During endocytosis, energy is invested to narrow the necks of cargo-containing plasma membrane invaginations to radii at which the opposing segments spontaneously coalesce, thereby leading to the detachment by scission of endocytic uptake carriers1. In the clathrin pathway, dynamin uses mechanical energy from GTP hydrolysis to this effect2,3,4, assisted by the BIN/amphiphysin/Rvs (BAR) domain-containing protein endophilin5,6. Clathrin-independent endocytic events are often less reliant on dynamin7, and whether in these cases BAR domain proteins such as endophilin contribute to scission has remained unexplored. Here we show, in human and other mammalian cell lines, that endophilin-A2 (endoA2) specifically and functionally associates with very early uptake structures that are induced by the bacterial Shiga and cholera toxins, which are both clathrin-independent endocytic cargoes8. In controlled in vitro systems, endoA2 reshapes membranes before scission. Furthermore, we demonstrate that endoA2, dynamin and actin contribute in parallel to the scission of Shiga-toxin-induced tubules. Our results establish a novel function of endoA2 in clathrin-independent endocytosis. They document that distinct scission factors operate in an additive manner, and predict that specificity within a given uptake process arises from defined combinations of universal modules. Our findings highlight a previously unnoticed link between membrane scaffolding by endoA2 and pulling-force-driven dynamic scission.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Multivalent interactions between molecular components involved in fast endophilin mediated endocytosis drive protein phase separation
Nature Communications Open Access 26 August 2022
Cellular and Molecular Life Sciences Open Access 27 May 2022
Scientific Reports Open Access 10 May 2022
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Campelo, F. & Malhotra, V. Membrane fission: the biogenesis of transport carriers. Annu. Rev. Biochem. 81, 407–427 (2012)
Shnyrova, A. V. et al. Geometric catalysis of membrane fission driven by flexible dynamin rings. Science 339, 1433–1436 (2013)
Morlot, S. et al. Membrane shape at the edge of the dynamin helix sets location and duration of the fission reaction. Cell 151, 619–629 (2012)
Faelber, K. et al. Crystal structure of nucleotide-free dynamin. Nature 477, 556–560 (2011)
Sundborger, A. et al. An endophilin-dynamin complex promotes budding of clathrin-coated vesicles during synaptic vesicle recycling. J. Cell Sci. 124, 133–143 (2011)
Neumann, S. & Schmid, S. L. Dual role of BAR domain-containing proteins in regulating vesicle release catalyzed by the GTPase, dynamin-2. J. Biol. Chem. 288, 25119–25128 (2013)
Howes, M. T., Mayor, S. & Parton, R. G. Molecules, mechanisms, and cellular roles of clathrin-independent endocytosis. Curr. Opin. Cell Biol. 22, 519–527 (2010)
Römer, W. et al. Shiga toxin induces tubular membrane invaginations for its uptake into cells. Nature 450, 670–675 (2007)
Mim, C. & Unger, V. M. Membrane curvature and its generation by BAR proteins. Trends Biochem. Sci. 37, 526–533 (2012)
Römer, W. et al. Actin dynamics drive membrane reorganization and scission in clathrin-independent endocytosis. Cell 140, 540–553 (2010)
Ewers, H. et al. GM1 structure determines SV40-induced membrane invagination and infection. Nature Cell Biol. 12, 11–18 (2010)
Milosevic, I. et al. Recruitment of endophilin to clathrin-coated pit necks is required for efficient vesicle uncoating after fission. Neuron 72, 587–601 (2011)
Llobet, A. et al. Endophilin drives the fast mode of vesicle retrieval in a ribbon synapse. J. Neurosci. 31, 8512–8519 (2011)
Kononenko, N. L. et al. Clathrin/AP-2 mediate synaptic vesicle reformation from endosome-like vacuoles but are not essential for membrane retrieval at central synapses. Neuron 82, 981–988 (2014)
