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ATG16L1 induces the formation of phagophore-like membrane cups

Abstract

The hallmark of non-selective autophagy is the formation of cup-shaped phagophores that capture bulk cytoplasm. The process is accompanied by the conjugation of LC3B to phagophores by an E3 ligase complex comprising ATG12–ATG5 and ATG16L1. Here we combined two complementary reconstitution approaches to reveal the function of LC3B and its ligase complex during phagophore expansion. We found that LC3B forms together with ATG12–ATG5–ATG16L1 a membrane coat that remodels flat membranes into cups that closely resemble phagophores. Mechanistically, we revealed that cup formation strictly depends on a close collaboration between LC3B and ATG16L1. Moreover, only LC3B, but no other member of the ATG8 protein family, promotes cup formation. ATG16L1 truncates that lacked the C-terminal membrane binding domain catalyzed LC3B lipidation but failed to assemble coats, did not promote cup formation and inhibited the biogenesis of non-selective autophagosomes. Our results thus demonstrate that ATG16L1 and LC3B induce and stabilize the characteristic cup-like shape of phagophores.

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Fig. 1: Conjugation of LC3B to model membranes in vitro.
Fig. 2: Scaffold formation by LC3B and ATG12–ATG5–ATG16L1 on model membranes in vitro.
Fig. 3: Cup formation by ATG16L1 on SLBs in vitro.
Fig. 4: WIPI2-CAAX recruits ATG16L1 and LC3B to the PM independently of the upstream autophagy core machinery.
Fig. 5: Reconstitution of membrane remodeling and cup formation at the plasma membrane.
Fig. 6: Generation of membrane cups by LC3B and ATG12–ATG5–ATG16L1 in vivo.
Fig. 7: Localization of ATG16L1 variants at cellular membranes revealed by CLEM.

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All data are provided in the main text, and Extended Data Figures and Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We thank F. Lafont and S. Janel (Center for Infection and Immunity of Lille, Institut Pasteur de Lille, Lille, France) for access to atomic force microscopes, scientific and technical support and fruitful discussions. We thank A. Canette (Institut de Biologie Paris-Seine (IBPS) Electron Microscopy facility) for help with metal coaters, and J. Lainé (Sorbonne Université, INSERM, Institut de Myologie) for her expertise in preparing thin sections for EM. We also thank S. Tooze (The Francis Crick Institute, London, UK) for the WIPI2-CAAX construct and fruitful discussions. We thank M. Lazarou (Monash University, Melbourne) for sharing his Hexa-KO cells. We thank A. Gazi, O. Gorgette and G. Pehau-Arnaudet at the Ultrastructural BioImaging Platform (UTechS UBI, Institut Pasteur Paris) for access to electron microscopy and sample-preparation equipment, as well as for technical support and training. We thank J. Krijnse Locker (Paul-Ehrlich-Institut and formerly at the Institut Pasteur Paris) for helpful discussions and support. We thank A. Echard and C. Zurzolo (Institut Pasteur, Paris) for commenting on the manuscript. UTechS PBI is part of the France–BioImaging infrastructure network (FBI) supported by the French National Research Agency (ANR-10-INBS-04; Investments for the Future), and acknowledges support from Institut Pasteur, ANR/FBI, the Région Ile-de-France (grant on infectious diseases DIM1HEALTH), and the French Government Investissement d’Avenir Programme—Laboratoire d’Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (ANR-10-LABX-62-IBEID) for the use of ELYRA7 microscope (Carl Zeiss). We are grateful for the support of A. Mallet (UBI) and financial support for UBI equipment from the French Government Programme Investissements d’Avenir France BioImaging (FBI, N° ANR-10-INSB-04-01) and the French gouvernement (Agence Nationale de la Recherche) Investissement d’Avenir programme, Laboratoire d’Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (ANR-10-LABX-62-IBEID). This work was supported by the ERC-Starting Grant GA 638603 (Autophagy in vitro), by the Agence Nationale de la Recherche (grants ANR-22-CE13-0007-01, AUTOBEND) and by intramural funds of the Institut Pasteur to T.W.; and by Sorbonne Université, INSERM, Association Institut de Myologie core funding and the Agence Nationale de la Recherche (grants ANR-20-CE13-0024-02 and ANR-21-CE13-0018-01) to S.V.

