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.

Spatiotemporal dynamics of RhoA activity in migrating cells

Nature volume 440, pages 1069–1072 (2006)Cite this article

Abstract

Rho family GTPases regulate the actin and adhesion dynamics that control cell migration. Current models postulate that Rac promotes membrane protrusion at the leading edge and that RhoA regulates contractility in the cell body1,2. However, there is evidence that RhoA also regulates membrane protrusion3,4. Here we use a fluorescent biosensor, based on a novel design preserving reversible membrane interactions, to visualize the spatiotemporal dynamics of RhoA activity during cell migration. In randomly migrating cells, RhoA activity is concentrated in a sharp band directly at the edge of protrusions. It is observed sporadically in retracting tails, and is low in the cell body. RhoA activity is also associated with peripheral ruffles and pinocytic vesicles, but not with dorsal ruffles induced by platelet-derived growth factor (PDGF). In contrast to randomly migrating cells, PDGF-induced membrane protrusions have low RhoA activity, potentially because PDGF strongly activates Rac, which has previously been shown to antagonize RhoA activity5,6. Our data therefore show that different extracellular cues induce distinct patterns of RhoA signalling during membrane protrusion.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Figure 1: Design and characterization of the RhoA biosensor.
Figure 2: RhoA activity in randomly migrating MEFs.
Figure 3: RhoA activity at ruffles and pinosomes.
Figure 4: PDGF-induced membrane protrusions.

References

  1. Raftopoulou, M. & Hall, A. Cell migration: Rho GTPases lead the way. Dev. Biol. 265, 23–32 (2004)

    Article  CAS  Google Scholar 

  2. Burridge, K. & Wennerberg, K. Rho and Rac take center stage. Cell 116, 167–179 (2004)

    Article  CAS  Google Scholar 

  3. Fukata, Y. et al. Phosphorylation of adducin by Rho-kinase plays a crucial role in cell motility. J. Cell Biol. 145, 347–361 (1999)

    Article  CAS  Google Scholar 

  4. Palazzo, A. F., Cook, T. A., Alberts, A. S. & Gundersen, G. G. mDia mediates Rho-regulated formation and orientation of stable microtubules. Nature Cell Biol. 3, 723–729 (2001)

    Article  CAS  Google Scholar 

  5. Nimnual, A. S., Taylor, L. J. & Bar-Sagi, D. Redox-dependent downregulation of Rho by Rac. Nature Cell Biol. 5, 236–241 (2003)

    Article  CAS  Google Scholar 

  6. Sander, E. E., ten Klooster, J. P., van Delft, S., van der Kammen, R. A. & Collard, J. G. Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behaviour. J. Cell Biol. 147, 1009–1022 (1999)

    Article  CAS  Google Scholar 

  7. Ren, X. D., Kiosses, W. B. & Schwartz, M. A. Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J. 18, 578–585 (1999)

    Article  CAS  Google Scholar 

  8. Griesbeck, O., Baird, G. S., Campbell, R. E., Zacharias, D. A. & Tsien, R. Y. Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J. Biol. Chem. 276, 29188–29194 (2001)

    Article  CAS  Google Scholar 

  9. Miyawaki, A. & Tsien, R. Y. Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. Methods Enzymol. 327, 472–500 (2000)

    Article  CAS  Google Scholar 

  10. Hoffman, G. R., Nassar, N. & Cerione, R. A. Structure of the Rho family GTP-binding protein Cdc42 in complex with the multifunctional regulator RhoGDI. Cell 100, 345–356 (2000)

    Article  CAS  Google Scholar 

  11. Miura, K. et al. ARAP1: a point of convergence for Arf and Rho signaling. Mol. Cell 9, 109–119 (2002)

    Article  CAS  Google Scholar 

  12. Norman, J. C. et al. ARF1 mediates paxillin recruitment to focal adhesions and potentiates Rho-stimulated stress fiber formation in intact and permeabilized Swiss 3T3 fibroblasts. J. Cell Biol. 143, 1981–1995 (1998)

    Article  CAS  Google Scholar 

  13. Cho, S. Y. & Klemke, R. L. Purification of pseudopodia from polarized cells reveals redistribution and activation of Rac through assembly of a CAS/Crk scaffold. J. Cell Biol. 156, 725–736 (2002)

    Article  CAS  Google Scholar 

  14. Matsumura, F., Ono, S., Yamakita, Y., Totsukawa, G. & Yamashiro, S. Specific localization of serine 19 phosphorylated myosin II during cell locomotion and mitosis of cultured cells. J. Cell Biol. 140, 119–129 (1998)

    Article  CAS  Google Scholar 

  15. Worthylake, R. A., Lemoine, S., Watson, J. M. & Burridge, K. RhoA is required for monocyte tail retraction during transendothelial migration. J. Cell Biol. 154, 147–160 (2001)

    Article  CAS  Google Scholar 

  16. Kurokawa, K. & Matsuda, M. Localized RhoA activation as a requirement for the induction of membrane ruffling. Mol. Biol. Cell 16, 4294–4303 (2005)

    Article  CAS  Google Scholar 

  17. Shaw, R. J., Henry, M., Solomon, F. & Jacks, T. RhoA-dependent phosphorylation and relocalization of ERM proteins into apical membrane/actin protrusions in fibroblasts. Mol. Biol. Cell 9, 403–419 (1998)

    Article  CAS  Google Scholar 

  18. Fernandez-Borja, M., Janssen, L., Verwoerd, D., Hordijk, P. & Neefjes, J. RhoB regulates endosome transport by promoting actin assembly on endosomal membranes through Dia1. J. Cell Sci. 118, 2661–2670 (2005)

    Article  CAS  Google Scholar 

  19. Merrifield, C. J. et al. Endocytic vesicles move at the tips of actin tails in cultured mast cells. Nature Cell Biol. 1, 72–74 (1999)

    Article  CAS  Google Scholar 

  20. Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D. & Hall, A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70, 401–410 (1992)

    Article  CAS  Google Scholar 

  21. Hodgson, L., Nalbant, P., Shen, F. & Hahn, K. M. Imaging and photobleach correction of Mero-CBD, sensor for endogenous Cdc42 activation. Methods Enzymol. 406, 140–156 (2006)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to G. Bokoch, P. Sun, M. Schwartz, R. Tsien, C. Der and E. Sahai for reagents, and to F. Shen for help with image analysis. This work was supported by grants from the Swiss National Science Foundation, Roche Research Foundation, Novartis and Philip Morris to O.P., and from the National Institutes of Health to K.M.H. and R.L.K.

Author information

Author notes

  1. Olivier Pertz & Richard L. Klemke

    Present address: Department of Pathology and Moores Cancer Center, Basic Science Building, University of California at San Diego, Room 1040, 9500 Gilman Drive, MC 0612, La Jolla, California, 92093, USA

  2. Louis Hodgson & Klaus M. Hahn

    Present address: Department of Pharmacology and Lineberger Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599, USA

Authors and Affiliations

  1. Department of Pharmacology and Lineberger Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599, USA

    Olivier Pertz, Louis Hodgson & Klaus M. Hahn

  2. Department of Cell Biology, The Scripps Research Institute, La Jolla, 10550 North Torrey Pines Road, California, 92037, USA

    Olivier Pertz, Louis Hodgson, Richard L. Klemke & Klaus M. Hahn

  3. Department of Pathology and Moores Cancer Center, Basic Science Building, University of California at San Diego, Room 1040, 9500 Gilman Drive, MC 0612, La Jolla, California, 92093, USA

    Louis Hodgson & Klaus M. Hahn

Authors
  1. Olivier Pertz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Louis Hodgson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Richard L. Klemke
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Klaus M. Hahn
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Olivier Pertz or Klaus M. Hahn.

Ethics declarations

Competing interests

K.M.H. is a paid consultant of Panomics Corporation, which markets reagents for high-content microscopy screening.

Supplementary information

Supplementary Notes

This file contains Supplementary Methods and Supplementary Figures 1–6. (PDF 582 kb)

Supplementary Movie 1

Phase contrast timelapse movie comparing MEFs with or without biosensor expression (induced versus non-induced). (MOV 5924 kb)

Supplementary Movie 2

Timelapse movie showing YFP and ratio images of a MEF fibroblast randomly migrating on fibronectin (as in Fig. 2a). (MOV 3638 kb)

Supplementary Movie 3

Timelapse movie showing ratio images of a MEF fibroblast randomly migrating on fibronectin (as in Fig. 2b). (MOV 1028 kb)

Supplementary Movie 4

Timelapse movie of a peripheral ruffle. (MOV 3633 kb)

Supplementary Movie 5

Timelapse movie of PDGF-stimulated pinocytosis. (MOV 7683 kb)

Supplementary Movie 6

Timelapse movie showing robust PDGF stimulation of protrusions in the MEF cells, as shown in Fig.4a. (MOV 5446 kb)

Supplementary Movie 7

Timelapse movie showing PDGF induction of membrane protrusions in MEF cells expressing T19N DN biosensor. (MOV 7177 kb)

Supplementary Movie 8

Timelapse movie of a MEF migrating out of a wounded monolayer, then stimulated with PDGF. (MOV 5463 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Pertz, O., Hodgson, L., Klemke, R. et al. Spatiotemporal dynamics of RhoA activity in migrating cells. Nature 440, 1069–1072 (2006). https://doi.org/10.1038/nature04665

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature04665

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