Amessou, M. et al. Syntaxin 16 and syntaxin 5 control retrograde transport of several exogenous and endogenous cargo proteins. J. Cell Sci. 120, 1457–1468 (2007)
Draper, R. K., Goda, Y., Brodsky, F. M. & Pfeffer, S. R. Antibodies to clathrin inhibit endocytosis but not recycling to the trans Golgi network in vitro. Science 248, 1539–1541 (1990)
Nesterov, A., Carter, R. E., Sorkina, T., Gill, G. N. & Sorkin, A. Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant μ2 subunit and its effects on endocytosis. EMBO J. 18, 2489–2499 (1999)
Robinson, M. S., Sahlender, D. A. & Foster, S. D. Rapid inactivation of proteins by rapamycin-induced rerouting to mitochondria. Dev. Cell 18, 324–331 (2010)
Campillo, C. et al. Unexpected membrane dynamics unveiled by membrane nanotube extrusion. Biophys. J. 104, 1248–1256 (2013)
Cuvelier, D., Derenyi, I., Bassereau, P. & Nassoy, P. Coalescence of membrane tethers: experiments, theory, and applications. Biophys. J. 88, 2714–2726 (2005)
Evans, E. & Yeung, A. Hidden dynamics in rapid changes of bilayer shape. Chem. Phys. Lipids 73, 39–56 (1994)
Toba, S., Watanabe, T. M., Yamaguchi-Okimoto, L., Toyoshima, Y. Y. & Higuchi, H. Overlapping hand-over-hand mechanism of single molecular motility of cytoplasmic dynein. Proc. Natl Acad. Sci. USA 103, 5741–5745 (2006)
Hehnly, H., Sheff, D. & Stamnes, M. Shiga toxin facilitates its retrograde transport by modifying microtubule dynamics. Mol. Biol. Cell 17, 4379–4389 (2006)
Meinecke, M. et al. Cooperative recruitment of dynamin and BIN/amphiphysin/Rvs (BAR) domain-containing proteins leads to GTP-dependent membrane scission. J. Biol. Chem. 288, 6651–6661 (2013)
Johannes, L. & Römer, W. Shiga toxins - from cell biology to biomedical applications. Nature Rev. Microbiol. 8, 105–116 (2010)
Boucrot, E. et al. Endophilin marks and controls a clathrin-independent endocytic pathway. Nature http://dx.doi.org/10.1038/nature14067 (this issue)
Itoh, T. et al. Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins. Dev. Cell 9, 791–804 (2005)
Roux, A., Uyhazi, K., Frost, A. & De Camilli, P. GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission. Nature 441, 528–531 (2006)
Zha, X. et al. Sphingomyelinase treatment induces ATP-independent endocytosis. J. Cell Biol. 140, 39–47 (1998)
Lizárraga, F. et al. Diaphanous-related formins are required for invadopodia formation and invasion of breast tumor cells. Cancer Res. 69, 2792–2800 (2009)
Riedl, J. et al. Lifeact: a versatile marker to visualize F-actin. Nature Methods 5, 605–607 (2008)
Jordan, M., Schallhorn, A. & Wurm, F. M. Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res. 24, 596–601 (1996)
Montagnac, G. et al. Decoupling of activation and effector binding underlies ARF6 priming of fast endocytic recycling. Curr. Biol. 21, 574–579 (2011)
Mallard, F. & Johannes, L. in Shiga Toxin Methods and Protocols Vol. 73 (eds Philpott, D. & Ebel, F. ) Ch. 17 209–220 (Humana Press, 2003)
Gortat, A., San-Roman, M. J., Vannier, C. & Schmidt, A. A. Single point mutation in Bin/Amphiphysin/Rvs (BAR) sequence of endophilin impairs dimerization, membrane shaping, and Src homology 3 domain-mediated partnership. J. Biol. Chem. 287, 4232–4247 (2012)
Amessou, M., Popoff, V., Yelamos, B., Saint-Pol, A. & Johannes, L. Measuring retrograde transport to the trans-Golgi network. Curr. Protoc. Cell. Biol. Chapter 15, Unit–15.10 (2006)
Singh, R. D. et al. Selective caveolin-1-dependent endocytosis of glycosphingolipids. Mol. Biol. Cell 14, 3254–3265 (2003)
Wolf, A. A. et al. Ganglioside structure dictates signal transduction by cholera toxin and association with caveolae-like membrane domains in polarized epithelia. J. Cell Biol. 141, 917–927 (1998)
Stechmann, B. et al. Inhibition of retrograde transport protects mice from lethal ricin challenges. Cell 141, 231–242 (2010)
Robinson, M. S. & Hirst, J. Rapid inactivation of proteins by knocksideways. Curr. Protoc. Cell Biol. 61, 15.20.1–15.20.7 (2013)
Boncompain, G. & Perez, F. Synchronizing protein transport in the secretory pathway. Curr. Protoc. Cell Biol. Chapter 15, Unit–15.19 (2012)
Pautot, S., Frisken, B. J. & Weitz, D. A. Engineering asymmetric vesicles. Proc. Natl Acad. Sci. USA 100, 10718–10721 (2003)
Pontani, L. L. et al. Reconstitution of an actin cortex inside a liposome. Biophys. J. 96, 192–198 (2009)
Sorre, B. et al. Nature of curvature coupling of amphiphysin with membranes depends on its bound density. Proc. Natl Acad. Sci. USA 109, 173–178 (2012)
Bolte, S. & Cordelieres, F. P. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213–232 (2006)
We would like to acknowledge the following people for help in experiments and providing materials or expertise: A. Berthier, L. Cabanié, K. Carvalho, P. de Camilli, C. Day, D. Drubin, A. El Marjou, V. Fraisier, A. Gautreau, T. Kirchhausen, L. Leconte, C. Merrifield, G. Montagnac, P. Paul-Gilloteaux, M. S. Robinson, L. Sengmanivong and E. Smythe. The facilities as well as scientific and technical assistance from staff in the PICT-IBiSA/Nikon Imaging Centre at Institut Curie-CNRS and the France-BioImaging infrastructure (ANR-10-INSB-04) are acknowledged. This work was supported by grants from the Agence Nationale pour la Recherche (ANR-09-BLAN-283 to L.J. and C.S., ANR-10-LBX-0038 to C.L., ANR-11 BSV2 014 03 to L.J. and P.B., ANR-12-BSV5-0014 to C.S.), the Indo-French Centre for the Promotion of Advanced Science (project no. 3803, L.J.), Marie Curie Actions — Networks for Initial Training (FP7-PEOPLE-2010-ITN, L.J.), European Research Council advanced grant (project 340485, L.J.), Marie Curie International Reintegration Grant (FP7-RG-277078, C.W.), the Royal Society (RG120481, E.B.), Fondation ARC pour la Recherche sur le Cancer (DEQ20120323737, C.S.), National Institutes of Health (RO1 GM106720, A.K.K.), La Ligue contre le Cancer, Comité de Paris (RS08/75-89, A.A.S.), and by fellowships from Fondation ARC pour la Recherche sur le Cancer (H.-F.R., J.L. and M.-D.G.-C.), AXA Research Funds (J.L. and M.-D.G.-C.), the Biological Sciences Research Council (David Phillips Research Fellowship to E.B.), Chateaubriand fellowship and the France and Chicago Collaborating in the Sciences grant (M.S.). The Johannes, Lamaze, Sykes and Bassereau teams are members of Labex CelTisPhyBio (11-LBX-0038) and of Idex Paris Sciences et Lettres (ANR-10-IDEX-0001-02 PSL). P.B.’s group belongs to the French research consortium CellTiss.
The authors declare no competing financial interests.
Extended data figures and tables
a, Listing of BAR domain proteins that were tested in our localization screen. Toca-1, Toca-3 and amphiphysin-2 (yellow underlay) colocalized with STxB on tubular structures (not shown). See below for endoA2 (green underlay). b, At variance with the expected phenotype, expression of GFP-tagged endoA2 (green) led to the disappearance of long STxB-induced plasma membrane invaginations (red). Tubule length was quantified in non-transfected control cells (n = 50) and endoA2–GFP-expressing cells (n = 59). Quantifications show mean ± s.e.m. of two independent experiments. ***P < 0.001 (two-tailed Mann–Whitney U test). c, STxB–Cy3 (50 nM, red) was incubated for 45 min at 37 °C with cells expressing GFP (i), endoA2–GFP (ii) or Rab5(Q79L)–GFP (iii). Expression of endoA2–GFP did not affect STxB trafficking to the Golgi/TGN membranes. n = 48 for GFP-expressing cells, n = 43 for endoA2–GFP-expressing cells, and n = 46 for Rab5(Q79L)–GFP expressing cells; two independent experiments. Quantifications show mean ± s.e.m. NS, non significant; ***P < 0.001 (Bonferroni’s multiple comparison test). Scale bars, 10 μm.
a–c, STxB is internalized in endoA2–GFP-positive vesicles and induces the recruitment of endoA2–GFP to the plasma membrane. HeLaM cells stably expressing endoA2–GFP–FKBP (a, c), or HeLa cells transiently expressing endoA2–GFP (b) were incubated continuously at 37 °C respectively with 0.2 µM STxB–Cy5 or 0.5 µM STxB–Cy3. Observation by live-cell imaging using a spinning disk microscope (a, c) or TIRFM (b). a, STxB endocytosis. Image series of 170 s (for the complete sequence, see Supplementary Video 1). Arrows show the formation in the cell periphery of STxB–Cy5 and endoA2–GFP-positive vesicles that move into cells. b, Kymographs of HeLa cells transiently expressing endoA2–GFP in the absence (−) or presence (+) of STxB–Cy3. Quantification from TIRFM recordings of endoA2–GFP spot lifetime (−STxB, n = 1,768 events; +STxB, n = 144 events; three independent experiments. ***P < 0.001 (two-tailed Mann–Whitney U test). c, Plasma membrane recruitment of endoA2. STxB–Cy5 was added 15 s after the beginning of image acquisition. Two time points are shown (for the complete sequence, see Supplementary Video 2): before STxB addition (−STxB, 0 s) and after STxB addition (+STxB, +80 s). A striking recruitment of endoA2–GFP to the plasma membrane was observed (white arrowheads). Fluorescence intensities along plasma membrane segments of endoA2–GFP and STxB–Cy5 were followed over time (means ± s.e.m., n = 6 cells, three independent experiments). Note that both rose in a similar manner. d–i, EndoA2 and Shiga toxin poorly colocalize with markers of clathrin-mediated endocytosis. All images show live-cell TIRFM recordings. d, HeLa cells were transfected with plasmids expressing endoA2–GFP and μ2–mCherry. The overlap between both markers was very small. e, The genome-edited cell line SK-MEL-2 expressing LCa–mRFP was transfected transiently with the endoA2–GFP plasmid. Again, both markers showed very little overlap. f, HeLa cells were transfected with plasmids expressing endoA2–GFP and DNM2–mRFP. A substantial overlap was observed between both markers. For d–f, quantifications are reported in Fig. 1d. g–i, HeLa cells transiently expressing mRFP–LCa (g) or μ2–mCherry (h), and the genome-edited cell line SK-MEL-2 expressing LCa–mRFP (i) were continuously incubated with 0.5 μM STxB-Alexa488 for 5 min at 37 °C. Note the weak overlap between STxB and the other markers. j, Quantification of colocalization for g–i. Mean ± s.e.m. of the following numbers of cells: mRFP–LCa, n = 11; μ2–mCherry, n = 6; LCa–mRFP, n = 19; two independent experiments. ***P < 0.001 (Bonferroni’s multiple comparison test). Scale bars, 2 μm.
Extended Data Figure 3 Effect of endoA2 depletion on Shiga toxin endocytosis at early times of uptake.
a, After binding on ice for 30 min, STxB–Cy3 (50 nM, red) was incubated for 5 min at 37 °C with HeLa cells that had been transfected with negative control siRNAs (18 images), or siRNAs against endoA2 (23 images). After fixation, Golgi membranes were labelled with antibodies against giantin (green). Note the presence of short STxB-containing tubules (arrowheads) in the endoA2-depletion condition. The western blot documents the efficiency of endoA2 depletion. Clathin heavy chain (CHC) was used as a loading control. b, c, Characterization of STxB conjugates with monofunctionalized nanogold. b, HeLa cells were incubated for 45 min at 37 °C in the indicated conditions, fixed, treated for silver enhancement, and viewed by transmission light microscopy. Note the strong perinuclear signal (arrowheads) that was visible only when cells were incubated with nanogold-coupled STxB (113 images). c, HeLa cells were incubated for 45 min at 37 °C with nanogold-coupled STxB, fixed, treated for silver enhancement, and viewed by electron microscopy after sectioning (32 images). Note the strong signal in the Golgi region (G), which validated the functionality of the conjugate. Some STxB-nanogold could still be seen at the plasma membrane (PM) and in endosomes (E). N, nucleus. d, HeLa cells were transfected with negative control siRNAs, or siRNAs against endoA2, incubated for 30 min on ice with nanogold-coupled STxB, shifted for 5 min to 37 °C, and then fixed. Silver enhancement was used to enlarge nanogold particles. Note that STxB-containing invaginations (arrowheads) were much longer in endoA2-depleted cells than in cells transfected with negative control siRNA. Quantification is shown in Fig. 2b (see legend for cell numbers). Scale bars, 10 μm (a, b), 200 nm (c), 100 nm (d).
Extended Data Figure 4 Effect of endoA2 depletion on STxB and CTxB uptake, and rescue with endoA2 mutants.
a, Efficiency of endoA2 depletion with different siRNA sequences. HeLa cells were transfected with three different siRNA sequences against human endoA2 (5, 7 and 8). The efficiency of endoA2 depletion was monitored by western blotting with antibodies against endoA2. Cells transfected with negative control siRNA were used for comparison. The western signal for CHC served as loading control. Quantification shows mean ± s.e.m.; three independent experiments. NS, non significant; **P < 0.01, ***P < 0.001 (Bonferroni’s multiple comparison test). Trafficking studies on these cells are reported in Fig. 2c. b, Effect of endoA2 depletion on STxB binding to cells. HeLa cells were transfected with siRNAs as in a, detached, incubated for 30 min on ice with 1 μM STxB-Alexa488 and 10 μg ml−1 Tf-Alexa647, washed and analysed by FACS. Cells treated with the glycosylceramide synthase inhibitor PPMP were used as a control for signal specificity. Mean ± s.e.m. of three independent experiments are shown (except for PPMP, which was done twice). The increased STxB binding with siRNA 8 is not explained at this stage. These cell surface binding data serve as controls for the sulfation experiment of Fig. 2c. c, EndoA2 functions in CTxB uptake. CTxB (5 nM) uptake assay (three independent experiments) after 10 min at 37 °C in conditions of endoA2 depletion. *P < 0.05 (two-tailed t-test). As opposed to STxB, CTxB could be removed from the plasma membrane by acid washes, and endocytosis could therefore be measured directly using a plate-reader assay (see Supplementary Methods). d, STxB trafficking rescue experiment under endoA2-depletion conditions. HeLa cells were transfected with negative control siRNAs, or siRNAs against endoA2. After 48 h, endoA2-depleted cells were transfected for 24 h with siRNA-resistant expression vectors encoding: GFP-tagged H0 helix deletion mutant (endoA2ΔH0–GFP or ΔH0), SH3 domain deletion mutant (endoA2ΔSH3–GFP or ΔSH3), or full-length GFP-tagged wild-type endoA2 (FL). STxB–Cy3 (50 nM, red) was incubated with these cells for 45 min at 37 °C (for quantification and statistical data, see Fig. 2d). The H0 helix deletion mutant did not rescue STxB transport to perinuclear Golgi/TGN membranes, as opposed to wild-type endoA2 and endoA2ΔSH3. The western blot documents the efficiency of endoA2 depletion in siRNA-transfected cells. CHC was used as a loading control. e, Effect of endoA2 mutants on STxB-induced membrane invaginations. Non-transfected HeLa cells (ctrl) or cells expressing full-length endoA2 (FL), H0 helix deletion mutant (ΔH0), or SH3 domain deletion mutant (ΔSH3) were ATP-depleted and incubated for 10 min at 37 °C with 0.5 μM STxB–Cy3. Tubule length was quantified (control, n = 277 in 102 cells; full-length, n = 90 in 15 cells; ΔH0, n = 183 in 48 cells; ΔSH3, n = 164 in 36 cells); two independent experiments. Quantifications show mean ± s.e.m. ***P < 0.001 (Bonferroni’s multiple comparison test). Scale bars, 10 µm.
