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Visualizing the mechanical activation of Src

Nature volume 434, pages 1040–1045 (2005)Cite this article

Abstract

The mechanical environment crucially influences many cell functions1. However, it remains largely mysterious how mechanical stimuli are transmitted into biochemical signals. Src is known to regulate the integrin–cytoskeleton interaction2, which is essential for the transduction of mechanical stimuli3,4,5. Using fluorescent resonance energy transfer (FRET), here we develop a genetically encoded Src reporter that enables the imaging and quantification of spatio-temporal activation of Src in live cells. We introduced a local mechanical stimulation to human umbilical vein endothelial cells (HUVECs) by applying laser-tweezer traction on fibronectin-coated beads adhering to the cells. Using the Src reporter, we observed a rapid distal Src activation and a slower directional wave propagation of Src activation along the plasma membrane. This wave propagated away from the stimulation site with a speed (mean ± s.e.m.) of 18.1 ± 1.7 nm s-1. This force-induced directional and long-range activation of Src was abolished by the disruption of actin filaments or microtubules. Our reporter has thus made it possible to monitor mechanotransduction in live cells with spatio-temporal characterization. We find that the transmission of mechanically induced Src activation is a dynamic process that directs signals via the cytoskeleton to spatial destinations.

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Figure 1: The domain structure, schematic representation and in vitro characterizations of the Src reporter.
Figure 2: Characterization of the Src reporters.
Figure 3: Directional and long-range propagation of Src induced by mechanical force.
Figure 4: Actin filaments and microtubules are essential for the polarized and long-range Src activation.

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References

  1. Ingber, D. E. Tensegrity. II. How structural networks influence cellular information processing networks. J. Cell. Sci. 116, 1397–1408 (2003)

    Article  CAS  Google Scholar 

  2. Felsenfeld, D. P., Schwartzberg, P. L., Venegas, A., Tse, R. & Sheetz, M. P. Selective regulation of integrin–cytoskeleton interactions by the tyrosine kinase Src. Nature Cell Biol. 1, 200–206 (1999)

    Article  CAS  Google Scholar 

  3. Critchley, D. R. Focal adhesions—the cytoskeletal connection. Curr. Opin. Cell Biol. 12, 133–139 (2000)

    Article  CAS  Google Scholar 

  4. Chicurel, M. E., Singer, R. H., Meyer, C. J. & Ingber, D. E. Integrin binding and mechanical tension induce movement of mRNA and ribosomes to focal adhesions. Nature 392, 730–733 (1998)

    Article  ADS  CAS  Google Scholar 

  5. Jiang, G., Giannone, G., Critchley, D. R., Fukumoto, E. & Sheetz, M. P. Two-piconewton slip bond between fibronectin and the cytoskeleton depends on talin. Nature 424, 334–337 (2003)

    Article  ADS  CAS  Google Scholar 

  6. Alenghat, F. J. & Ingber, D. E. Mechanotransduction: all signals point to cytoskeleton, matrix, and integrins. Sci. STKE 2002, E6 (2002)

    Google Scholar 

  7. Arias-Salgado, E. G. et al. Src kinase activation by direct interaction with the integrin beta cytoplasmic domain. Proc. Natl Acad. Sci. USA 100, 13298–13302 (2003)

    Article  ADS  CAS  Google Scholar 

  8. Fincham, V. J., Brunton, V. G. & Frame, M. C. The SH3 domain directs acto-myosin-dependent targeting of v-Src to focal adhesions via phosphatidylinositol 3-kinase. Mol. Cell Biol. 20, 6518–6536 (2000)

    Article  CAS  Google Scholar 

  9. Kaplan, G. et al. Identification of a surface glycoprotein on African green monkey kidney cells as a receptor for hepatitis A virus. EMBO J. 15, 4282–4296 (1996)

    Article  CAS  Google Scholar 

  10. Ting, A. Y., Kain, K. H., Klemke, R. L. & Tsien, R. Y. Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells. Proc. Natl Acad. Sci. USA 98, 15003–15008 (2001)

    Article  ADS  CAS  Google Scholar 

  11. Hamasaki, K. et al. Src kinase plays an essential role in integrin-mediated tyrosine phosphorylation of Crk-associated substrate p130Cas. Biochem. Biophys. Res. Commun. 222, 338–343 (1996)