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Authors and Affiliations

Authors

Contributions

J.M. purified proteins, did all in vitro reconstitutions, performed FRAP and AFM experiments, performed autophagy experiments (ATG16L1 puncta, membrane binding, LC3B conjugation), generated truncated ATG16L1 variants and prepared samples for PREM (in vitro and in vivo). S.B.M. generated constructs, performed confocal imaging of ATG16L1 plasma membrane targeting, provided samples for in vivo PREM studies and performed CLEM–PREM. C.G.-B. performed TEM and CLEM experiments, including segmentation and quantification. S.V. conceptualized, performed and analyzed all PREM experiments, acquired funding and co-supervised S.B.M. D.C. performed colocalization experiments with ATG14, ATG9 and ULK1 and autophagy-inhibition and siRNA experiments with support from C.N. S.B. generated stable cell lines and performed flux experiments. S.R. performed in vivo characterization of ATG16L1 variants together with J.M. A.S. performed 3D-STORM experiments. E.P. and C.G.B. performed FIB–SEM and serial sectioning TEM experiments. T.W. conceptualized, supervised and administered the study, analyzed data, wrote the paper draft, provided resources and acquired funding. All authors analyzed and discussed the results and revised the paper.

Corresponding authors

Correspondence to Stéphane Vassilopoulos or Thomas Wollert.

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Nature Structural & Molecular Biology thanks Sharon Tooze, Helen Walden and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Reconstitution of LC3B lipidation in vitro.

(a) SDS-PAGE gel of purified components of the Ub-like conjugation system used for reconstitutions. MW = molecular weight in kDa. (b) SDS-PAGE gel of thioester intermediates between LC3BG120 and the E1-like enzyme ATG7 and the E2-like enzyme ATG3 as indicated. MW = molecular weight in kDa. (c) FRAP experiment of Atto 590 labeled LC3BG120 (yellow) that was conjugated to fluorescently labeled GUVs (red) in the presence of Alexa488 labeled ATG16L1Alexa488 (green). The figure corresponds to Fig. 1f. (d, e) FRAP experiment of LC3BAlexa488 (green) conjugated to GUVs (red) in the presence of ATG16L1FL (d) or ATG16L1NT (e). FRAP images show GUVs before (-1:00), immediately after (0:00) and seven minutes after photobleaching. The fluorescence was bleached in the indicated area (arrowhead). Scale bars = 10 µm. Related to Fig. 1e.

Extended Data Fig. 2 Scaffold formation by LC3B and ATG12–ATG5-ATG16L1.

(a) FRAP experiments of labelled LC3B Alexa488 (green) enzymatically conjugated to a Supported Lipid Bilayer (SLB, stained with Lissamine-rhodamine PE and shown in red) in the presence of ATG12–ATG5-ATG16L1. Confocal images show the SLB directly after photobleaching (upper panel) and 30 min after photobleaching (lower panel). Scale bar = 5 µm. (b) Representative confocal images of GUVs (membrane in red) to which Alexa488 labeled LC3BG120 (green) was enzymatically conjugated in the presence of ATG12–ATG5-ATG16L1 complexes comprising different ATG16L1 variants as indicated. Fluorescence of approximately 10 % of the total membrane area was bleached (area indicated by arrowheads) and images were taken seven minutes after photobleaching. Scale bars = 10µm. (c) AFM height profiles of SLBs to which LC3B was enzymatically conjugated in the presence of ATG12–ATG5-ATG16L1307. The line fitted image was averaged and a Gaussian filter was applied for better visualization. The colors correspond to heights as indicated in the corresponding height scale. The chart shows the line profile taken from the indicated region (white line) of the height profile. The dotted red lines indicate the membrane height (set to zero) and the average protein height used to calculate the thickness of the protein coat. Scale bar = 0.5 µm. Related to Fig. 2. (d) Examples for definitions of rims, premature cups and mature cups from in vitro experiments. Intensity profiles of the grey values in PREM micrographs have been plotted along the cross sections of the structures (indicated by the yellow lines) using ImageJ. The distribution and length of intensity maxima (indicated by red lines) have been used to define rims, premature cups and expanded cups. For details see Methods section. Related to Fig. 3b.

Extended Data Fig. 3 WIPI2-CAAX recruits ATG16L1 and LC3B to the plasma membrane independently of the upstream autophagy core machinery.

