Skip to main content
Biophysical Journal logoLink to Biophysical Journal
. 2001 Apr;80(4):1744–1757. doi: 10.1016/s0006-3495(01)76145-0

Traction force microscopy of migrating normal and H-ras transformed 3T3 fibroblasts.

S Munevar 1, Y Wang 1, M Dembo 1
PMCID: PMC1301364  PMID: 11259288

Abstract

Mechanical interactions between cell and substrate are involved in vital cellular functions from migration to signal transduction. A newly developed technique, traction force microscopy, makes it possible to visualize the dynamic characteristics of mechanical forces exerted by fibroblasts, including the magnitude, direction, and shear. In the present study such analysis is applied to migrating normal and transformed 3T3 cells. For normal cells, the lamellipodium provides almost all the forces for forward locomotion. A zone of high shear separates the lamellipodium from the cell body, suggesting that they are mechanically distinct entities. Timing and distribution of tractions at the leading edge bear no apparent relationship to local protrusive activities. However, changes in the pattern of traction forces often precede changes in the direction of migration. These observations suggest a frontal towing mechanism for cell migration, where dynamic traction forces at the leading edge actively pull the cell body forward. For H-ras transformed cells, pockets of weak, transient traction scatter among small pseudopods and appear to act against one another. The shear pattern suggests multiple disorganized mechanical domains. The weak, poorly coordinated traction forces, coupled with weak cell-substrate adhesions, are likely responsible for the abnormal motile behavior of H-ras transformed cells.

Full Text

The Full Text of this article is available as a PDF (6.7 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Albrecht-Buehler G. Autonomous movements of cytoplasmic fragments. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6639–6643. doi: 10.1073/pnas.77.11.6639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bondy G. P., Wilson S., Chambers A. F. Experimental metastatic ability of H-ras-transformed NIH3T3 cells. Cancer Res. 1985 Dec;45(12 Pt 1):6005–6009. [PubMed] [Google Scholar]
  3. Boudreau N. J., Jones P. L. Extracellular matrix and integrin signalling: the shape of things to come. Biochem J. 1999 May 1;339(Pt 3):481–488. [PMC free article] [PubMed] [Google Scholar]
  4. Brown A. F., Dugina V., Dunn G. A., Vasiliev J. M. A quantitative analysis of alterations in the shape of cultured fibroblasts induced by tumour-promoting phorbol ester. Cell Biol Int Rep. 1989 Apr;13(4):357–366. doi: 10.1016/0309-1651(89)90162-8. [DOI] [PubMed] [Google Scholar]
  5. Burton K., Park J. H., Taylor D. L. Keratocytes generate traction forces in two phases. Mol Biol Cell. 1999 Nov;10(11):3745–3769. doi: 10.1091/mbc.10.11.3745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burton K., Taylor D. L. Traction forces of cytokinesis measured with optically modified elastic substrata. Nature. 1997 Jan 30;385(6615):450–454. doi: 10.1038/385450a0. [DOI] [PubMed] [Google Scholar]
  7. Byers H. R., Etoh T., Doherty J. R., Sober A. J., Mihm M. C., Jr Cell migration and actin organization in cultured human primary, recurrent cutaneous and metastatic melanoma. Time-lapse and image analysis. Am J Pathol. 1991 Aug;139(2):423–435. [PMC free article] [PubMed] [Google Scholar]
