Several cell surface molecules including signalling receptors are internalized by clathrin-independent endocytosis. How this process is initiated, how cargo proteins are sorted and membranes are bent remains unknown. Here, we found that a carbohydrate-binding protein, galectin-3 (Gal3), triggered the glycosphingolipid (GSL)-dependent biogenesis of a morphologically distinct class of endocytic structures, termed clathrin-independent carriers (CLICs). Super-resolution and reconstitution studies showed that Gal3 required GSLs for clustering and membrane bending. Gal3 interacted with a defined set of cargo proteins. Cellular uptake of the CLIC cargo CD44 was dependent on Gal3, GSLs and branched N-glycosylation. Endocytosis of β1-integrin was also reliant on Gal3. Analysis of different galectins revealed a distinct profile of cargoes and uptake structures, suggesting the existence of different CLIC populations. We conclude that Gal3 functionally integrates carbohydrate specificity on cargo proteins with the capacity of GSLs to drive clathrin-independent plasma membrane bending as a first step of CLIC biogenesis.
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We would like to thank the following people for help in experiments, providing materials or expertise: P. Bassereau, L. Cabanié, P. Chavrier, B. Hofmann, H. Ideo, A. Raz, W. Römer, C. Schiff and T. Wollert. The facilities as well as scientific and technical assistance from staff in the Australian Microscopy and Microanalysis Facility (AMMRF) at the Centre for Microscopy and Microanalysis at The University of Queensland, the Australian Cancer Research Foundation (ACRF)-Institute for Molecular Bioscience Dynamic Imaging Facility for Cancer Biology, the Biomedical Imaging Facility at UNSW and the PICT-IBiSA-Nikon Imaging Centre of Institut Curie with support from FRM (AAP ‘Grand Equipement’ 2011 number DGE20111123020), Inca (Number 2011-1-Label-SALAMERO IC 4) and the ‘CanNoli project’ supported by the DIM Canceropole-IdF (number 2012-2-EML-04) are acknowledged. This work was supported by grants from the National Health and Medical Research Council of Australia (1037320, R.G.P. and K.G.; 1045092, R.G.P., N.A. and M.T.H.), the Agence Nationale pour la Recherche (ANR-09-BLAN-283 and ANR-11 BSV2 014 03, L.J.), the Indo-French Centre for the Promotion of Advanced Science (project number 3803, L.J.), Marie Curie Actions—Networks for Initial Training (FP7-PEOPLE-2010-ITN, L.J.), European Research Council (project 340485, L.J.), fellowships from Association pour la Recherche sur le Cancer (R.L.), Marie Curie International Reintegration Grant (FP7-RG-277078, C.W.), and Deutsche Forschungsgemeinschaft (U.B.). The L.J. team is a member of Labex CelTisPhyBio.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 GSLs are required for CD44 uptake.
(a) Control or PPMP-treated MEFs were incubated at 37 °C with anti-CD44 antibodies (green) and Tf (red) (both at 10 μg ml−1) for 2 or 10 min, and cell surface exposed material was removed by acid washes. CD44 uptake was strongly inhibited in PPMP-treated cells. Quantification is shown in Fig. 1c. (b) The indicated types of cells were incubated for 2 min with anti-CD44 antibodies, and cell surface exposed material was removed by acid washes. CD44 uptake was strongly inhibited in GSL-deficient GM95 cells. Quantification is shown in Fig. 1d. (c) Control or PPMP-treated MEFs were incubated on ice with anti-CD44 antibodies (green) and CTxB (red). Quantification on 15–20 cells per condition (means ± s.e.m., n = 3 independent experiments). PPMP treatment significantly reduced CTxB binding, but not that of anti-CD44 