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Coordination of Rho GTPase activities during cell protrusion

Nature volume 461, pages 99–103 (2009)Cite this article

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Abstract

The GTPases Rac1, RhoA and Cdc42 act together to control cytoskeleton dynamics1,2,3. Recent biosensor studies have shown that all three GTPases are activated at the front of migrating cells4,5,6,7, and biochemical evidence suggests that they may regulate one another: Cdc42 can activate Rac1 (ref. 8), and Rac1 and RhoA are mutually inhibitory9,10,11,12. However, their spatiotemporal coordination, at the seconds and single-micrometre dimensions typical of individual protrusion events, remains unknown. Here we examine GTPase coordination in mouse embryonic fibroblasts both through simultaneous visualization of two GTPase biosensors and using a ‘computational multiplexing’ approach capable of defining the relationships between multiple protein activities visualized in separate experiments. We found that RhoA is activated at the cell edge synchronous with edge advancement, whereas Cdc42 and Rac1 are activated 2 μm behind the edge with a delay of 40 s. This indicates that Rac1 and RhoA operate antagonistically through spatial separation and precise timing, and that RhoA has a role in the initial events of protrusion, whereas Rac1 and Cdc42 activate pathways implicated in reinforcement and stabilization of newly expanded protrusions.

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Figure 1: Activation of Rho GTPases in migrating MEFs.
Figure 2: Dynamics of cell edge morphology and GTPase activation.
Figure 3: Correlation of Rho GTPase activation and cell edge velocity as a function of time and space.
Figure 4: Spatiotemporal coordination of Rac1, Cdc42 and RhoA activation.

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Change history

  • 03 September 2009

    Author affiliations for P.N. were changed on 3 September 2009.

References

  1. Jaffe, A. B. & Hall, A. Rho GTPases: biochemistry and biology. Annu. Rev. Cell Dev. Biol. 21, 247–269 (2005)

    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. Ridley, A. J. et al. Cell migration: integrating signals from front to back. Science 302, 1704–1709 (2003)

    Article  ADS  CAS  Google Scholar 

  4. Kraynov, V. et al. Localized Rac activation dynamics visualized in living cells. Science 290, 333–337 (2000)

    Article  ADS  CAS  Google Scholar 

  5. Nalbant, P., Hodgson, L., Kraynov, V., Toutchkine, A. & Hahn, K. M. Activation of endogenous Cdc42 visualized in living cells. Science 305, 1615–1619 (2004)

    Article  ADS  CAS  Google Scholar 

  6. Pertz, O., Hodgson, L., Klemke, R. L. & Hahn, K. M. Spatiotemporal dynamics of RhoA activity in migrating cells. Nature 440, 1069–1072 (2006)

    Article  ADS  CAS  Google Scholar 

  7. 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 

  8. Nobes, C. D. & Hall, A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81, 53–62 (1995)

    Article  CAS  Google Scholar 

  9. Rottner, K., Hall, A. & Small, J. V. Interplay between Rac and Rho in the control of substrate contact dynamics. Curr. Biol. 9, 640–649 (1999)

    Article  CAS  Google Scholar 

  10. 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 

  11. Arthur, W. T., Burridge, K. & Rho, A. Inactivation by p190RhoGAP regulates cell spreading and migration by promoting membrane protrusion and polarity. Mol. Biol. Cell 12, 2711–2720 (2001)

    Article  CAS  Google Scholar 

  12. Ohta, Y., Hartwig, J. H. & Stossel, T. P. FilGAP, a Rho- and ROCK-regulated GAP for Rac binds filamin A to control actin remodelling. Nature Cell Biol. 8, 803–814 (2006)

    Article  CAS  Google Scholar 

  13. Machacek, M. & Danuser, G. Morphodynamic profiling of protrusion phenotypes. Biophys. J. 90, 1439–1452 (2006)

    Article  ADS  CAS  Google Scholar 

  14. Marguet, D., Lenne, P. F., Rigneault, H. & He, H. T. Dynamics in the plasma membrane: how to combine fluidity and order. EMBO J. 25, 3446–3457 (2006)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. Zaidel-Bar, R., Ballestrem, C., Kam, Z. & Geiger, B. Early molecular events in the assembly of matrix adhesions at the leading edge of migrating cells. J. Cell Sci. 116, 4605–4613 (2003)

    Article  CAS  Google Scholar 

  17. Del Pozo, M. A. et al. Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI. Nature Cell Biol. 4, 232–239 (2002)

    Article  CAS  Google Scholar 

  18. Nayal, A. et al. Paxillin phosphorylation at ser273 localizes a GIT1-PIX-PAK complex and regulates adhesion and protrusion dynamics. J. Cell Biol. 173, 587–599 (2006)

    Article  CAS  Google Scholar 

  19. ten Klooster, J. P., Jaffer, Z. M., Chernoff, J. & Hordijk, P. L. Targeting and activation of Rac1 are mediated by the exchange factor β-Pix. J. Cell Biol. 172, 759–769 (2006)

    Article  CAS  Google Scholar 

  20. Ji, L., Lim, J. & Danuser, G. Fluctuations of intracellular forces during cell protrusion. Nature Cell Biol. 10, 1393–1400 (2008)

    Article  CAS  Google Scholar 

  21. Choi, C. K. et al. Actin and alpha-actinin orchestrate the assembly and maturation of nascent adhesions in a myosin II motor-independent manner. Nature Cell Biol 10, 1039–1050

  22. Narumiya, S., Ishizaki, T. & Watanabe, N. Rho effectors and reorganization of actin cytoskeleton. FEBS Lett. 410, 68–72 (1997)

    Article  CAS  Google Scholar 

  23. Yamana, N. et al. The Rho-mDia1 pathway regulates cell polarity and focal adhesion turnover in migrating cells through mobilizing Apc and c-Src. Mol. Cell. Biol. 26, 6844–6858 (2006)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  25. Rodriguez, O. C. et al. Conserved microtubule-actin interactions in cell movement and morphogenesis. Nature Cell Biol. 5, 599–609 (2003)

    Article  CAS  Google Scholar 

  26. Del Pozo, M. A., Price, L. S., Alderson, N. B., Ren, X. D. & Schwartz, M. A. Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK. EMBO J. 19, 2008–2014 (2000)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  28. Hodgson, L., Shen, F. & Hahn, K. Biosensors for characterizing the dynamics of Rho family GTPases in living cells. Curr. Protoc. Cell Biol. (in the press)

Download references

Acknowledgements

We acknowledge funding from the Swiss National Science Foundation and the Novartis Foundation, formerly the Ciba-Geigy Jubilee Foundation (M.M.), NIH T32 GM008719 and NIH F30 HL094020 (C.W.), NIH R01 GM57464 (K.M.H.), NIH R01 GM71868 (G.D.), and the Cell Migration Consortium, grant U54 GM064346 from NIGMS (G.D. and K.M.H.).

Author Contributions M.M. initiated the project, conceptualized the idea of computational multiplexing, wrote all image analysis software pertinent to multiplexing, and contributed to the writing of the manuscript; L.H. developed simultaneous imaging of RhoA and Cdc42, including the modification and validation of the meroCBD probe, developed the intermolecular RhoA sensor, including controls and validation, studying the effects of biosensor stoichiometry and expression level, developed the new version of the Rac biosensor, and contributed to writing of the manuscript; C.W. produced stable cell lines of the intermolecular RhoA biosensor and conducted imaging experiments for the comparison of intra- and intermolecular biosensor designs; H.E. contributed simulations for validation of the correlation analysis and assisted with image processing; O.P. and P.N. contributed image data of RhoA and Cdc42 activity, respectively; A.A. and G.L.J. contributed valuable advice and unpublished reagents; K.M.H. and G.D. coordinated the study and wrote the final version of the manuscript and supplement.

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Author notes

  1. Matthias Machacek, Louis Hodgson & Olivier Pertz

    Present address: Present addresses: Novartis Pharma AG, Lichtstrasse 35, CH-4056 Basel, Switzerland (M.M.); Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Ave, Bronx, New York 10461, USA (L.H.); Department of Biomedicine, University of Basel, Mattenstrasse 28, CH-4058 Basel, Switzerland (O.P.).,

  2. Matthias Machacek, Louis Hodgson and Christopher Welch: These authors contributed equally to this work.

Authors and Affiliations

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

    Matthias Machacek, Hunter Elliott, Olivier Pertz & Gaudenz Danuser

  2. Departments of Pharmacology, Medicinal Chemistry and Lineberger Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA,

    Louis Hodgson, Christopher Welch, Amy Abell, Gary L. Johnson & Klaus M. Hahn

  3. Department of Molecular Cell Biology, Center for Medical Biotechnology, University of Duisburg-Essen, 45117 Essen, Germany

    Perihan Nalbant

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Correspondence to Klaus M. Hahn or Gaudenz Danuser.

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary Data, Supplementary Figures S1-S10 with Legends and Supplementary References. Please note that the Movies appeared online in the wrong order and were corrected on 16th April 2010. (PDF 939 kb)

Supplementary Movie 1

This Movie shows the Rac1 activation measured using the FRET/CyPet ratio to monitor interaction between Rac1 and the p21-binding domain from PAK1 (see Figs. 1a, S1a). Left panel: corresponding time points imaged by DIC. Frame interval: 10 s. Replay: 10 frames/s. Duration of original sequence: 20 min. Magnification 40x, 2x2 binning. Scale bar: 20 µm. Colour-bar defines the dynamic range of the corrected FRET/CyPet ratio. (MOV 8200 kb)

Supplementary Movie 2

This Movie shows the zoom of the protrusive region of Movie 1. This sector of the cell edge is analyzed in Fig. 2. (MOV 4531 kb)

Supplementary Movie 3

This Movie shows the Cdc42 activation measured using the meroCBD biosensor (see Figs. 1b, S1b). Left panel: corresponding time points imaged by DIC. Frame interval: 10 s. Duration of original sequence: 13 min. Replay: 10 frames/s. Magnification 40x, 2x2 binning. Scale bar: 20 µm. Colour-bar defines the dynamic range of the merocyanine dye/EGFP ratio. (MOV 4778 kb)

Supplementary Movie 4

This Movie shows the zoom of the protrusive region of Movie 3. This sector of the cell edge is analyzed in Fig. 2. (MOV 4656 kb)

Supplementary Movie 5

This Movie shows the RhoA activation measured using the single chain, intramolecular FRET biosensor (see Figs. 1c, S1c). Left panel: corresponding time points imaged by DIC. Frame interval: 10 s. Replay: 10 frames/s. Duration of original sequence: 10 min. Magnification 40x, 2x2 binning. Scale bar: 20 µm. Colour-bar defines the dynamic range of the FRET/CFP ratio. (MOV 4506 kb)

Supplementary Movie 6

This Movie shows the zoom of the protrusive region of Movie 5. This sector of the cell edge is analyzed in Fig. 2. (MOV 5456 kb)

Supplementary Movie 7

This Movie shows sampling windows of 1.8 µm width and 0.9 µm depth placed at 0 µm and 2.5 µm from the edge follow the cell morphology. Windows are overlaid on the time lapse sequence shown in Movie 4. Frame interval: 10s. Replay: 10 frames/s. (MOV 3343 kb)

Supplementary Movie 8

This Movie shows the RhoA activation measured using the intermolecular dual chain FRET biosensor (see Figs. S1d, S10). Frame interval: 10 s. Replay: 10 frames/s. Magnification 40x, 2x2 binning. Scale bar: 20 µm. Colour-bar defines the dynamic range of the corrected FRET/CyPet ratio. (MOV 2775 kb)

Supplementary Movie 9

This Movie shows the zoomed view of protrusion and retraction activity of a randomly migrating MEF imaged simultaneously using Cdc42 and RhoA biosensors (see Fig. S7 for snapshots of the full view time sequence). Frame interval: 10s. Replay: 10 frames/s. Magnification 40x, 2x2 binning. Scale-bar: 15 μm. Colour-bar defines the dynamic range of the Cdc42 and RhoA signals. (MOV 7432 kb)

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Machacek, M., Hodgson, L., Welch, C. et al. Coordination of Rho GTPase activities during cell protrusion. Nature 461, 99–103 (2009). https://doi.org/10.1038/nature08242

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