Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Shiga toxin induces tubular membrane invaginations for its uptake into cells

Nature volume 450, pages 670–675 (2007)Cite this article

Abstract

Clathrin seems to be dispensable for some endocytic processes and, in several instances, no cytosolic coat protein complexes could be detected at sites of membrane invagination. Hence, new principles must in these cases be invoked to account for the mechanical force driving membrane shape changes. Here we show that the Gb3 (glycolipid)-binding B-subunit of bacterial Shiga toxin induces narrow tubular membrane invaginations in human and mouse cells and model membranes. In cells, tubule occurrence increases on energy depletion and inhibition of dynamin or actin functions. Our data thus demonstrate that active cellular processes are needed for tubule scission rather than tubule formation. We conclude that the B-subunit induces lipid reorganization that favours negative membrane curvature, which drives the formation of inward membrane tubules. Our findings support a model in which the lateral growth of B-subunit–Gb3 microdomains is limited by the invagination process, which itself is regulated by membrane tension. The physical principles underlying this basic cargo-induced membrane uptake may also be relevant to other internalization processes, creating a rationale for conceptualizing the perplexing diversity of endocytic routes.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: STxB is found on endocytic membrane invaginations.
Figure 2: STxB induces tubular invaginations on cells.
Figure 3: Reconstitution of tubule formation on GUVs.
Figure 4: STxB-induced domain formation.

Similar content being viewed by others

References

  1. Mayor, S. & Pagano, R. E. Pathways of clathrin-independent endocytosis. Nature Rev. Mol. Cell Biol. 8, 603–612 (2007)

    Article  CAS  Google Scholar 

  2. Kirkham, M. & Parton, R. G. Clathrin-independent endocytosis: new insights into caveolae and non-caveolar lipid raft carriers. Biochim. Biophys. Acta 1745, 273–286 (2005)

    Article  CAS  PubMed  Google Scholar 

  3. Pelkmans, L. & Helenius, A. Endocytosis via caveolae. Traffic 3, 311–320 (2002)

    Article  CAS  PubMed  Google Scholar 

  4. Conner, S. D. & Schmid, S. L. Regulated portals of entry into the cell. Nature 422, 37–44 (2003)

    Article  ADS  CAS  PubMed  Google Scholar 

  5. Johannes, L. & Lamaze, C. Clathrin-dependent or not: Is it still the question? Traffic 3, 443–451 (2002)

    Article  CAS  PubMed  Google Scholar 

  6. Nichols, B. J. & Lippincott-Schwartz, J. Endocytosis without clathrin coats. Trends Cell Biol. 11, 406–412 (2001)

    Article  CAS  PubMed  Google Scholar 

  7. Nichols, B. J. et al. Rapid cycling of lipid raft markers between the cell surface and Golgi complex. J. Cell Biol. 153, 529–541 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Lauvrak, S. U., Torgersen, M. L. & Sandvig, K. Efficient endosome-to-Golgi transport of Shiga toxin is dependent on dynamin and clathrin. J. Cell Sci. 117, 2321–2331 (2004)

    Article  CAS  PubMed  Google Scholar 

  9. Saint-Pol, A. et al. Clathrin adaptor epsinR is required for retrograde sorting on early endosomal membranes. Dev. Cell 6, 525–538 (2004)

    Article  CAS  PubMed  Google Scholar 

  10. Praefcke, G. J. & McMahon, H. T. The dynamin superfamily: universal membrane tubulation and fission molecules? Nature Rev. Mol. Cell Biol. 5, 133–147 (2004)

    Article  CAS  Google Scholar 

  11. Macia, E. et al. Dynasore, a cell-permeable inhibitor of dynamin. Dev. Cell 10, 839–850 (2006)

    Article  CAS  PubMed  Google Scholar 

  12. Sabharanjak, S., Sharma, P., Parton, R. G. & Mayor, S. GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway. Dev. Cell 2, 411–423 (2002)

    Article  CAS  PubMed  Google Scholar 

  13. Kirkham, M. et al. Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles. J. Cell Biol. 168, 465–476 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Glebov, O. O., Bright, N. A. & Nichols, B. J. Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells. Nature Cell Biol. 8, 46–54 (2006)

    Article  CAS  PubMed  Google Scholar 

  15. 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)

    Article  ADS  CAS  PubMed  Google Scholar 

  16. Falguières, T. et al. Targeting of Shiga toxin B-subunit to retrograde transport route in association with detergent resistant membranes. Mol. Biol. Cell 12, 2453–2468 (2001)

    Article  PubMed  PubMed Central  Google Scholar 

  17. Bast, D. J., Banerjee, L., Clark, C., Read, R. J. & Brunton, J. L. The identification of three biologically relevant globotriaosyl ceramide receptor binding sites on the Verotoxin 1 B subunit. Mol. Microbiol. 32, 953–960 (1999)

    Article  CAS  PubMed  Google Scholar 

  18. Henley, J. R., Krueger, E. W., Oswald, B. J. & McNiven, M. A. Dynamin-mediated internalization of caveolae. J. Cell Biol. 141, 85–99 (1998)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Farsad, K. & De Camilli, P. Mechanisms of membrane deformation. Curr. Opin. Cell Biol. 15, 372–381 (2003)

    Article  CAS  PubMed  Google Scholar 

  20. McMahon, H. T. & Gallop, J. L. Membrane curvature and mechanisms of dynamic cell membrane remodelling. Nature 438, 590–596 (2005)

    Article  ADS  CAS  PubMed  Google Scholar 

  21. Zimmerberg, J. & Kozlov, M. M. How proteins produce cellular membrane curvature. Nature Rev. Mol. Cell Biol. 7, 9–19 (2006)

    Article  CAS  Google Scholar 

  22. Stein, P. E., Boodhoo, A., Tyrrell, G. J., Brunton, J. L. & Read, R. J. Crystal structure of the cell-binding B oligomer of verotoxin-1 from E. coli. . Nature 355, 748–750 (1992)

    Article  ADS  CAS  PubMed  Google Scholar 

  23. Hegnerelle, X. et al. Two-dimensional structures of the Shiga toxin B-subunit and of a chimera bound to the glycolipid receptor Gb3. J. Struct. Biol. 139, 113–121 (2002)

    Article  CAS  Google Scholar 

  24. Israelachvili, J. Intramolecular and Surface Forces. 2nd edn, pt 3 (Academic Press, 1991)

    Google Scholar 

  25. Ling, H. et al. Structure of Shiga-like toxin I B-pentamer complexed with an analogue of its receptor Gb3. Biochemistry 37, 1777–1788 (1998)

    Article  CAS  PubMed  Google Scholar 

  26. Sens, P. & Turner, M. S. Theoretical model for the formation of caveolae and similar membrane invaginations. Biophys. J. 86, 2049–2057 (2004)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  27. Fraser, M. E., Chernaia, M. M., Kozlov, Y. V. & James, M. N. Crystal structure of the holotoxin from Shigella dysenteriae at 2.5 Å resolution. Nature Struct. Biol. 1, 59–64 (1994)

    Article  CAS  PubMed  Google Scholar 

  28. Sens, P. & Safran, S. A. Inclusions induced phase separation in mixed lipid film. Eur. Phys. J. E 1, 237–248 (2000)

    Article  CAS  Google Scholar 

  29. Jensen, M. H., Morris, E. J. & Simonsen, A. C. Domain shapes, coarsening, and random patterns in ternary membranes. Langmuir 23, 8135–8141 (2007)

    Article  CAS  PubMed  Google Scholar 

  30. Gaus, K. et al. Visualizing lipid structure and raft domains in living cells with two-photon microscopy. Proc. Natl Acad. Sci. USA 100, 15554–15559 (2003)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  31. Gaus, K., Zech, T. & Harder, T. Visualizing membrane microdomains by Laurdan 2-photon microscopy. Mol. Membr. Biol. 23, 41–48 (2006)

    Article  CAS  PubMed  Google Scholar 

  32. Julicher, F. Domain induced budding of vesicles. Phys. Rev. Lett. 70, 2964–2967 (1993)

    Article  ADS  CAS  PubMed  Google Scholar 

  33. Baumgart, T., Hess, S. T. & Webb, W. W. Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension. Nature 425, 821–824 (2003)

    Article  ADS  CAS  PubMed  Google Scholar 

  34. Bacia, K., Schwille, P. & Kurzchalia, T. Sterol structure determines the separation of phases and the curvature of the liquid-ordered phase in model membranes. Proc. Natl Acad. Sci. USA 102, 3272–3277 (2005)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  35. Antonny, B. Membrane deformation by protein coats. Curr. Opin. Cell Biol. 18, 386–394 (2006)

    Article  CAS  PubMed  Google Scholar 

  36. Reynwar, B. J. et al. Aggregation and vesiculation of membrane proteins by curvature-mediated interactions. Nature 447, 461–464 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  37. Oda, R., Huc, I., Schmutz, M., Candau, S. J. & MacKintosh, F. C. Tuning bilayer twist using chiral counterions. Nature 399, 566–569 (1999)