antibody. (d) The indicated types of cells were incubated as in c. Similar CD44 surface levels were observed on all cells. Quantification on 38-49 cells assessed from 4 fields (means, n = 2 independent experiments). (e) Biochemical CD44 uptake assay. Fluorescently labelled anti-CD44 antibodies at 10 μg ml−1 were incubated for 10 min at 37 °C with MEFs in the indicated conditions (±PPMP), or with MEB4, GM95, or CG1 cells. After acid washes, the cells were lysed and lysates analysed by SDS-PAGE and fluorescence detection. Means ± s.d. are shown, n = 4 (upper graph) and n = 5 (lower graph) independent experiments. Note that uptake efficiency was strongly dependent on GSL expression. The main GSL, GM3, was 35-fold less expressed in CG1 cells than in MEB4 cells, likely explaining the partial rescue of the CD44 uptake phenotype. A representative gel is shown for the ±PPMP conditions. (f) Dose-response of Gal3–HRP CLIC formation. MEFs were incubated for 2 min at 37 °C with the indicated concentrations of Gal3–HRP. The diaminobenzidine reaction for imaging HRP was performed on ice in the presence of ascorbic acid, and cells were processed for EM. Gal3-CLIC (tubules/rings) were observed with Gal3–HRP concentrations as low as 10 ng ml−1. (g) Control or PPMP-treated TECs were incubated for 2 min at 37 °C with Gal3–HRP. The diaminobenzidine reaction for imaging HRP was performed on ice in the presence of ascorbic acid, and cells were processed for EM. Gal3-CLIC (tubules/rings) formation was significantly inhibited on PPMP-treated cells. Quantification is shown in Fig. 1f. Statistical analysis in this figure: Student, unpaired t-test. ∗∗P < 0.01. Scale bars, 10 μm in a–d, and 200 nm in f,g.
Supplementary Figure 2 dSTORM imaging and cluster analysis of Gal3-Alexa647 in control and PPMP-treated HeLa cells.
(a) dSTORM images of Gal3-Alexa647 in HeLa cells. Cells were pre-treated or not with PPMP, and incubated with 4 μg ml−1 of Gal3-Alexa647 for 20 min, fixed and imaged under total internal reflection fluorescence (TIRF) illumination. (b) Color-coded cluster maps retrieved from local point pattern analysis of regions (red squares in a). Colour indicates level of clustering, L(r), with low to high clustering coloured blue to red. (c) Maps of Gal3-Alexa647 clusters and molecules inside clusters after applying a threshold to color-coded maps shown in b. (d–g) Density of Gal3-Alexa647 molecules (d), density of Gal3-Alexa647 clusters (e), radii of Gal3-Alexa647 clusters in nm (f), area covered by Gal3-Alexa647 clusters (g) were obtained from threshold cluster maps. Means ± s.e.m. from 10-20 cells, n = 3 independent experiments for each panel. (h–k) Analysis for the effects of photoblinking. Max of L(r) − r values (h) of Gal3-Alexa647 molecules adhered to coverslips under cell-free conditions compared to corresponding data of Gal3-Alexa647 on control and PPMP-treated HeLa cells, shown in Fig. 1g, h. To correct for repeated excitation of the same fluorophores, the number of localized Galectin3-Alexa647 molecules in control (i) and PPMP-treated (j) HeLa cells as well as Alexa647-Gal3 molecules adhered to coverslips (k) was plotted versus the off-gap. Data (round symbols) was fitted (solid line) to equation 4 (see Supplementary Note 1). The off-gap threshold was at 40 frames, illustrating that in three examples, >97.5% of over-counted molecules were removed. i–k are representative experiments, n = 3 independent experiments with 10-20 cells examined per experiment. Scale bars: a, 6 μm; b,c, 500 nm. Student, unpaired t-test, ∗∗P < 0.01;∗∗∗P < 0.001;∗∗∗∗P < 0.0001.
Supplementary Figure 3 Gal3 endocytosis under perturbation conditions.
