Journal: EMBO Reports

Article Title: Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1

doi: 10.15252/embr.202153429

Figure Lengend Snippet: A Schematic representation of the sequence of Rabaptin5. Coiled‐coil (CC) segments are shown in yellow. Colored backgrounds highlight the segments shown to interact with Rab4, Rab5, Rabex5, and the GAE and GAT domains of GGAs (Golgi‐localizing, γ‐adaptin ear homology domain, ARF‐binding proteins). Below, the segments used to test yeast two‐hybrid interaction with residues 257–444 of FIP200 are shown with their number (#) and the observed interaction (+ or –). B Yeast two‐hybrid analysis for interaction between the above‐shown Rabaptin5 segments (Rbpt5#, fused to LexA on the bait plasmid) and residues 257–444 of FIP200 (FIP, fused to the Gal4 activation domain on the prey plasmid) to drive HIS3 expression. Three different clones each were replica‐plated on medium with His or without His, but containing 3‐amino‐1,2,4‐triazole (3AT; an inhibitor of His synthesis to increase stringency) and grown in the absence of Trp and leucine as a control. As negative controls, empty bait or prey plasmids were used. The asterisk indicates a clone invalidated by recombination. C Schematic representation of the sequence of FIP200 with its coiled‐coil segments in yellow. Residues 281–439 (gray) indicate the minimal sequence identified to interact with Rabaptin5 in the yeast two‐hybrid screen. D FIP200 was immunoprecipitated (IP) from lysates of HeLa or HEK293A cells and probed for FIP200, Rabaptin5 (Rbpt5), and EEA1 (early endosome antigen 1) by immunoblotting. Input lysate (10%) was immunoblotted blotted parallel. As a negative control, the immunoprecipitation was performed using an anti‐GAPDH antibody. E–H Lysates of HeLa cells transiently transfected with full‐length FIP200‐mCherry (FIP200‐mCh) or a deletion mutant lacking the segment interacting with Rabaptin5 (∆280–440) were immunoprecipitated with anti‐mCherry (IP FIP200‐mCh) or, as a control, with anti‐FLAG antibodies (IP FLAG). Immunoprecipitates and input lysates (10%) were immunoblotted for mCherry and Rabaptin5 (E), ATG13 (F), or ULK1 (G). Co‐immunoprecipitation of Rabaptin5, ATG13, and ULK1 with FIP200∆280–440 (FIP∆) was quantified in comparison with that with wild‐type FIP200 (H; signals normalized to that of the immunoprecipitated protein; mean ± SD of three independent experiments each).

Article Snippet: The cDNA of human FIP200 was purchased from OriGene (SC114884), pmCherry_Gal3 was a gift from Hemmo Meyer (Addgene plasmid #85662) (Papadopoulos et al , ), mRuby3‐Gal8 (PB‐CAG‐mRuby3‐Gal8‐P2A‐Zeo) was obtained from Addgene (#150815), pSpCas9(BB)‐2A‐GFP (PX458) was a gift from Feng Zhang (Addgene plasmid #48138) (Ran et al , ), and mCherry‐ATG16L1 was a kind gift from Sharon Tooze (Francis Crick Institute).

Techniques: Sequencing, Binding Assay, Plasmid Preparation, Activation Assay, Expressing, Clone Assay, Control, Two Hybrid Screening, Immunoprecipitation, Western Blot, Negative Control, Transfection, Mutagenesis, Comparison

Journal: EMBO Reports

Article Title: Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1

doi: 10.15252/embr.202153429

Figure Lengend Snippet: A–E HeLa cells were transfected with FLAG‐tagged FIP200 alone (A) or together with Rabaptin5 (Rbpt5‐wt) (B), GFP‐Rab4 (C), RFP‐Rab5 (D), or Citrine‐Rab7 (E), fixed after 24 h, and subjected to immunofluorescence microscopy. Scale bar, 10 µm.

Article Snippet: The cDNA of human FIP200 was purchased from OriGene (SC114884), pmCherry_Gal3 was a gift from Hemmo Meyer (Addgene plasmid #85662) (Papadopoulos et al , ), mRuby3‐Gal8 (PB‐CAG‐mRuby3‐Gal8‐P2A‐Zeo) was obtained from Addgene (#150815), pSpCas9(BB)‐2A‐GFP (PX458) was a gift from Feng Zhang (Addgene plasmid #48138) (Ran et al , ), and mCherry‐ATG16L1 was a kind gift from Sharon Tooze (Francis Crick Institute).

Techniques: Transfection, Immunofluorescence, Microscopy

Journal: EMBO Reports

Article Title: Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1

doi: 10.15252/embr.202153429

Figure Lengend Snippet: A–I To more easily visualize Rabaptin5, a stable HEK293A cell line overexpressing Rabaptin5 (HEK +Rbpt5 ) was generated. Immunofluorescence microscopy of Rabaptin5 and transferrin receptor (TfR) showed swelling of early endosomes upon treatment with 60 µM chloroquine for 30 min (+CQ, panel A) compared with untreated cells (–CQ, A'). Rabaptin5 levels were analyzed by immunoblotting in comparison with parental HEK293A cells (panel B). HEK +Rbpt5 cells, untransfected or 24 h after transfection with mCherry‐galectin 3 (mCh‐Gal3), mRuby3‐galectin 8 (mRuby‐Gal8), mCherry‐FIP200, or mCherry‐ATG16L1 were analyzed upon chloroquine treatment by immunofluorescence microscopy for Rabaptin5 and mCherry‐galectin 3 or mRuby3‐galectin 8 (C), ubiquitin (Ub; D), p62 (E), mCherry‐FIP200 (F), WIPI2 (G), mCherry‐ATG16L1 (H), or LC3B (I). Scale bar, 10 µm. In the enlarged insets, arrowheads point out chloroquine‐induced enlarged, ring‐like early endosomes. Rabaptin5‐positive enlarged endosomes positive for mCherry‐galectin 3 (Gal3) or mRuby3‐galectin 8 (Gal8) were quantified (C'; mean ± SD and individual values of three independent experiments counting > 45 structures each). J, K HEK +Rbpt5 cells, untransfected or 24 h after transfection with mCherry‐ATG16L1, were treated with 60 µM chloroquine for 0, 15, and 30 min and stained for Rabaptin5 and either WIPI2 or mCherry‐ATG16L1. Manders’ colocalization coefficients were determined, M1 showing the fraction of Rabaptin5‐positive structures also positive for WIPI2 (J) or mCherry‐ATG16L1 (K), and M2 showing the respective inverse (mean ± SD of three independent experiments; ANOVA: * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The cDNA of human FIP200 was purchased from OriGene (SC114884), pmCherry_Gal3 was a gift from Hemmo Meyer (Addgene plasmid #85662) (Papadopoulos et al , ), mRuby3‐Gal8 (PB‐CAG‐mRuby3‐Gal8‐P2A‐Zeo) was obtained from Addgene (#150815), pSpCas9(BB)‐2A‐GFP (PX458) was a gift from Feng Zhang (Addgene plasmid #48138) (Ran et al , ), and mCherry‐ATG16L1 was a kind gift from Sharon Tooze (Francis Crick Institute).

Techniques: Generated, Immunofluorescence, Microscopy, Western Blot, Comparison, Transfection, Ubiquitin Proteomics, Staining

Journal: EMBO Reports

Article Title: Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1

doi: 10.15252/embr.202153429

Figure Lengend Snippet: HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing Rabaptin5 (siRbpt5) or FIP200 (siFIP200) for 72 h and treated without (–) or with 60 µM chloroquine (+CQ) or 250 nM Torin1 for 150 min. Scale bar, 10 µm. Below, the efficiency of Rabaptin5 and FIP200 knockdown was assayed by immunoblotting using tubulin (Tub) as a loading control. WIPI2 or LC3B puncta per cell were quantified for each condition (mean ± SD of three independent experiments; ANOVA: * P < 0.05, ** P < 0.01). HEK293A cells were transfected with siCtr or siRbpt5 as in A and incubated without or with 280 µM LLOMe for 150 min to induce lysophagy. Cells were fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. WIPI2 or LC3B puncta per cell were quantified (mean ± SD of three independent experiments).

Article Snippet: The cDNA of human FIP200 was purchased from OriGene (SC114884), pmCherry_Gal3 was a gift from Hemmo Meyer (Addgene plasmid #85662) (Papadopoulos et al , ), mRuby3‐Gal8 (PB‐CAG‐mRuby3‐Gal8‐P2A‐Zeo) was obtained from Addgene (#150815), pSpCas9(BB)‐2A‐GFP (PX458) was a gift from Feng Zhang (Addgene plasmid #48138) (Ran et al , ), and mCherry‐ATG16L1 was a kind gift from Sharon Tooze (Francis Crick Institute).

Techniques: Transfection, Knockdown, Western Blot, Control, Incubation

Journal: EMBO Reports

Article Title: Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1

doi: 10.15252/embr.202153429

Figure Lengend Snippet: A HeLa cells transiently transfected with full‐length mCherry‐ATG16L1 were treated with 60 µM chloroquine (CQ) for 0, 30, or 120 min, lysed, and immunoprecipitated with anti‐Rabaptin5 (IP: Rbpt5) or, as a control, with anti‐FLAG antibodies (IP FLAG). Immunoprecipitates and input lysates (10%) were immunoblotted for Rabaptin5 and ATG16L1. Signals were quantified and the ratios of mCherry‐ATG16L1/Rabaptin5 normalized to that without (0 min) chloroquine treatment (mean ± S of six independent experiments; two‐tailed Student’s t ‐test: * P < 0.05, ** P < 0.001). B Co‐immunoprecipitation was performed as in panel A using parental HEK293A cells and CRISPR/Cas9 knockout cells lacking FIP200 (FIP‐KO). Anti‐HA antibodies were used as a control (IP HA). On the right, HEK293A‐ and FIP200‐knockout cells were immunoblotted for FIP200 and as a loading control of tubulin (Tub). Signals were quantified and the ratios of mCherry‐ATG16L1/Rabaptin5 normalized to that of HEK293A cells without chloroquine treatment (mean ± SD of four independent experiments; two‐tailed Student’s t ‐test: * P < 0.05, ** P < 0.001). C, D Lysates of HEK293A or HeLa cells transiently transfected with full‐length mCherry‐ATG16L1 (wt) or a mutant lacking the WD domain (∆WD; residues 1–319 of ATG16L1, precisely deleting only the WD40 repeats residues 320–607) were immunoprecipitated with anti‐Rabaptin5 or anti‐FLAG antibodies, and immunoblotted for Rabaptin5 and mCherry‐ATG16L1 (C), or immunoprecipitated with anti‐Rabaptin5 or anti‐HA antibodies, and immunoblotted for Rabaptin5 and mCherry‐ATG16L1 (D). In panel D, cells were incubated with or without 60 µM chloroquine for 30 min before analysis. Co‐immunoprecipitation of ATG16L1∆WD with Rabaptin5 (C) was reduced to 6.6 ± 2.1 and 3.4 ± 2.1% in HEK293A and HeLa cells, respectively, relative to that of full‐length ATG16L1 (signals normalized to the immunoprecipitated protein; mean ± SD deviation of three independent experiments each). E The consensus sequence of the ATG16L1 interaction motifs of TMEM59, NOD2, and TLR2 (above; Boada‐Romero et al, ) is shown together with the matching sequence in Rabaptin5 (below). The three point mutations to alanine to produce the AAA mutant of Rabaptin5 are indicated. F Lysates of HeLa cells transiently transfected with myc‐tagged wild‐type Rabaptin5 (wt) or triple‐alanine mutant (AAA) were immunoprecipitated with anti‐myc (IP myc) or anti‐FLAG antibodies (IP FLAG), and immunoblotted for myc and ATG16L1. Co‐immunoprecipitation of ATG16L1 with Rabaptin5‐AAA triple mutant was reduced to 1.5 ± 1.2% relative to that with wild‐type Rabaptin5 (signals normalized to that of the immunoprecipitated protein; mean ± SD of three independent experiments).