  8. Davies P. F. Flow-mediated endothelial mechanotransduction. Physiol Rev. 1995 Jul;75(3):519–560. doi: 10.1152/physrev.1995.75.3.519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. DePasquale J. A., Izzard C. S. Evidence for an actin-containing cytoplasmic precursor of the focal contact and the timing of incorporation of vinculin at the focal contact. J Cell Biol. 1987 Dec;105(6 Pt 1):2803–2809. doi: 10.1083/jcb.105.6.2803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dembo M., Wang Y. L. Stresses at the cell-to-substrate interface during locomotion of fibroblasts. Biophys J. 1999 Apr;76(4):2307–2316. doi: 10.1016/S0006-3495(99)77386-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. DiMilla P. A., Barbee K., Lauffenburger D. A. Mathematical model for the effects of adhesion and mechanics on cell migration speed. Biophys J. 1991 Jul;60(1):15–37. doi: 10.1016/S0006-3495(91)82027-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Egan S. E., McClarty G. A., Jarolim L., Wright J. A., Spiro I., Hager G., Greenberg A. H. Expression of H-ras correlates with metastatic potential: evidence for direct regulation of the metastatic phenotype in 10T1/2 and NIH 3T3 cells. Mol Cell Biol. 1987 Feb;7(2):830–837. doi: 10.1128/mcb.7.2.830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Elson E. L., Felder S. F., Jay P. Y., Kolodney M. S., Pasternak C. Forces in cell locomotion. Biochem Soc Symp. 1999;65:299–314. [PubMed] [Google Scholar]
  14. Galbraith C. G., Sheetz M. P. A micromachined device provides a new bend on fibroblast traction forces. Proc Natl Acad Sci U S A. 1997 Aug 19;94(17):9114–9118. doi: 10.1073/pnas.94.17.9114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Galbraith C. G., Sheetz M. P. Forces on adhesive contacts affect cell function. Curr Opin Cell Biol. 1998 Oct;10(5):566–571. doi: 10.1016/s0955-0674(98)80030-6. [DOI] [PubMed] [Google Scholar]
  16. Giancotti F. G., Ruoslahti E. Integrin signaling. Science. 1999 Aug 13;285(5430):1028–1032. doi: 10.1126/science.285.5430.1028. [DOI] [PubMed] [Google Scholar]
  17. Goldschmidt-Clermont P. J., Galbraith R. M., Emerson D. L., Marsot F., Nel A. E., Arnaud P. Distinct sites on the G-actin molecule bind group-specific component and deoxyribonuclease I. Biochem J. 1985 Jun 1;228(2):471–477. doi: 10.1042/bj2280471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Grinnell F., Zhu M., Carlson M. A., Abrams J. M. Release of mechanical tension triggers apoptosis of human fibroblasts in a model of regressing granulation tissue. Exp Cell Res. 1999 May 1;248(2):608–619. doi: 10.1006/excr.1999.4440. [DOI] [PubMed] [Google Scholar]
  19. Gumbiner B. M. Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell. 1996 Feb 9;84(3):345–357. doi: 10.1016/s0092-8674(00)81279-9. [DOI] [PubMed] [Google Scholar]
  20. Hall A. Rho GTPases and the actin cytoskeleton. Science. 1998 Jan 23;279(5350):509–514. doi: 10.1126/science.279.5350.509. [DOI] [PubMed] [Google Scholar]
  21. Harris A. K., Stopak D., Wild P. Fibroblast traction as a mechanism for collagen morphogenesis. Nature. 1981 Mar 19;290(5803):249–251. doi: 10.1038/290249a0. [DOI] [PubMed] [Google Scholar]
  22. Harris A. K., Wild P., Stopak D. Silicone rubber substrata: a new wrinkle in the study of cell locomotion. Science. 1980 Apr 11;208(4440):177–179. doi: 10.1126/science.6987736. [DOI] [PubMed] [Google Scholar]