(a,b) Gal3 uptake in cells with perturbed clathrin function. Incubation of Gal3 and Tf for 15 min at 37 °C with HeLa cells (a) or TECs (b) that expressed dominant negative GFP-tagged mutant of Eps15 (EH2-1). Cells were lactose and acid washed to remove plasma membrane accessible material. Tf endocytosis was strongly inhibited in mutant expressing HeLa cells and TECs, in contrast to Gal3. (c,d) Gal3 uptake in cells with perturbed dynamin function. Incubation of Gal3 and Tf for 15 min at 37 °C with HeLa cells (c) or TECs (d) that expressed dominant negative GFP-tagged mutant dynamin (K44A). Cells were lactose and acid washed to remove plasma membrane accessible material. Gal3 endocytosis was not significantly perturbed in K44A expressing cells, while Tf endocytosis was strongly inhibited. Statistical analysis in this figure: Means ± s.d., n = 3 independent experiments, 25–30 cells each. Student, unpaired t-test, ∗P < 0.05, ∗∗∗P < 0.001. Scale bars, 10 μm.
Supplementary Figure 4 Analysis of Gal3 uptake determinants.
(a) Gal3 surface binding on GSL-depleted cells. Control or PPMP-treated TECs were incubated on ice with Gal3 (red) and CTxB (green). On PPMP-treated cells, the binding of CTxB was strongly reduced, while Gal3 binding was not affected. Quantification of data on 12–15 cells per condition (means ± s.e.m.; n = 3 independent experiments). (b) Protein determinants are required for Gal3 binding to cells. TECs were treated with proteinase K (right panel) or not (left panel) and incubated with Gal3 (red) and CTxB (green). Gal3 did not bind to proteinase K-treated cells, as opposed to CTxB. A representative result is shown, n = 3. (c) Gal3 needs N-glycans for binding to cells. TECs were incubated or not for 24 h with 6 μM tunicamycin or 48 h with 90 μg ml−1 1-deoxymannojirimycin, put on ice and incubated for 20 min with Gal3 (red) and CTxB (green). Gal3 binding was strongly impaired, but not that of CTxB. Quantification of data on 19–26 cells assessed from 4 fields per condition (means ± s.d., n = 3 independent experiments). (d) Sampling of Gal3 and Gal4 interacting partners according to intra and extracellular localizations. Protein identities are given in Supplementary Table 1. Statistical analysis in this figure: Students, unpaired t-test. ∗P < 0.5, ∗∗∗P < 0.001. Scale bars, 10 μm.
Supplementary Figure 5 CD44, 1-integrin, and fluid phase uptake in Gal3-depleted cells.
(a) Depletion of Gal3 on TECs using siRNA sequence #3. Western blot analysis in which tubulin served as a loading control. A representative result is shown. Corresponding cells were used for uptake experiments of Fig. 4d. Cells were tested for efficient knockdown in each experiment. (b) CD44 cell surface levels on MEFs were not significantly altered on Gal3 depletion. Untreated, scrambled siRNA transfected, or Gal3 depleted MEFs were incubated on ice with anti-CD44 antibodies, and after fixation with fluorescently labelled secondary antibodies. Means ± s.d., n = 3 independent experiments, 15-35 cells assessed from 5 fields each. Corresponding cells were used for uptake experiments of Fig. 4d. (c) Gal3 is required for CD44 uptake. Tf (blue) and anti-CD44 antibodies (green) were concomitantly incubated for the indicated times and in the indicated conditions with MEFs that were complemented or not with exogenous Gal3 (red). Cells were acid washed to remove plasma membrane accessible material. CD44 uptake was specifically reduced in Gal3-depleted MEFs, and exogenous Gal3 rescued this phenotype. Quantification is shown in Fig. 4c. (d) Western blotting analysis of proteins expression in the indicated depletion conditions. A representative result is shown. Corresponding cells were used for uptake experiments of Fig. 4g. Cells were tested for efficient knockdowns in each experiment. (e) Examples of cells from the internalization analysis of Fig. 4g, using 9EG7 antibody against the active form of 1-integrin. (f) 1-integrin cell surface levels in the indicated conditions, as determined by antibody binding on ice. Means ± s.d., n = 3 independent experiments, 15-35 cells assessed from 5 fields each. Corresponds to experiments shown in Fig. 4g. (g) EM analysis of HRP uptake in Gal3-depleted cells. Untransfected, scrambled siRNA, or Gal3 siRNA transfected MEFs were pulsed for 2 min with HRP (10 mg ml−1) and processed for EM. In control conditions (untransfected or scrambled siRNA transfected cells), HRP-labeled tubular and ring-shaped structures were observed at early times of uptake (2 min, arrows). Some vesicular structures of different diameters were also labelled (arrowheads), likely representing clathrin-coated vesicles (80–120 nm) or detached caveolae (40–60 nm). On Gal3 siRNA transfected cells, the tubular and ring-shaped structures were strongly reduced, and mostly vesicular profiles were visualized. Quantification is shown in Fig. 4h. Statistical analysis: Student, unpaired t-test. ∗∗P < 0.01. Scale bars: c,e, 10 μm; g, 200 nm.
Supplementary Figure 6 CD44 uptake and fluid phase CLIC formation on cells incubated in the presence of lactose.
(a) Galectin function is required for CD44 uptake. MEFs were incubated with Tf (red) and anti-CD44 antibodies (green) for the indicated times in the presence of 100 mM sucrose or lactose. Cells were acid washed to remove plasma membrane accessible material. (b) Labelling intensities of experiments as in a were quantified on 10 to 12 cells per condition (Means ± s.e.m., n = 3 independent experiments) and normalized to the Sucrose 2 min sample average for each marker, separately. Note that lactose specifically inhibited CD44 uptake. (c) CD44 cell surface levels on MEFs were not significantly altered on lactose incubation. MEFs in conditions as in a were incubated on ice with anti-CD44 antibodies, and after fixation with fluorescently labelled secondary antibodies. Means ± s.d. of 15 to 25 cells assessed from 5 fields per experiment, n = 3 independent experiments. (d) Fluid phase uptake is sensitive to galectin function. Lactose or sucrose-treated MEFs were pulsed for 2 min with HRP (10 mg ml−1) and processed for EM. In control conditions (sucrose), HRP labelled tubular and ring-shaped CLIC structures at early times of uptake (arrows). Some vesicular structures of different diameters were also labelled (arrowheads), likely representing clathrin-coated vesicles (80–120 nm) and detached caveolae (40–60 nm). In the presence of lactose, the occurrence of tubular and ring-shaped CLIC structures was largely reduced, and mostly vesicular profiles were visualized. (e) Quantification of experiments as in d on 20–24 cells per condition (means ± s.e.m., n = 3 independent experiments) confirmed a significant decrease of tubular and ring-shaped structures on lactose treatment, while vesicles were not significantly affected. (f) Fluid phase dextran uptake experiment. MEFs were incubated for 5 min with 1 mg ml−1 of FITC-labelled dextran in the presence of the indicated sugars, fixed, and analysed for labelling intensity. Lactose significantly inhibited fluid phase endocytosis. g, Quantification of experiments as in f. Means ± s.d. of 17 to 27 cells per experiment, n = 3 independent experiments. Scale bars, a,f, 10 μm; d, 200 nm. Statistical analysis in this figure: Student, unpaired t-test. ∗∗P < 0.01.
Supplementary Figure 7 N-glycosylation in CD44 clustering and uptake.
(a) Clustering of Alexa-647-labeled anti-CD44 antibodies in control and 1-deoxymannojirimycin-treated MEFs. Mean Ripley’s K-function curves, L(r) − r, derived from single molecule localizations obtained with dSTORM imaging, plotted against radius, r, of concentric circles centred on each molecule relative to random distributions (99% confidence interval (CI) of simulated data). Averages of 15–20 cells, n = 3 independent experiments. (b) Maxima of Ripley’s K-function curves. Each symbol represents one image region; small horizontal lines indicate mean (±s.e.m., n = 3 independent experiments). (c) CD44 uptake in MEFs in which the biogenesis of complex type N-glycosylation was perturbed or prevented with the indicated inhibitors. Cells were incubated for 2 min at 37 °C with anti-CD44 antibodies. Quantification of uptake is shown in Fig. 5c. (d,f) Cell surface levels of endocytic markers. MEFs in the indicated conditions were incubated on ice with anti-CD44 antibodies (revealed with appropriate fluorophore-labelled secondary antibodies) and Gal3-Alexa488 (d). (e,g) Fluorescence was quantified after washing and fixation for data as in d and f, respectively. Means ± s.d., n = 3 independent experiments with 21–28 cells assessed from 5 fields (e), n = 4 independent experiments with 21–25 cells assessed from 7 fields (g). Statistical analysis in this figure: Student, unpaired t-test. ∗∗P < 0.01, ∗∗∗∗P < 0.0001.
Supplementary Figure 8 Scans of uncropped blots.
(a–d) Corresponds to Fig. 3b. Western blotting analysis for Gal3 binding proteins. Blots were probed with anti-CD44 (a), anti-IFNAR2 (b), anti-1 integrin (c) or anti-actin antibodies (d). (e,f) Corresponds to Supplementary Fig. 1e. Biochemical CD44 uptake assay. Cell lysates were analysed for anti-CD44-FITC by SDS-PAGE and fluorescence detection. (g) Corresponds to Supplementary Fig. 5a. Cell lysates were probed against Gal3 and tubulin. (h,i) Corresponds to Supplementary Fig. 5d. Cell lysates were probed against clathrin, actin (h) and Gal3 (i). kDa: Kilodalton.
Supplementary Information (PDF 2657 kb)
Supplementary Table 1
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Supplementary Table 2
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Supplementary Table 3
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Supplementary Table 4
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Supplementary Table 5
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Gal3 is localized to CLICs in unperturbed MEFs.
MEFs were incubated with Gal3–HRP for 2 minutes at 37 °C, cooled on ice, labelled with DAB in the presence of ascorbic acid, fixed and processed for electron tomography. Movie displays the captured tilt series before highlighting the segmented Gal3–HRP positive carrier (green). Note the presence of invaginations in the Gal3–HRP carrier, as well as connected tubular extensions, similar to that previously described for CLICs. Image series was captured in IMOD and processed with ImageJ to 10 frames per second. Corresponds to Fig. 1e. (AVI 4501 kb)
Gal3 is localized to CLICs in unperturbed TECs.
TECs were incubated with Gal3–HRP for 2 min at 37 °C, cooled on ice, labelled with DAB in the presence of ascorbic acid, fixed and processed for electron tomography. Movie displays the captured tilt series before highlighting the segmented Gal3–HRP positive carrier (green). Image series was captured in IMOD and processed with ImageJ to 10 frames per second. (AVI 4437 kb)
Gal3 induces cisternal CLIC-like invaginations in ATP-depleted MEFs.
ATP-depleted MEFs were incubated with Gal3–HRP and processed for electron tomography. Movie displays the captured tilt series before highlighting the segmented Gal3–HRP positive carriers (green and yellow), microtubules (orange) and actin microfilaments (blue). Complex, basket and ring shaped structures were observed with morphologies that were strikingly similar to previously captured tomography data of CLICs in unperturbed cells. Note the close proximity of actin and microtubules to Gal3–HRP positive invaginations. Image series was captured in IMOD and processed with ImageJ to 10 frames per second. Corresponds to Fig. 6c. (AVI 4703 kb)
Supplementary Video 4
Gal3 induces tubular invaginations on GSL-containing GUVs, even in the presence of α-galactosidase. GUVs were prepared with a plasma membrane-like lipid composition containing Ni-lipids and a mix of GSLs, were treated with α-galactosidase for 30 min, subsequently incubated with 200 nM Gal3–His at 21 °C, and imaged at the equatorial plane by confocal microscopy. Gal3–His induced invaginations after 30 min. Note: No invaginations formed on GUVs treated with β-galactosidase. Corresponds to Fig. 7f. (MOV 525 kb)
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Lakshminarayan, R., Wunder, C., Becken, U. et al. Galectin-3 drives glycosphingolipid-dependent biogenesis of clathrin-independent carriers. Nat Cell Biol 16, 592–603 (2014). https://doi.org/10.1038/ncb2970
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