Article Snippet: The cDNA of human FIP200 was purchased from OriGene (SC114884), pmCherry_Gal3 was a gift from Hemmo Meyer (Addgene plasmid #85662) (Papadopoulos et al , ), mRuby3‐Gal8 (PB‐CAG‐mRuby3‐Gal8‐P2A‐Zeo) was obtained from Addgene (#150815), pSpCas9(BB)‐2A‐GFP (PX458) was a gift from Feng Zhang (Addgene plasmid #48138) (Ran et al , ), and mCherry‐ATG16L1 was a kind gift from Sharon Tooze (Francis Crick Institute).

Techniques: Transfection, Immunoprecipitation, Control, Two Tailed Test, CRISPR, Knock-Out, Mutagenesis, Incubation, Sequencing

Journal: EMBO Reports

Article Title: Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1

doi: 10.15252/embr.202153429

Figure Lengend Snippet: HeLa cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing Rabaptin5 (siRbpt5) or FIP200 (siFIP200) for 72 h. The cells were infected with Salmonella by centrifugation at 500 × g for 5 min at 37°C and incubation for 10 min at 37°C, washed three times, and incubated in fresh culture medium containing gentamicin to prevent growth of extracellular bacteria for 0, 1, 3, or 6 h before lysis of the host cells and plating of the bacteria on LB agar plates at various dilutions to determine the number of live bacteria at the different time points, shown as a percentage of internalized cells after infection (mean ± SD of three independent experiments). The fractions of internalized bacteria alive 1 h after infection are shown separately in the middle (mean ± SD of three independent experiments; ANOVA: * P < 0.05). On the right, the fraction of infected cells was determined for HeLa cells transfected with siRNAs and infected as above with Salmonella expressing GFP, washed, and immediately fixed for fluorescence microscopy and stained with anti‐transferrin receptor and anti‐LC3B as cellular markers. Z ‐stacks for > 5,000 cells/sample were acquired and analyzed in Fiji to determine the fraction of infected cells (mean ± SD of three independent experiments). The average number of bacteria per infected cell was identical (2.16, 2.11, and 2.12 bacteria per cell transfected with siCtr, siRbpt5, and siFIP200, respectively). Wild‐type HEK293A, HEK +Rbpt5 , Rbpt5‐KO, Rbpt5‐KO+wt, Rbpt5‐KO+AAA, and FIP200‐KO cells were infected with Salmonella and treated and analyzed as in panels A (mean ± SD of three independent experiments; ANOVA: * P < 0.05, **** P < 0.0001). Wild‐type HEK293A, HEK +Rbpt5 , Rbpt5‐KO, Rbpt5‐KO+AAA, and FIP200‐KO cells were infected with Salmonella expressing GFP as in panel B, incubated in fresh culture medium containing gentamicin for 0, 5, 15, 30, and 60 min, fixed with methanol, and immunostained for transferrin receptor (TfR) as a marker of early endosomes and for LC3B as a marker of autophagy. Salmonella were classified according to their association with a TfR‐ and/or LC3B‐positive compartment—as illustrated on the top left (scale bar, 2 µm)—during the first hour after infection. In the absence of Rabaptin5, LC3‐positive SCVs with early endosomal characteristics (containing TfR) were strongly reduced. (mean ± SD of three independent experiments, analyzing >50 bacteria for each time point.)

Article Snippet: The cDNA of human FIP200 was purchased from OriGene (SC114884), pmCherry_Gal3 was a gift from Hemmo Meyer (Addgene plasmid #85662) (Papadopoulos et al , ), mRuby3‐Gal8 (PB‐CAG‐mRuby3‐Gal8‐P2A‐Zeo) was obtained from Addgene (#150815), pSpCas9(BB)‐2A‐GFP (PX458) was a gift from Feng Zhang (Addgene plasmid #48138) (Ran et al , ), and mCherry‐ATG16L1 was a kind gift from Sharon Tooze (Francis Crick Institute).

Techniques: Transfection, Infection, Centrifugation, Incubation, Bacteria, Lysis, Expressing, Fluorescence, Microscopy, Staining, Marker