(a) Confocal images of HeLa cells expressing WIPI2-CAAX, ATG16L1, GFP-LC3B and either mKate-ATG14, ULK1 or RFP-ATG9 as indicated (corresponding to Fig. 4b). X,Y distribution refers to confocal images of GFP-LC3B with either ATG14, ULK1 or ATG9, polar distributions have been generated from confocal images using the center of the cell (marked by an asterisk) as a reference position. Distribution diagrams (right panels): Radial intensity profiles of the merged channels, from the cell centroid (value = 0) to the cell edge (value = 100). Radial intensity profile and polar distributions were produced with the Clock Scan plug-in of the ImageJ software. (b, c) Western blots showing the knockdown efficiency for ULK1 (b) and ATG14 (c). Control = scrambled siRNA. Actin was used as a loading control and to normalize protein levels. Data are shown as mean ± S.D. of N = 3 independent experiments. Statistical significance was calculated using an unpaired, two-tailored Student’s t-test with p-values p = 0.0026 for ULK1 and p = 0.0012 for ATG14. MW = molecular weight in kDa. (d) Representative confocal image of HeLa cells expressing WIPI2-CAAX, ATG16L1, GFP-LC3B (green in the merged image) and the phosphatidylinositol (3) phosphate sensor RFP-2xFYVE (red in the merged image). Scale bars = 10 µm. Related to Fig. 4.

Source data

Extended Data Fig. 4 WIPI2-CAAX induce membrane compartment.

(a, b) PREM images of unroofed ATG16L1 knock out HeLa cells transfected with untagged WIPI2, scale bar = 1µm (a) and of a cell that has been treated with transfection reagents in the absence of plasmids as transfection control, scale bar = 200 nm (b). Related to Fig. 5a.

Extended Data Fig. 5 Remodeling of the plasma membrane by ATG16L1 in cells expressing WIPI2-CAAX.

(a-b) Correlative SMLM and PREM image of an unroofed cell expressing WIPI2-CAAX, HA-ATG16L1 (red) and LC3B (green). Insets show areas with consecutively increased magnifications (from left to right) of areas indicated by the white rectangles. Scale bar of overview = 10 µm, left inset = 5 µm, middle inset = 2 µm and right inset = 0.5 µm. (a) Correlated fluorescent and PREM images of a cell with strong recruitment of ATG16L1 but limited conjugation of LC3B show membrane remodeling by ATG16L1 independently of LC3B. (b) Correlated fluorescent and PREM images of a cell with strong colocalization of LC3B and ATG16L1 show membrane remodeling by the synergistic action of the two proteins. Related to Fig. 6b. (c) Representative confocal image of ATG16L1 knockout cells expressing WIPI-CAAX, GFP-LC3B and mCherry-ATG16L1207 (scale bar = 10 µm) with corresponding PREM view (bottom, scale bar = 200 nm, arrowheads indicate clathrin coated pits). (d) Confocal images of single channels corresponding to merged images shown in Fig. 6d–g of ATG16L1 knockout cells expressing WIPI-CAAX, GFP-LC3B and mCherry-ATG16L1 variants as indicated. Scale bar = 5µm. (e) Correlative super resolution (spinning disk) microscopy and PREM image of an unroofed cell expressing WIPI2-CAAX (green) and HA-ATG16L1 (red) in which LC3B was knocked down by siRNA. The inset shows the area indicated by the white rectangle. Scale bar of overview = 1 µm and of inset = 0.2 µm. Western blot confirms efficient knock-down of LC3B by siRNA. (f) Correlative SMLM and PREM image of an unroofed cell expressing WIPI2-CAAX (green) and ATG16L1. Endogenous LC3B was detected by CLEM (red). Scale bar of overview = 2 µm and of inset = 1 µm. (g, h) Representative PREM views of ATG8 Hexa knockout cells expressing WIPI2-CAAX and ATG16L1 (g) and complemented with LC3B (h). The encircled area in (g) indicates extended ER-PM contact sites from which phagophore-like cups would be formed. (h) Asterisks indicate clathrin coated pits, arrows indicate cups. Scale bars in overviews 500 nm and in insets 200 nm.

Source data

Extended Data Fig. 6 Autophagic activity of ATG16L1 variants.

