Letter | Published:

Differentially oriented populations of actin filaments generated in lamellipodia collaborate in pushing and pausing at the cell front

Nature Cell Biology volume 10, pages 306313 (2008) | Download Citation

Subjects

Abstract

Eukaryotic cells advance in phases of protrusion, pause and withdrawal1. Protrusion occurs in lamellipodia, which are composed of diagonal networks of actin filaments, and withdrawal terminates with the formation of actin bundles parallel to the cell edge. Using correlated live-cell imaging and electron microscopy, we have shown that actin filaments in protruding lamellipodia subtend angles from 15–90° to the front, and that transitions from protrusion to pause are associated with a proportional increase in filaments oriented more parallel to the cell edge. Microspike bundles of actin filaments also showed a wide angular distribution and correspondingly variable bilateral polymerization rates along the cell front. We propose that the angular shift of filaments in lamellipodia serves in adapting to slower protrusion rates while maintaining the filament densities required for structural support; further, we suggest that single filaments and microspike bundles contribute to the construction of the lamella behind and to the formation of the cell edge when protrusion ceases. Our findings provide an explanation for the variable turnover dynamics of actin filaments in lamellipodia observed by fluorescence speckle microscopy2 and are inconsistent with a current model of lamellipodia structure that features actin filaments branching at 70° in a dendritic array3.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , & The locomotion of fibroblasts in culture. II. 'Ruffling'. Exp. Cell Res. 60, 437–444 (1970).

  2. 2.

    , , , & Two distinct actin networks drive the protrusion of migrating cells. Science 305, 1782–1786 (2004).

  3. 3.

    & 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).

  4. 4.

    , , & The lamellipodium: where motility begins. Trends Cell Biol. 12, 112–20 (2002).

  5. 5.

    , & Polarity of actin at the leading edge of cultured cells. Nature 272, 638–639 (1978).

  6. 6.

    , & Actin filament organization in the fish keratocyte lamellipodium. J. Cell Biol. 129, 1275–1286 (1995).

  7. 7.

    & Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453–465 (2003).

  8. 8.

    Regulation of actin filament assembly by arp2/3 complex and formins. Annu. Rev. Biophys. Biomol. Struct. 36, 451–477 (2007).

  9. 9.

    & On the mechanisms of cortical actin flow and its role in cytoskeletal organisation of fibroblasts. Symp. Soc. Exp. Biol. 47, 35–56 (1993).

  10. 10.

    , , & VASP dynamics during lamellipodia protrusion. Nature Cell Biol. 1, 321–322 (1999).

  11. 11.

    & Intravital imaging of cell movement in tumours. Nature Rev. Cancer 3, 921–930 (2003).

  12. 12.

    & The comings and goings of actin: coupling protrusion and retraction in cell motility. Curr. Opin. Cell Biol. 17, 517–523 (2005).

  13. 13.

    , & Myosin II functions in actin-bundle turnover in neuronal growth cones. Nature Cell Biol. 8, 215–226 (2006).

  14. 14.

    The actin cytoskeleton. Electron Microsc. Rev. 1, 155–174 (1988).

  15. 15.

    , , & The actin-based nanomachine at the leading edge of migrating cells. Biophys. J. 77, 1721–1732 (1999).

  16. 16.

    & Processive capping by formin suggests a force-driven mechanism of actin polymerization. J. Cell Biol. 167, 1011–1017 (2004).

  17. 17.

    & Control of actin assembly dynamics in cell motility. J. Biol. Chem. 282, 23005–23009 (2007).

  18. 18.

    On the edge: modeling protrusion. Curr. Opin. Cell Biol. 18, 32–39 (2006).

  19. 19.

    , , & Centripetal transport of cytoplasm, actin, and the cell surface in lamellipodia of fibroblasts. Cell Motil. Cytoskeleton 11, 235–247 (1988).

  20. 20.

    , , , & Tracking retrograde flow in keratocytes: news from the front. Mol. Biol. Cell 16, 1223–1231 (2005).

  21. 21.

    & Stress fibers are generated by two distinct actin assembly mechanisms in motile cells. J. Cell Biol. 173, 383–394 (2006).

  22. 22.

    , & Inhibition of the Arp2/3 complex-nucleated actin polymerization and branch formation by tropomyosin. Curr. Biol. 11, 1300–1304 (2001).

  23. 23.

    , & Actin dynamics. J. Cell Sci. 114, 3–4 (2001).

  24. 24.

    & Direct real-time observation of actin filament branching mediated by Arp2/3 complex using total internal reflection fluorescence microscopy. Proc. Natl Acad. Sci. USA. 98, 15009–15013 (2001).

  25. 25.

    , & The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments. Proc. Natl Acad. Sci. USA. 95, 6181–6186 (1998).

  26. 26.

    , , , & Pure F-actin networks are distorted and branched by steps in the critical-point drying method. J. Struct. Biol. 137, 305–312 (2002).

  27. 27.

    , , , , & Macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography. Science. 298, 1209–1213 (2002).

  28. 28.

    et al. Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin. J. Cell Biol. 168, 619–631 (2005).

  29. 29.

    et al. Lamellipodial actin mechanically links myosin activity with adhesion-site formation. Cell 128, 561–575 (2007).

  30. 30.

    , & Spatiotemporal image correlation spectroscopy (STICS) theory, verification, and application to protein velocity mapping in living CHO cells. Biophys. J. 88, 3601–3614 (2005).

Download references

Acknowledgements

The authors thank the Human Frontier Science Program Organization (HFSPO), The Austrian Science Research Council (FWF) and the Vienna Science Research and Technology Fund (WWTF) as well as the City of Vienna/Zentrum für Innovation und Technologie via the Spot of Excellence grant 'Center of Molecular and Cellular Nanostructure' for financial support. K.R. was supported in part by grants from the Deutsche Forschungsgemeinschaft (SPP1150 and FOR629). We also thank Guenter Resch for the electron microscope facility management and advice with image processing, Tibor Kulcsar and Hannes Tkadletz for graphics and Natalia Andreyeva for helpful comments. The authors thank Roger Tsien, Annette Muller-Taubenberger, Malgorzata Szczodrak, George Patterson, Jennifer Lippincott-Schwarz and Rex Chisholm for probes, and Jeff Segall and Bob van de Water for MTLn3 cells.

Author information

Affiliations

  1. Institute of Molecular Biotechnology, Austrian Academy of Sciences, Dr. Bohr-Gasse 3, 1030, Vienna, Austria.

    • Stefan A. Koestler
    • , Sonja Auinger
    • , Marlene Vinzenz
    •  & J. Victor Small
  2. Cytoskeleton Dynamics Group, Helmholtz Centre for Infection Research (HZI), Inhoffen Strasse 7, D-38124 Braunschweig, Germany.

    • Klemens Rottner

Authors

  1. Search for Stefan A. Koestler in:

  2. Search for Sonja Auinger in:

  3. Search for Marlene Vinzenz in:

  4. Search for Klemens Rottner in:

  5. Search for J. Victor Small in:

Corresponding author

Correspondence to J. Victor Small.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary figures S1, S2 and S3

Videos

  1. 1.

    Supplementary Information

    Supplementary Information Movie 1

  2. 2.

    Supplementary Information

    Supplementary Information Movie 2

  3. 3.

    Supplementary Information

    Supplementary Information Movie 3

  4. 4.

    Supplementary Information

    Supplementary Information Movie 4

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/ncb1692

Further reading