  23. Heath J. P. Behaviour and structure of the leading lamella in moving fibroblasts. I. Occurrence and centripetal movement of arc-shaped microfilament bundles beneath the dorsal cell surface. J Cell Sci. 1983 Mar;60:331–354. doi: 10.1242/jcs.60.1.331. [DOI] [PubMed] [Google Scholar]
  24. Hill S. A., Wilson S., Chambers A. F. Clonal heterogeneity, experimental metastatic ability, and p21 expression in H-ras-transformed NIH 3T3 cells. J Natl Cancer Inst. 1988 Jun 1;80(7):484–490. doi: 10.1093/jnci/80.7.484. [DOI] [PubMed] [Google Scholar]
  25. Ilić D., Damsky C. H., Yamamoto T. Focal adhesion kinase: at the crossroads of signal transduction. J Cell Sci. 1997 Feb;110(Pt 4):401–407. doi: 10.1242/jcs.110.4.401. [DOI] [PubMed] [Google Scholar]
  26. Ishihara A., Holifield B., Jacobson K. Analysis of lateral redistribution of a plasma membrane glycoprotein-monoclonal antibody complex [corrected]. J Cell Biol. 1988 Feb;106(2):329–343. doi: 10.1083/jcb.106.2.329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Katz B. Z., Yamada K. M. Integrins in morphogenesis and signaling. Biochimie. 1997 Sep;79(8):467–476. doi: 10.1016/s0300-9084(97)82738-1. [DOI] [PubMed] [Google Scholar]
  28. Lauffenburger D. A., Horwitz A. F. Cell migration: a physically integrated molecular process. Cell. 1996 Feb 9;84(3):359–369. doi: 10.1016/s0092-8674(00)81280-5. [DOI] [PubMed] [Google Scholar]
  29. Leader W. M., Stopak D., Harris A. K. Increased contractile strength and tightened adhesions to the substratum result from reverse transformation of CHO cells by dibutyryl cyclic adenosine monophosphate. J Cell Sci. 1983 Nov;64:1–11. doi: 10.1242/jcs.64.1.1. [DOI] [PubMed] [Google Scholar]
  30. Lee W. M., Galbraith R. M. The extracellular actin-scavenger system and actin toxicity. N Engl J Med. 1992 May 14;326(20):1335–1341. doi: 10.1056/NEJM199205143262006. [DOI] [PubMed] [Google Scholar]
  31. Lukashev M. E., Werb Z. ECM signalling: orchestrating cell behaviour and misbehaviour. Trends Cell Biol. 1998 Nov;8(11):437–441. doi: 10.1016/s0962-8924(98)01362-2. [DOI] [PubMed] [Google Scholar]
  32. Malawista S. E., De Boisfleury Chevance A. The cytokineplast: purified, stable, and functional motile machinery from human blood polymorphonuclear leukocytes. J Cell Biol. 1982 Dec;95(3):960–973. doi: 10.1083/jcb.95.3.960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Narumiya S., Ishizaki T., Watanabe N. Rho effectors and reorganization of actin cytoskeleton. FEBS Lett. 1997 Jun 23;410(1):68–72. doi: 10.1016/s0014-5793(97)00317-7. [DOI] [PubMed] [Google Scholar]
  34. Oliver T., Dembo M., Jacobson K. Separation of propulsive and adhesive traction stresses in locomoting keratocytes. J Cell Biol. 1999 May 3;145(3):589–604. doi: 10.1083/jcb.145.3.589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Oliver T., Jacobson K., Dembo M. Design and use of substrata to measure traction forces exerted by cultured cells. Methods Enzymol. 1998;298:497–521. doi: 10.1016/s0076-6879(98)98042-9. [DOI] [PubMed] [Google Scholar]
  36. Parsons J. T. Integrin-mediated signalling: regulation by protein tyrosine kinases and small GTP-binding proteins. Curr Opin Cell Biol. 1996 Apr;8(2):146–152. doi: 10.1016/s0955-0674(96)80059-7. [DOI] [PubMed] [Google Scholar]
  37. Pelham R. J., Jr, Wang Y. l. High resolution detection of mechanical forces exerted by locomoting fibroblasts on the substrate. Mol Biol Cell. 1999 Apr;10(4):935–945. doi: 10.1091/mbc.10.4.935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Radmacher M., Tillamnn R. W., Fritz M., Gaub H. E. From molecules to cells: imaging soft samples with the atomic force microscope. Science. 1992 Sep 25;257(5078):1900–1905. doi: 10.1126/science.1411505. [DOI] [PubMed] [Google Scholar]
  39. Rottner K., Hall A., Small J. V. Interplay between Rac and Rho in the control of substrate contact dynamics. Curr Biol. 1999 Jun 17;9(12):640–648. doi: 10.1016/s0960-9822(99)80286-3. [DOI] [PubMed] [Google Scholar]
  40. Sadoshima J., Izumo S. Mechanical stretch rapidly activates multiple signal transduction pathways in cardiac myocytes: potential involvement of an autocrine/paracrine mechanism. EMBO J. 1993 Apr;12(4):1681–1692. doi: 10.1002/j.1460-2075.1993.tb05813.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schoenwaelder S. M., Burridge K. Bidirectional signaling between the cytoskeleton and integrins. Curr Opin Cell Biol. 1999 Apr;11(2):274–286. doi: 10.1016/s0955-0674(99)80037-4. [DOI] [PubMed] [Google Scholar]
  42. Schwartz M. A., Baron V. Interactions between mitogenic stimuli, or, a thousand and one connections. Curr Opin Cell Biol. 1999 Apr;11(2):197–202. doi: 10.1016/s0955-0674(99)80026-x. [DOI] [PubMed] [Google Scholar]
  43. Sheetz M. P., Felsenfeld D. P., Galbraith C. G. Cell migration: regulation of force on extracellular-matrix-integrin complexes. Trends Cell Biol. 1998 Feb;8(2):51–54. doi: 10.1016/s0962-8924(98)80005-6. [DOI] [PubMed] [Google Scholar]
  44. Shin E. Y., Lee J. Y., Park M. K., Jeong G. B., Kim E. G., Kim S. Y. H-Ras is a negative regulator of alpha3beta1 integrin expression in ECV304 endothelial cells. Biochem Biophys Res Commun. 1999 Apr 2;257(1):95–99. doi: 10.1006/bbrc.1999.0302. [DOI] [PubMed] [Google Scholar]
  45. Small J. V., Rottner K., Kaverina I., Anderson K. I. Assembling an actin cytoskeleton for cell attachment and movement. Biochim Biophys Acta. 1998 Sep 16;1404(3):271–281. doi: 10.1016/s0167-4889(98)00080-9. [DOI] [PubMed] [Google Scholar]
  46. Svitkina T. M., Verkhovsky A. B., McQuade K. M., Borisy G. G. Analysis of the actin-myosin II system in fish epidermal keratocytes: mechanism of cell body translocation. J Cell Biol. 1997 Oct 20;139(2):397–415. doi: 10.1083/jcb.139.2.397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Van Baelen H., Bouillon R., De Moor P. Vitamin D-binding protein (Gc-globulin) binds actin. J Biol Chem. 1980 Mar 25;255(6):2270–2272. [PubMed] [Google Scholar]
  48. Varani J., Fligiel S. E., Wilson B. Motility of rasH oncogene transformed NIH-3T3 cells. Invasion Metastasis. 1986;6(6):335–346. [PubMed] [Google Scholar]
  49. Verkhovsky A. B., Svitkina T. M., Borisy G. G. Self-polarization and directional motility of cytoplasm. Curr Biol. 1999 Jan 14;9(1):11–20. doi: 10.1016/s0960-9822(99)80042-6. [DOI] [PubMed] [Google Scholar]
  50. Wang Y. L. Exchange of actin subunits at the leading edge of living fibroblasts: possible role of treadmilling. J Cell Biol. 1985 Aug;101(2):597–602. doi: 10.1083/jcb.101.2.597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wang Y. L., Pelham R. J., Jr Preparation of a flexible, porous polyacrylamide substrate for mechanical studies of cultured cells. Methods Enzymol. 1998;298:489–496. doi: 10.1016/s0076-6879(98)98041-7. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES