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.

  • Letter
  • Published:

Reconstitution of actin-based motility of Listeria and Shigella using pure proteins

Nature volume 401, pages 613–616 (1999)Cite this article

Abstract

Actin polymerization is essential for cell locomotion and is thought to generate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to signalling1,2,3,4,5,6. The bacteria Listeria and Shigella bypass the signalling pathway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells7,8,9,10. However, the Arp2/3 complex alone is insufficient to promote movement. Here we have used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing factor (ADF, or cofilin) and capping protein are also required for motility as they maintain a high steady-state level of G-actin, which controls the rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement is more effective when profilin, α-actinin and VASP (for Listeria) are also included. These results have implications for our understanding of the mechanism of actin-based motility in cells.

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: Reconstitution of actin-based movement of E. coli (IcsA) and L. monocytogenes with pure components.
Figure 2: SDS–PAGE pattern of proteins used in the reconstitution assay of actin-based motility.

Similar content being viewed by others

References

  1. Machesky,L. M. & Insall,R. H. Scar1 and the related Wiskott-Aldrich syndrome protein WASP regulate the actin cytoskeleton through the Arp2/3 complex. Curr. Biol. 8, 1347–1356 (1998).

    Article  CAS  Google Scholar 

  2. Machesky,L. M. et al. Scar, a WASP-related protein, activates dendritic nucleation of actin filaments by the Arp2/3 complex. Proc. Natl Acad. Sci. USA 96, 3739–3744 (1999).

    Article  ADS  CAS  Google Scholar 

  3. Rohatgi,R. et al. The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly. Cell 97, 221–231 (1999).

    Article  CAS  Google Scholar 

  4. Winter,D., Lechler,T. & Li,R. Activation of the Arp2/3 complex by Bee 1p, a WASP-family protein. Curr. Biol. 9, 501–504 (1999).

    Article  CAS  Google Scholar 

  5. Yarar,D., To,W., Abo,A. & Welch,M. D. The Wiskott-Aldrich syndrome protein directs actin-based motility by stimulating actin nucleation with the Arp2/3 complex. Curr. Biol. 9, 555–558 (1999).

    Article  CAS  Google Scholar 

  6. Svitkina,T. M. & Borisy,G. G. Arp2/3 complex and Actin Depolymerizing Factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia. J. Cell Biol. 145, 1009–1026 (1999).

    Article  CAS  Google Scholar 

  7. Theriot,J. A., Mitchison,T. J., Tilney,L. G. & Portnoy,D. A. The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerisation. Nature 357, 257–260 (1992).

    Article  ADS  CAS  Google Scholar 

  8. Welch,M. D., Iwamatsu,A. & Mitchison,T. J. Actin polymerization is induced by Arp2/3 protein complex at the surface of Listeria monocytogenes. Nature 385, 265–269 (1997).

    Article  ADS  CAS  Google Scholar 

  9. Welch,M. D., Rosenblatt,J., Skoble,J., Portnoy,D. A. & Mitchison,T. J. Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation. Science 281, 105–108 (1998).

    Article  ADS  CAS  Google Scholar 

  10. Egile,C. et al. Activation of the Cdc42 effector N-WASP by the Shigella IcsA protein promotes actin nucleation by Arp2/3 complex and bacterial actin-based motility J. Cell Biol. 146, 1319–1332 (1999).

    Article  CAS  Google Scholar 

  11. Miki,H., Sasaki,T., Takai,Y. & Takenawa,T. Induction of filopodium formation by a N-WASP-related actin-depolymerizing factor. Nature 391, 93–96 (1998).

    Article  ADS  CAS  Google Scholar 

  12. Suzuki,T., Miki,H., Takenawa,T. & Sasakawa,C. Neural Wiskott-Aldrich syndrome protein is implicated in the actin-based motility of Shigella flezneri. EMBO J. 17, 2767–2776 (1998).

    Article  CAS  Google Scholar 

  13. Laurent,V. et al. Role of the proteins of the Ena/VASP family in actin-based motility of Listeria monocytogenes. J. Cell Biol. 144, 1245–1258 (1999).

    Article  CAS  Google Scholar 

  14. Small,J. V. Getting the actin filaments straight: nucleation-release or treadmilling. Trends Cell Biol. 5, 52–55 (1995).

    Article  CAS  Google Scholar 

  15. Carlier, M.-F. & Pantaloni,D. Control of actin dynamics in cell motility. J. Mol. Biol. 269, 459–467 (1997).

    Article  Google Scholar 

  16. Carlier, M.-F. Control of actin dynamics. Curr. Opin. Cell Biol. 10, 45–51 (1998).

    Article  Google Scholar 

  17. Carlier, M.-F. et al. Actin Depolymerising Factor (ADF/cofilin) enhances the rate of filament turnover: implication in actin-based motility. J. Cell Biol. 136, 1307–1322 (1997).

    Article  Google Scholar 

  18. Didry,D., Carlier, M.-F. & Pantaloni,D. Synergy between ADF and profilin in enhancing actin filament turnover. J. Biol. Chem. 273, 25602–25611 (1998).

    Article  CAS  Google Scholar 

  19. Ressad,F., Didry,D., Egile,C., Pantaloni,D. & Carlier, M.-F. Control of actin filament length and turnover by actin depolymerizing factor (ADF/cofilin) in the presence of capping proteins and Arp2/3 complex. J. Biol. Chem. 274, 20970–20976 (1999).

    Article  CAS  Google Scholar 

  20. Nanavati,D., Ashton,F. T., Sanger,J. M. & Sanger,J. W. Dynamics of actin and α-actinin in the tails of Listeria monocytogenes in infected PtK2 cells. Cell Motil. Cytoskel. 28, 346–358 (1994).

    Article  CAS  Google Scholar 

  21. Smith,G. A., Theriot,J. A. & Portnoy,D. A. The tandem repeat domain in the Listeria monocytogenes ActA protein controls the rate of actin-based motility, the percentage of moving bacteria and the localization of VASP and profilin. J. Cell Biol. 35, 647–660 (1996).

    Article  Google Scholar 

  22. Lasa,I. et al. Identification of two regions in the N-terminal domain of ActA involved in the actin tail formation by Listeria monocytogenes. EMBO J. 16, 1531–1540 (1997).

    Article  CAS  Google Scholar 

  23. Goldberg,M. & Theriot,J. A. Shigella flexneri surface protein IcsA is sufficient to direct actin-based motility. Proc. Natl Acad. Sci. USA 92, 6572–7576 (1995).

    Article  ADS  CAS  Google Scholar 

  24. Kocks,C. et al. The unrelated surface proteins ActA of Listeria monocytogenes and IcsA of Shigella flexneri are sufficient to confer actin-based motility on Listeria innocua and E. coli respectively. Mol. Microbiol. 18, 413–423 (1995).

    Article  CAS  Google Scholar 

  25. Cunningham,C., Stossel,T. P. & Kwiatkowski,D. Enhanced motility in NIH 3T3 fibroblasts that overexpress gelsolin. Science 251, 1233–1236 (1991).

    Article  ADS  CAS  Google Scholar 

  26. Hug,C. et al. Capping protein levels influence actin assembly and cell motility in Dictyostelium. Cell 81, 591–600 (1995).

    Article  CAS  Google Scholar 

  27. Sun,H., Kwiatkowska,K., Wooten,D. & Yin,H. Effects of CapG overexpression on agonist-induced motility and second messenger generation. J. Cell Biol. 129, 147–156 (1995).

    Article  CAS  Google Scholar 

  28. Marchand, J.-B. et al. Actin-based movement of Listeria monocytogenes: actin assembly results from the local maintenance of uncapped filament barbed ends at the bacterium surface. J. Cell Biol. 130, 331–343 (1995).

    Article  Google Scholar 

  29. Rottner,K., Behrendt,B., Small,J. V. & Wehland,J. VASP dynamics during lamellipodia protrusion. Nature Cell Biol. 1, 321–322 (1999).

    Article  CAS  Google Scholar 

  30. Goldberg,M. Shigella actin-based motility in the absence of vinculin. Cell Motil. Cytoskel. 37, 44–53 (1997).

    Article  CAS  Google Scholar 

  31. Kuhlmann,P. A. & Fowler,V. M. Purification and characterization of an α1β2 isoform of CapZ from human erythrocytes: cytosolic location and inability to bind to Mg++ ghosts suggest that erythrocyte actin filaments are capped by adducin. Biochemistry 36, 13461–13472 (1997).

    Article  Google Scholar 

Download references

Acknowledgements

We thank V. Laurent and C. Egile for discussion, D. Didry for Arp2/3 complex purification, and F. Ressad for human ADF. We acknowledge partial support from the Association pour la Recherche contre le Cancer, the Association Française contre les Myopathies and a Human Frontier in Science grant. T.P.L. is supported by a fellowship from the Natural Sciences and Engineering Research Council of Canada.

Author information

Authors and Affiliations

  1. Dynamique du Cytosquelette, LEBS, CNRS, Gif-sur-Yvette, France

    Thomas P. Loisel, Rajaa Boujemaa, Dominique Pantaloni & Marie-France Carlier

Authors
  1. Thomas P. Loisel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rajaa Boujemaa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dominique Pantaloni
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marie-France Carlier
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Marie-France Carlier.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Loisel, T., Boujemaa, R., Pantaloni, D. et al. Reconstitution of actin-based motility of Listeria and Shigella using pure proteins. Nature 401, 613–616 (1999). https://doi.org/10.1038/44183

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/44183

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