    Article  ADS  CAS  PubMed  Google Scholar 

  38. Sarasij, R. C. & Rao, M. Tilt texture domains on a membrane and chirality induced budding. Phys. Rev. Lett. 88, 088101 (2002)

    Article  ADS  CAS  Google Scholar 

  39. Sarasij, R. C., Mayor, S. & Rao, M. Chirality induced budding: a raft-mediated mechanism for endocytosis and morphology of caveolae? Biophys. J. 92, 3140–3158 (2007)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  40. Hancock, J. F. Lipid rafts: contentious only from simplistic standpoints. Nature Rev. Mol. Cell Biol. 7, 456–462 (2006)

    Article  CAS  Google Scholar 

  41. Johannes, L., Tenza, D., Antony, C. & Goud, B. Retrograde transport of KDEL-bearing B-fragment of Shiga toxin. J. Biol. Chem. 272, 19554–19561 (1997)

    Article  CAS  PubMed  Google Scholar 

  42. Merrifield, C. J., Perrais, D. & Zenisek, D. Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells. Cell 121, 593–606 (2005)

    Article  CAS  PubMed  Google Scholar 

  43. Zha, X. et al. Sphingomyelinase treatment induces ATP-independent endocytosis. J. Cell Biol. 140, 39–47 (1998)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Mathivet, L., Cribier, S. & Devaux, P. F. Shape change and physical properties of giant phospholipid vesicles prepared in the presence of an AC electric field. Biophys. J. 70, 1112–1121 (1996)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  45. Menke, M., Gerke, V. & Steinem, C. Phosphatidylserine membrane domain clustering induced by annexin A2/S100A10 heterotetramer. Biochemistry 44, 15296–15303 (2005)

    Article  CAS  PubMed  Google Scholar 

  46. Schmidt, R. R. & Zimmermann, P. Synthesis of D-erythro-sphingosines. Tetrahedr. Lett. 27, 481–484 (1986)

    Article  CAS  Google Scholar 

  47. Manzoni, L., Lay, L. & Schmidt, R. R. Synthesis of Lewis A and Lewis X pentasaccharides based on N-trichloroethoxycarbonyl protection. J. Carbohydr. Chem. 17, 739–758 (1998)

    Article  CAS  Google Scholar 

  48. Aly, M. R., Rochaix, P., Amessou, M., Johannes, L. & Florent, J. C. Efficient Synthesis of globo- and isoglobotriosides bearing a cinnamoylphenyl tag as novel electrophilic thiol-specific carbohydrate reagents. Carbohydr. Res. 341, 2026–2036 (2006)

    Article  CAS  PubMed  Google Scholar 

  49. Qiu, D. & Schmidt, R. R. Glycosyl imidates. LII. Synthesis of globotriaosylceramide (Gb3) and isoglobotriaosylceramide (isoGb3). Liebigs Ann. Chem. 3, 217–224 (1992)

    Article  Google Scholar 

  50. Figueroa-Perez, S. & Schmidt, R. R. Total synthesis of α-galactosyl cerebroside. Carbohydr. Res. 328, 95–102 (2000)

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank S. Mayor and M. Rao for helpful discussions and sharing unpublished data, M. McNiven for advice, and J.-B. Sibarita and B. Stechmann for assistance with experiments. The following colleagues are acknowledged for providing materials: T. Kirchhausen, M. Bornens, M. McNiven and A. Smith. Our laboratories were supported by: Ligue Nationale contre le Cancer, Association de Recherche Contre le Cancer, Curie Institute (PIC Vectorisation), European Commission (SoftComp), CNRS (ACI Dynamique et réactivité des assemblages biologiques) and the Human Frontier Science Program Organization. W.R. holds a postdoctoral fellowship from the CNRS, and L.B. is supported by a grant from the Direction Générale pour l'Armement (DGA).

Author information

Authors and Affiliations

  1. Institut Curie, Centre de Recherche, Laboratoire Trafic, Signalisation et Ciblage Intracellulaires,,

    Winfried Römer, Valérie Chambon, Christophe Lamaze & Ludger Johannes

  2. Laboratoire Physico-Chimie,,

    Ludwig Berland & Patricia Bassereau

  3. Laboratoire Structure et Compartiments Membranaires,,

    Danièle Tenza & Graça Raposo

  4. Laboratoire Chimie Organique (Vectorisation), 26 rue d’Ulm, 75248 Paris Cedex 05, France ,

    Mohamed R. E. Aly & Jean-Claude Florent

  5. CNRS UMR144,,

    Winfried Römer, Valérie Chambon, Danièle Tenza, Vincent Fraisier, Christophe Lamaze, Graça Raposo & Ludger Johannes

  6. Université P. et M. Curie/CNRS UMR168,,

    Ludwig Berland & Patricia Bassereau

  7. CNRS UMR176,,

    Mohamed R. E. Aly & Jean-Claude Florent

  8. Centre for Vascular Research, University of New South Wales, 2052 Sydney, Australia ,

    Katharina Gaus

  9. Department of Haematology, Prince of Wales Hospital, 2031 Sydney, Australia

    Katharina Gaus

  10. Institut für Organische und Biomolekulare Chemie, Georg-August Universität, Tammannstr. 2, 37077 Göttingen, Germany ,

    Barbara Windschiegl & Claudia Steinem

  11. Laboratoire de Physiologie Cellulaire de la Synapse, CNRS UMR 5091 et Université Bordeaux 2, Institut François Magendie, 33077 Bordeaux, France

    David Perrais

  12. UMR Gulliver CNRS-ESPCI 7083, 10 rue Vauquelin, 75231 Paris Cedex 05, France ,

    Pierre Sens

Authors
  1. Winfried Römer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ludwig Berland
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Valérie Chambon
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Katharina Gaus
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Barbara Windschiegl
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Danièle Tenza
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mohamed R. E. Aly
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Vincent Fraisier
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jean-Claude Florent
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. David Perrais
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Christophe Lamaze
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Graça Raposo
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Claudia Steinem
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Pierre Sens
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Patricia Bassereau
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Ludger Johannes
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ludger Johannes.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-14 with Legends, Legends for Supplementary Movies 1-11 and additional references. (PDF 8199 kb)

Supplementary Movie 1

The file contains Supplementary Movie 1 which shows confocal live cell imaging of tubule growth after injection of 20 nM Alexa488-labeled STxB onto energy-depleted HeLa cells at 37°C. (MOV 1382 kb)

Supplementary Movie 2

The file contains Supplementary Movie 2 which shows confocal live cell imaging of tubule growth at 37°C following binding of 20 nM Alexa488-labeled STxB onto energy-depleted HeLa cells at 4°C. (MOV 1702 kb)

Supplementary Movie 3

The file contains Supplementary Movie 3 which shows confocal live cell imaging of tubule growth at 37°C following binding of 20 nM Alexa488-labeled STxB onto energy-depleted HeLa cells at 4°C. (MOV 3982 kb)

Supplementary Movie 4

The file contains Supplementary Movie 4 which shows evanescent field live cell imaging of tubule scission at 37°C after wash out of energy poisons. (MOV 2105 kb)

Supplementary Movie 5

The file contains Supplementary Movie 5 which shows confocal real time imaging experiment showing tubule formation on a GUV composed of DOPC/cholesterol/Gb3 after injection of Cy3-labeled STxB. (MOV 6177 kb)

Supplementary Movie 6

The file contains Supplementary Movie 6 which shows confocal real time imaging showing tubule dynamics at the equatorial plane of a GUV composed of DOPC/cholesterol/Gb3 at steady state. (MOV 668 kb)

Supplementary Movie 7

The file contains Supplementary Movie 7 which shows spinning-disk confocal real time imaging experiment showing tubule dynamics at steady state in 3D-projection at the equatorial plane of a GUV composed of DOPC/cholesterol/Gb3 during incubation with Cy3-labeled STxB. (MOV 1600 kb)

Supplementary Movie 8

The file contains Supplementary Movie 8 which shows confocal real time imaging experiment at the equatorial plane of a GUV composed of DOPC/cholesterol/Gb3 in the absence of STxB. (MOV 2159 kb)

Supplementary Movie 9

The file contains Supplementary Movie 9 which shows confocal real time imaging experiment on a GUV composed of DOPC/cholesterol/Gb3 onto which the Cy3-labeled W34A mutant was injected. (MOV 3433 kb)

Supplementary Movie 10

The file contains Supplementary Movie 10 which shows confocal real time imaging experiment on a GUV composed of DOPC/cholesterol and Gb3 with a single unsaturated C22:1 acyl chain. (MOV 2371 kb)

Supplementary Movie 11

The file contains Supplementary Movie 11 which shows confocal real time imaging experiment on a GUV composed of DOPC/cholesterol and Gb3 with a saturated C22:0 acyl chain. (MOV 1656 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Römer, W., Berland, L., Chambon, V. et al. Shiga toxin induces tubular membrane invaginations for its uptake into cells. Nature 450, 670–675 (2007). https://doi.org/10.1038/nature05996

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature05996

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing