indicate VI and p62 colocalizations. (C) Proportion of VI colocalization with NDP52 (n>30). (D) Proportion of VI colocalization with p62 (n>30). (E) U2OS control or tbk1 KO cells were infected with Ad WT and analyzed by immunofluorescence at 30 min pi using anti protein VI (green) and anti LC3 (red) antibodies. Representative confocal images and enlarged insets to the right on each panel are shown. (F) Quantification of LC3 dots from E (n>30). (G) Proportion of VI colocalization with LC3 (n>30). (H) U2OS control or tbk1 KO cells were pre-treated for 3 h with Bafilomycin A1 (BafA1) or DMSO. Cells were then infected with Ad WT for 45 min, and analyzed by western blot using anti TBK1, LC3 and actin antibodies. P values are based on Ordinary ONE-WAY ANOVA analysis and Dunnett’s multiple comparison test for F and unpaired t-test for C, D and G.
Fig 8.
ATG5 function is not required for TBK1 recruitment.
(A) U2OS control or atg5 KO cells were analyzed by western blot using anti ATG5 antibody. (B) U2OS control or atg5 KO cells were infected with 50 pp/cell of Ad WT (black) or M1 (red), both expressing GFP. Percentage of GFP expressing cells was determined 24 h pi by flow cytometry and normalized to Ad WT. (C) U2OS control and atg5 KO cells were infected with Ad WT and analyzed at 30 min pi by immunofluorescence using anti protein VI (green) and anti LC3 (red) antibodies. Representative confocal images and enlarged insets to the right of each panel are shown. White arrows represent VI and LC3 colocalizations. (D-E) Quantification of LC3 dots (n>30) (D) and proportion of VI colocalization with LC3 (n>30) (E). (F) U2OS control and atg5 KO cells were infected with fluorescently labeled Ad WT (cyan) and analyzed at 30 min pi by immunofluorescence using anti protein VI (green) and anti pTBK1 (magenta) antibodies. Representative confocal images and enlarged insets to the right of each panel are shown. White arrows represent VI and pTBK1 colocalizations. (G-H) Quantification of pTBK1 dots (n>30) (G) and proportion of VI colocalization with pTBK1 (n>30) (H). P values are based on unpaired t-test.
Fig 9.
Distinct damaged membranes detection relies on TBK1 activation and recruitment.
(A) U2OS cells were treated for 30 min with increasing amounts of LLOMe (0.1 mM to 1 mM). Immunofluorescence was performed using anti LC3 antibody (yellow) and representative confocal images are shown. (B) U2OS cells were treated with 1 mM of LLoMe for different times (30 min to 180 min) and analyzed by western blot as in Fig 1A. (C-F) U2OS cells were infected with Ad WT and analyzed at 30 min pi by immunofluorescence using anti pTBK1, anti Gal3 or anti Gal8 antibodies. Quantification of Gal3 and Gal8 colocalizations were normalized to Gal3 (C) or to Gal8 (D) (n>30). Quantification of pTBK1 and Gal3 (E) or pTBK1 and Gal8 (F) normalized to Gal3 and Gal8, respectively (n>30). P values are based on unpaired t-test.
Fig 10.
Model of autophagy activation via TBK1 at Ad endosome penetration sites.
Ad entry induces rupture of the endosomal membrane exposing intracellular glycans which are detected by Gal8 (I). This step creates a recruitment and activation hub for TBK1 to the Ad penetration site. Autophagy receptors (NDP52, p62) are recruited independently of TBK1 (II). TBK1 recruitment results in high local concentrations at the penetration site probably leading to its activation by auto-phosphorylation. Once activated, TBK1 promotes autophagy activation as well as phosphorylates autophagy receptors increasing their affinity to LC3 (III). Autophagy activation generates autophagosomal membranes with LC3 and autophagy receptor link damaged membranes into the forming autophagosome to induce degradation of the damaged membrane (including associated pathogens, i.e. Ad M1) to restore homeostasis (IV). In contrast, Ad WT stalls autophagosome formation and/or maturation and avoids degradation by escaping into the cytosol.
Table 1.
List of siRNA sequences used.
Table 2.
List of primers used for TBK1 mutagenesis.
Table 3.
List of antibodies used and their dilution.