a–c, EndoA2 depletion does not affect general trafficking processes. All experiments were performed on cells transfected with negative control siRNAs, and cells that were depleted for endoA2. a, Transferrin uptake (left, n = 3) and recycling (right, n = 4). EndoA2 depletion did not affect any of these processes. Quantifications show mean ± s.e.m. of the indicated numbers of independent experiments. b, Steady-state localization of TGN46 and CI-MPR (red), as analysed by immunofluorescence. Golgi membranes were labelled with antibodies against giantin (green). EndoA2 depletion did not affect the steady-state localization of these markers (TGN46 in siCtrl or siEndoA2 cells: 12 images; CI-MPR in siCtrl or siEndoA2 cells: 10 images; two independent experiments). c, Anterograde trafficking of E-cadherin. After release from endoplasmic reticulum, SBP-mCherry-E-cadherin protein was detected at the cell surface with anti-mCherry antibodies (0, 20 and 60 min time points). After 60 min, the relative means (±s.e.m.) of anti-mCherry fluorescence per unit area were quantified for control (6 images, 87 cells) and endoA2-depleted cells (6 images, 81 cells); three independent experiments. No significant difference was observed using a two-tailed t-test (P > 0.05). d–f, Depletion of α-adaptin does not affect Shiga toxin trafficking. All experiments were performed on negative control siRNA-transfected cells, and on cells that were depleted for the indicated proteins. Quantifications show mean ± s.e.m. d, Sulfation analysis of retrograde STxB transport (three independent experiments). HeLa cells in the indicated conditions were incubated for 20 min at 37 °C with STxB-Sulf2 in the presence of radioactive sulphate, and sulfated STxB-Sulf2 was measured by autoradiography. Note that α-adaptin depletion did not affect sulfation of STxB-Sulf2, whereas depletion of endoA2 or syntaxin-16 had a strong effect. NS, non significant; **P < 0.01 (Bonferroni’s multiple comparison test). e, Immunofluorescence analysis. HeLa cells in the indicated conditions were incubated for 45 min at 37 °C with 0.05 μM STxB–Cy3 (red). During the last 10 min, 10 μg ml−1 Tf-Alexa488 (green) were added in the growth medium. Cells were placed on ice, and cell-surface-exposed transferrin was removed by acid washes. After fixation, cells were labelled for giantin (blue). Note that α-adaptin depletion strongly inhibited transferrin uptake, but not retrograde transport of STxB to TGN/Golgi membranes. Transferrin uptake was quantified for control (5 images, 102 cells) and α-adaptin-depleted cells (5 images, 108 cells); two independent experiments. ***P < 0.001 (two-tailed t-test). f, siRNA-mediated depletion of endoA2 and of syntaxin-16 was analysed by western blotting. CHC was used as loading control. Scale bars, 10 μm.
a, Knocksideways. HeLaM cells stably expressing Mito–YFP–FRB and rat endoA2–GFP–FKBP (green) were transfected with negative control siRNAs (siCtrl), or siRNAs against human endoA2 (siEndoA2) that did not cross with the rat sequence. STxB–Cy3 (0.5 μM, red) was incubated for 15 min at 37 °C with ATP-depleted cells. The cells were then fixed at 37 °C, and viewed by confocal microscopy. Quantification of tubule formation and cell numbers are shown in Fig. 3a. Note that STxB-induced tubule length reversibly increased after endoA2–GFP–FKBP sequestration. The depletion of endogenous human endoA2, and the expression of GFP-FKBP-tagged rat endoA2 were assessed by western blotting with anti-endoA2 and anti-GFP antibodies, respectively. Western blotting against CHC was used as loading control. b, c, Interfering with microtubules or dynein motors strongly affects STxB-induced tubule length. b, HeLa cells were incubated for 1 h at 37 °C with DMSO or 10 μM nocodazole, ATP-depleted for 20 min, and then incubated for 10 min at 37 °C with 0.5 μM STxB–Cy3 (red) in the same conditions. Labelling with an antibody against α-tubulin (green) was used to visualize the efficiency of nocodazole treatment. Long tubular structures containing STxB could not be detected after incubation with nocodazole (−ATP/+DMSO: 18 images; −ATP/+nocodazole: 16 images; three independent experiments). c, Heavy chains of cytoplasmic dyneins (DYNC1H1 and DYNC2H1) were depleted from HeLa cells with siRNAs. Cells were ATP-depleted and then incubated for 10 min at 37 °C with 0.5 μM STxB–Cy3 (red). The presence of long STxB-induced tubules was strongly decreased under these conditions. Tubule length was quantified for negative-control-siRNA-treated cells (siCtrl, n = 188 tubules in 74 cells) and for DYNC2H1-depleted cells (siDYNC2H1, n = 165 tubules in 60 cells); two independent experiments. Quantifications show means ± SEM. ***P < 0.001, two-tailed Mann–Whitney U test. The western blot documents the efficiency of DYNC1H1 depletion. α-tubulin was used as a loading control. d, e, Model membranes experiments. d, Measurement of tube pulling force over time in the absence or presence of endoA2ΔH0 mutant (7 µM in injection pipette). Representative of six experiments. e, Scission experiments with tethers that were coated with endoA2ΔH0 (n = 5). Scale bars,10 μm (a, c) and 5 μm (b, e).