(a) Western blot of lysates from wild type or ATG16L1 knock out cells expressing ATG16L1NT, ATG16L1207, ATG16L1230, ATG16L1264, ATG16L1307 or ATG16L1FL as indicated. The upper blot shows ATG16L1 variants (using an anti-ATG16L1 primary antibody). Note that the N-terminal fragment ATG16L1NT is too small to be detected. The middle blot shows lipidated LC3B (LC3B-II), GAPDH serves as loading control. The chart shows the corresponding quantification of LC3B-II levels as mean ± S.D. from N = 3 independent experiments. Statistical significance was calculated using an unpaired two tailed t-test (comparing LC3B-II levels in ATG16L1 knock out cells complemented with ATG16L1 variants), with p values for Nt: 0.36; 207: 0.98; 230: 0.001; 264: 0.0054; 307: 0.00063; FL: 0.027. Source data are provided. MW = molecular weight in kDa. (b) Western blot of cytosolic (C) and membrane fractions (M) of ATG16L1 KO cells expressing ATG16L1207, ATG16L1230, ATG16L1264, ATG16L1307 or ATG16L1FL with quantification of relative protein levels. GAPDH serves as fractionation control and was analyzed on a separate blot using the same sample. The bar chart shows mean values of N = 2 independent experiments. Source data are provided. MW = molecular weight in kDa. (c) Representative confocal images of ATG16L1 puncta in ATG16L1 knock out HeLa cells complemented with HA-ATG16L1230, HA-ATG16L1264, HA-ATG16L1307 or HA-ATG16L1FL. Scale bars = 10 µm (overviews) and 2 µm (insets). (d, e) Quantification of ATG16L1 puncta with diameters of 0.1–0.7µm (d) or 0.7–2.0 µm (e) of images as shown in c. Bar charts represent mean values ± S.D. from N = 3 independent experiments. Statistical significance was calculated using unpaired two-tailed Student’s t-tests, p values (compared to ATG16L1FL) (d) 230: 0.00038; 264: 0.00051; 307: 0.32; and (e) 230: 0.0031; 264: 0.0041; 307: 0.00099461. p values (compared to ATG16L1307) (d) 230: 0.00037; 264: 0.00046 and (e) 230: 7.8E-05; 264: 9.4E-05. (f) Comparison of ATG16L1 vesicles in cells expressing ATG16L1FL or ATG16L1230. Images show segmentation of ATG16L1 vesicles in electron tomograms shown in Supplementary Movie 9. Related to Fig. 7a.

Source data

Extended Data Fig. 7 Functional analysis of ATG16L1 variants.

(a-c) Serial sectioning electron microscopy of cells expressing HA-tagged ATG16L1FL (a), ATG16L1307 (b) or ATG16L1230 (c). One representative slice of 10 to 15 consecutive slices is shown. Scale bars = 1 µm (overview) and 100 nm (insets). (d) GFP-RFP-LC3B assay to determine delivery of autophagosomes to lysosomes in wildtype HeLa cells, ATG16L1 KO cells, and in ATG16L1 KO cells expressing native levels of ATG16L1FL, ATG16L1307, ATG16L1264 or ATG16L1230. Micrographs show maximum intensity projects of z-stacks covering the entire cellular volume. Red puncta correspond to autolysosomes and yellow puncta to cytoplasmic phagophores/autophagosomes. Scale bars = 10 µm. (e) Quantification of autophagic flux from data shown in (d). The flux was determined by calculating the fraction of puncta that showed only red fluorescence (in % from total number of puncta). For each condition puncta from at least 75 cells of N = 3 independent experiments were quantified using Fiji and the ComDet (version 0.5.1) plugin. Statistical significance was calculated using unpaired two-tailed Student’s t-tests with p values (compared to wildtype, WT): ATG16L1 KO cell (KO): 0.22, 0.094, 0.012; Full length (FL): 0.91, 0.80, 0.038; ATG16L1 307: 0.35, 0.70 0.0083; ATG16L1 264: 0.18, 0.099, 0.0026 and ATG16L1 230: 0.19, 0.16, 0.0059.

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Supplementary Video 1

Conjugation of LC3B to GUVs induces membrane deformations and budding. Alexa-Fluor-488-labeled LC3BG120 (green) was conjugated to GUVs (red) in the presence of ATG7, ATG3, ATG12–ATG5 and ATG16L1. The conjugation reaction was observed for 180 min. Corresponding still images at defined time points are shown in Figure 1b. Scale bar, 10 µm.