    Article  CAS  Google Scholar 

  12. Vuori, K., Hirai, H., Aizawa, S. & Ruoslahti, E. Introduction of p130cas signaling complex formation upon integrin-mediated cell adhesion: a role for Src family kinases. Mol. Cell Biol. 16, 2606–2613 (1996)

    Article  CAS  Google Scholar 

  13. Songyang, Z. et al. Catalytic specificity of protein-tyrosine kinases is critical for selective signalling. Nature 373, 536–539 (1995)

    Article  ADS  CAS  Google Scholar 

  14. Yang, F., Liu, Y., Bixby, S. D., Friedman, J. D. & Shokat, K. M. Highly efficient green fluorescent protein-based kinase substrates. Anal. Biochem. 266, 167–173 (1999)

    Article  Google Scholar 

  15. Waksman, G. et al. Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides. Nature 358, 646–653 (1992)

    Article  ADS  CAS  Google Scholar 

  16. Bradshaw, J. M. & Waksman, G. Calorimetric examination of high-affinity Src SH2 domain-tyrosyl phosphopeptide binding: dissection of the phosphopeptide sequence specificity and coupling energetics. Biochemistry 38, 5147–5154 (1999)

    Article  CAS  Google Scholar 

  17. Yang, F., Moss, L. G. & Phillips, G. N. Jr The molecular structure of green fluorescent protein. Nature Biotechnol. 14, 1246–1251 (1996)

    Article  CAS  Google Scholar 

  18. Zacharias, D. A., Violin, J. D., Newton, A. C. & Tsien, R. Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002)

    Article  ADS  CAS  Google Scholar 

  19. Miyamoto, S. et al. Integrin function: molecular hierarchies of cytoskeletal and signaling molecules. J. Cell Biol. 131, 791–805 (1995)

    Article  CAS  Google Scholar 

  20. Tsien, R. Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998)

    Article  CAS  Google Scholar 

  21. Thomas, S. M. & Brugge, J. S. Cellular functions regulated by Src family kinases. Annu. Rev. Cell Dev. Biol. 13, 513–609 (1997)

    Article  CAS  Google Scholar 

  22. Heidermann, S. R., Kaech, S., Buxbaum, R. E. & Matus, A. Direct observations of the mechanical behaviors of the cytoskeleton in living fibroblasts. J. Cell Biol. 145, 109–122 (1999)

    Article  Google Scholar 

  23. Hu, S. et al. Intracellular stress tomography reveals stress focusing and structural anisotropy in cytoskeleton of living cells. Am. J. Physiol Cell Physiol. 285, C1082–C1090 (2003)

    Article  CAS  Google Scholar 

  24. Mack, P. J., Kaazempur-Mofrad, M. R., Karcher, H., Lee, R. T. & Kamm, R. D. Force-induced focal adhesion translocation: effects of force amplitude and frequency. Am. J. Physiol. Cell Physiol. 287, C954–C962 (2004)

    Article  CAS  Google Scholar 

  25. Riveline, D. et al. Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism. J. Cell Biol. 153, 1175–1186 (2001)

    Article  CAS  Google Scholar 

  26. Brugnera, E. et al. Unconventional Rac-GEF activity is mediated through the Dock180-ELMO complex. Nature Cell Biol. 4, 574–582 (2002)

    Article  CAS  Google Scholar 

  27. Miki, H., Suetsugu, S. & Takenawa, T. WAVE, a novel WASP-family protein involved in actin reorganization induced by Rac. EMBO J. 17, 6932–6941 (1998)

    Article  CAS  Google Scholar 

  28. Sandilands, E. et al. RhoB and actin polymerization coordinate Src activation with endosome-mediated delivery to the membrane. Dev. Cell 7, 855–869 (2004)

    Article  CAS  Google Scholar 

  29. Timpson, P., Jones, G. E., Frame, M. C. & Brunton, V. G. Coordination of cell polarization and migration by the Rho family GTPases requires Src tyrosine kinase activity. Curr. Biol. 11, 1836–1846 (2001)

    Article  CAS  Google Scholar 

  30. Otsu, N. Threshold selection method from Gray-level histograms. IEEE Trans. Syst. Man Cybern. 9, 62–66 (1979)

    Article  Google Scholar 

Download references

Acknowledgements

We thank J. Zhang and A. Y. Ting for their assistance and advice; J.-L. Guan, D. C. Flynn and J. Cooper for providing FAK protein, MEF cells, c-Yes plasmid and SYF cell lines. We also thank B. N. G. Giepmans and C. Hauser for their comments on the paper, and Q. Xiong for technical support. This work was supported by the National Institutes of Health (S.C., R.Y.T. and M.W.B.), the Howard Hughes Medical Institute (R.Y.T.), the Airforce Office of Scientific Research (M.W.B.), the Arnold and Mabel Beckman Foundation (E.L.B.) and the Alliance for Cellular Signaling Grant (R.Y.T., S.C. and Y.W.).

Author information

Authors and Affiliations

  1. Department of Bioengineering and the Whitaker Institute of Biomedical Engineering,

    Yingxiao Wang, Elliot L. Botvinick, Yihua Zhao, Michael W. Berns, Shunichi Usami & Shu Chien

  2. Department of Medicine,

    Shu Chien

  3. Howard Hughes Medical Institute and Departments of Pharmacology and Chemistry and Biochemistry, University of California at San Diego, La Jolla, California, 92093, USA

    Roger Y. Tsien

  4. Department of Biomedical Engineering,

    Michael W. Berns

  5. Beckman Laser Institute, University of California at Irvine, Irvine, California, 92697, USA

    Elliot L. Botvinick & Michael W. Berns

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Correspondence to Shu Chien.

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Supplementary information

Supplementary Materials

This file contains Supplementary Methods, Supplementary Figure Legends and Supplementary Movie Legends. (DOC 72 kb)

Supplementary Figures

This file contains Supplementary Figures S1-S7. (PPT 1203 kb)

Supplementary Movie S1

The EGF-induced FRET response in HeLa cells expressing the Src reporter. (MOV 207 kb)

Supplementary Movie S2

The effects of EGF stimulation and washout on FRET responses of the monomeric Src reporter in HeLa cells. (MOV 4949 kb)

Supplementary Movie S3

A fibronectin-coated bead induced local FRET responses in HUVECs. (MOV 3253 kb)

Supplementary Movie S4

PP1 reversed the local FRET response in HUVECs induced by fibronectin-coated beads. (MOV 2471 kb)

Supplementary Movie S5

The laser-tweezer-traction on a fibronectin-coated bead induced FRET responses of the cytosolic Src reporter in HUVECs. (MOV 4093 kb)

Supplementary Movie S6

The laser-tweezer-traction on a polylysine-coated bead did not induce FRET responses in HUVECs. (MOV 8073 kb)

Supplementary Movie S7

PP1 reversed the EGF-induced FRET responses of the membrane-targeted Src reporter in HeLa cells. (MOV 459 kb)

Supplementary Movie S8

Pretreatment with PP1 inhibited the EGF-induced FRET responses of the membrane-targeted Src reporter in HeLa cells. (MOV 1017 kb)

Supplementary Movie S9

The laser-tweezer-traction induced directional and long-range propagation of FRET responses of the membrane-targeted Src reporter in HUVECs. (MOV 6434 kb)

Supplementary Movie S10

The laser-tweezer traction on a fibronectin-coated bead did not induce FRET responses of the Y662F/Y664F mutant of the membrane-targeted Src reporter. (MOV 1759 kb)

Supplementary Movie S11

The laser-tweezer traction on a fibronectin-coated bead did not induce FRET responses of the R175V mutant of the membrane-targeted Src reporter. (MOV 1684 kb)

Supplementary Movie S12

A highlighted cell with a clear FRET wave propagation away from the stimulation site upon force application. (MOV 7469 kb)

Supplementary Movie S13

Cytochalasin D blocked the directional and long-range activation of Src in response to mechanical force. (MOV 6768 kb)

Supplementary Movie S14

Nocodazole blocked the directional and long-range activation of Src in response to mechanical force. (MOV 8909 kb)

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Wang, Y., Botvinick, E., Zhao, Y. et al. Visualizing the mechanical activation of Src. Nature 434, 1040–1045 (2005). https://doi.org/10.1038/nature03469

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