Extended Data Figure 7 Electron microscopy of STxB-nanogold on ATP-depleted cells following treatment with nocodazole.
HeLa cells were treated for 1 h at 37 °C with DMSO (top) or 10 μM nocodazole (bottom). During the last 20 min, ATP was depleted. Cells were then incubated for 10 min with STxB-nanogold in the continued presence of inhibitors, fixed, and prepared for electron microscopy. In the DMSO condition (top), long tubular structures connected to the cell surface were observed (magnified views in right insets, red arrowheads in C), as expected from light microscopy experiments. These structures were in close proximity with cytoskeletal elements, as indicated with blue arrowheads in magnifications A and B. In the nocodazole condition (bottom), STxB-induced plasma membrane invaginations were still present (arrowheads), but much shorter (mean length of 118.2 ± 7.0 nm, n = 109 invaginations; 0.90 ± 0.12 invaginations/μm of plasma membrane, n = 28 images; three independent experiments) than in the absence of the compound. Scale bar sizes are indicated.
a–c, EndoA2 codistribution with actin. a, HeLa cells transiently co-expressing endoA2-GFP and LifeAct-mCherry were observed by time-resolved TIRF microscopy. The panel shows acquisitions at the plasma membrane that were taken at 5-s intervals. Arrowheads point out examples of structures on which endoA2 and actin colocalize in a dynamic manner. Quantification of colocalization of endoA2-positive structures with LifeAct is presented as mean ± s.e.m. (n = 7 cells, 2 independent experiments). b, EndoA2-depleted HeLa cells were incubated continuously for 5 min at 37 °C with 0.5 μM STxB–Cy3. After fixation, actin filaments were stained with phalloidin-FITC. Arrowheads indicate STxB-induced tubules that are decorated by actin. c, HeLa cells transfected with negative control or endoA2 siRNAs and transiently expressing LifeAct-mCherry were observed by TIRF microscopy after addition of 0.5 μM STxB–Cy3 at 37 °C. Arrowheads point out examples of structures on which STxB and actin colocalize. Quantification of colocalization of STxB-positive structures with LifeAct is presented as means ± s.e.m. (n = 6 cells, two independent experiments). NS, non significant (two-tailed t-test). d, e, Analysis of STxB-induced plasma membrane invaginations in function of endoA2 depletion and/or actin perturbation. d, HeLa cells were transfected with negative control siRNAs or with siRNAs against endoA2, and treated or not with 0.5 μM latrunculin-A. The cells were then incubated continuously for 5 min at 37 °C in the presence of 0.5 μM STxB–Cy3 (red), fixed and labelled with phalloidin (green). The quantification of STxB tubule length is shown in Fig. 4a. Note that tubule length increased with combined treatments (siCtrl+DMSO: 22 images; siEndoA2+DMSO: 11 images; siCtrl+LatA: 14 images; siEndoA2+LatA: 14 images). e, Depletion of endoA2 was analysed by western blotting. CHC was used as loading control. f, Efficiency of ARPC2 depletion. HeLa cells were transfected for 72 h with a smartpool of four siRNA sequences against ARPC2. The efficiency of ARPC2 depletion was monitored by western blotting with antibodies against ARPC2. The western signal for CHC served as loading control. Corresponds to experiments in Fig. 4a. Scale bars, 2 μm (a, c), 5 μm (b) and 10 μm (d).
Extended Data Figure 9 Combined effects of interference with endoA2, dynamin and actin on STxB-induced membrane invaginations.
a–c, Endogenous endoA2 and dynamin are found on STxB-induced plasma membrane invaginations. a, Dynamin-2 was depleted from cells (siDNM2), which were then incubated continuously for 5 min at 37 °C in the presence or absence of 0.5 μM STxB–Cy3 (red), fixed, labelled for the indicated markers, and analysed by confocal microscopy. Note that endoA2-containing tubules (green) were seen only in the presence of STxB. No overlap was observed with clathrin (blue). –STxB: representative of 15 images; +STxB: representative of 35 images; two independent experiments. b, Experiment as in a on wild-type cells that were analysed by wide field microscopy. As above, endoA2 (green) was found on tubular structures only in the presence of STxB (red). The quantification shows mean ± s.e.m. of 3 independent experiments on 234 cells without STxB (−STxB) and 921 cells with STxB (+STxB). **P < 0.01, two-tailed t-test. c, Negative control siRNA transfected HeLa cells and endoA2-depleted cells were incubated for 5 min at 37 °C in the presence of 0.5 μM STxB–Cy3 (red). Endogenous endoA2 (blue) and dynamin (green, arrowheads) were labelled with specific antibodies, detected by immunofluorescence, and viewed by structured illumination microscopy. Note that dynamin localized in spots on STxB-induced invaginations, while endoA2 distributed in a continuous manner. d, Analysis of STxB-induced plasma membrane invaginations in function of endoA2 and dynamin-2 depletion, and actin perturbation. HeLa cells were depleted for dynamin-2 alone, dynamin-2 in combination with endoA2, or both depletions in combination with 0.25 μM latrunculin-A treatment, as indicated. These cells were then incubated continuously for 5 min at 37 °C with 0.5 μM STxB–Cy3 (red), fixed at 37 °C, and viewed by confocal microscopy. Note that the tubulation phenotype increased with each additional interference modality (siDNM2: 21 images; siEndoA2+siDNM2: 15 images; siEndoA2+siDNM2+LatA: 16 images; two independent experiments). The quantification of tubule length in the different experimental conditions of this figure is shown in Fig. 4a. The depletion of dynamin-2 and endoA2 was validated by immunoblotting. CHC was used as loading control. Scale bars, 5 μm (a, b), 0.5 μm (c) and 10 μm (d).
a–d, HeLa cells were depleted for endoA2 (a), dynamin-2 (b), incubated with 0.25 μM latrunculin-A (c), or submitted concomitantly to all three perturbations (d). These cells were then further incubated for 1 h in the presence of increasing concentrations of STx-1, at the end of which protein biosynthesis was measured. Note that only the triple treatment condition had a strong effect on cell intoxication. The protection factors determined on 4–13 independent experiments are shown in Fig. 4c. siEndoA2, n = 7; siDNM2, n = 4; latrunculin-A, n = 13; triple, n = 4. Error bars show the s.e.m.
This file contains Supplementary Methods and Supplementary References. (PDF 239 kb)
HeLaM cells stably expressing endoA2-GFP-FKBP were incubated continuously at 37°C with 0.2 µM STxBCy5, and observed by live cell imaging using a spinning disk microscope for 170 sec at 0.34 sec intervals (exposure time: 10 msec for each channel). Arrowheads indicate STxB and endoA2-GFP-positive vesicles in the cell periphery that move inwards. Scale bar, 2 µm. (MP4 9460 kb)
HeLaM cells stably expressing endoA2-GFP-FKBP were observed by live cell imaging at 37°C using a spinning disk microscope. STxB-Cy5 (0.2 µM) was added 15 sec after the beginning of image acquisition. Acquisition was performed for 80 sec at 0.84 sec intervals (exposure time: 80 msec for each channel). Scale bar, 2 µm. (MP4 2789 kb)
About this article
Cite this article
Renard, HF., Simunovic, M., Lemière, J. et al. Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis. Nature 517, 493–496 (2015). https://doi.org/10.1038/nature14064
This article is cited by
Multivalent interactions between molecular components involved in fast endophilin mediated endocytosis drive protein phase separation
Nature Communications (2022)
Integrating protein copy numbers with interaction networks to quantify stoichiometry in clathrin-mediated endocytosis
Scientific Reports (2022)
Scientific Reports (2022)
Cellular and Molecular Life Sciences (2022)
Inositol triphosphate-triggered calcium release blocks lipid exchange at endoplasmic reticulum-Golgi contact sites
Nature Communications (2021)