Supplementary Video 2

Membrane buckling on GUVs upon LC3B conjugation. 3D reconstruction of the GUV shown in Figure 1b and in Supplementary Video 1 at the end of the conjugation reaction form z-stacks. Related to Figure 1c.

Supplementary Video 3

Formation of membrane cups from supported lipid bilayers by ATG16L1FL in vitro. The video shows a PREM tomogram of a SLB to which LC3B was conjugated in the presence of the ATG12–ATG5–ATG16L1 complex. Related to Figure 3a.

Supplementary Video 4

Formation of membrane cups from supported lipid bilayers by ATG16L1307 in vitro. The video shows a PREM tomogram of a SLB to which LC3B was conjugated in the presence of the ATG12–ATG5–ATG16L1307 complex. A corresponding image at a tilt angle of 0° is shown in Figure 3d.

Supplementary Video 5

Formation of membrane cups from WIPI2-induced membrane compartments by ATG16L1 in vivo. The video shows a PREM tomogram of an unroofed ATG16-KO cell expressing WIPI2-CAAX, LC3B and ATG16L1. A corresponding image is shown in Figure 6g.

Supplementary Video 6

Correlation of ATG16L1 localization with membrane morphologies at WIPI2-induced membrane compartments. The video shows SMLM images of an ATG16L1-KO cell transfected with WIPI2-CAAX, HA-ATG16L1 and LC3B, along with the corresponding PREM view. ATG16L1 localization is highlighted in red. A corresponding image is shown in Extended Data Figure 5a.

Supplementary Video 7

Correlation of LC3B and ATG16L1 colocalization with membrane morphologies at WIPI2-induced membrane compartments. The video shows SMLM images of an ATG16L1-KO cell transfected with WIPI2-CAAX, HA-ATG16L1 and LC3B, along with the corresponding PREM view. ATG16L1 localization is highlighted in red and LC3B localization in green. Corresponding images are shown in Extended Data Figures 5b and 6b.

Supplementary Video 8

Formation of membrane rims on WIPI2-induced membrane compartments by ATG16L1230 in vivo. The video shows a PREM tomogram of a unroofed ATG16L1-KO cell expressing WIPI2-CAAX, LC3B and ATG16L1230. A corresponding image is shown in Figure 6d.

Supplementary Video 9

Electron tomography of ATG16L1 vesicles in cells expressing ATG16L1 variants. The tomograms show ATG16L1 vesicles in cells expressing HA-tagged ATG16L1FL (upper left), ATG16L1307 (upper right), ATG16L1264 (lower left) or ATG16L1230 (lower right). Cells were fluorescently abeled using anti-HA (primary) and Alexa-Fluor-488-tagged (secondary) antibodies. Tomograms were collected in regions selected by CLEM on the basis of the fluorescent signal from immunofluorescence. Corresponding images are shown in Extended Data Figure 6f.

Supplementary Video 10

FIB–SEM of a cell expressing ATG16L1307. The video shows scanning electron micrographs from a cell expressing ATG16L1307. After each scan, a thin layer of the embedded sample was milled using a focused ion beam. Related to Figure 7c.

Supplementary Video 11

FIB–SEM of a cell expressing ATG16L1230. The video shows scanning electron micrographs from a cell expressing ATG16L1230. After each scan, a thin layer of the embedded sample was milled using a focused ion beam. Related to Figure 7d.

Supplementary Video 12

Membrane intermediates during cup formation. The video shows a series of PREM tomograms of unroofed ATG16L1-KO cells expressing WIPI2-CAAX, LC3B and different ATG16L1 variants, as indicated. Characteristic membrane structures are highlighted in magenta. Thin cortical ER is present in cells expressing ATG16L1207, from which flat membrane rings are formed in ATG16L1230-expressing cells. Constriction of membrane rings might lead to the formation of membrane cups upon expression of ATG16L1FL. Related to Figure 7f.

Supplementary Table 1

Materials and reagents used in this study.

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Mohan, J., Moparthi, S.B., Girard-Blanc, C. et al. ATG16L1 induces the formation of phagophore-like membrane cups. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01300-y

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