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.

  • Article
  • Published:

Correlative cryo-ET identifies actin/tropomyosin filaments that mediate cell–substrate adhesion in cancer cells and mechanosensitivity of cell proliferation

Nature Materials volume 21, pages 120–128 (2022)Cite this article

Subjects

Abstract

The actin cytoskeleton is the primary driver of cellular adhesion and mechanosensing due to its ability to generate force and sense the stiffness of the environment. At the cell’s leading edge, severing of the protruding Arp2/3 actin network generates a specific actin/tropomyosin (Tpm) filament population that controls lamellipodial persistence. The interaction between these filaments and adhesion to the environment is unknown. Using cellular cryo-electron tomography we resolve the ultrastructure of the Tpm/actin copolymers and show that they specifically anchor to nascent adhesions and are essential for focal adhesion assembly. Re-expression of Tpm1.8/1.9 in transformed and cancer cells is sufficient to restore cell–substrate adhesions. We demonstrate that knock-out of Tpm1.8/1.9 disrupts the formation of dorsal actin bundles, hindering the recruitment of α-actinin and non-muscle myosin IIa, critical mechanosensors. This loss causes a force-generation and proliferation defect that is notably reversed when cells are grown on soft surfaces. We conclude that Tpm1.8/1.9 suppress the metastatic phenotype, which may explain why transformed cells naturally downregulate this Tpm subset during malignant transformation.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Fig. 1: Tropomyosins link cellular motility initiation with adhesion assembly.
Fig. 2: Ultrastructure of Tpm/actin filaments tethered to adhesions.
Fig. 3: Tropomyosins restore cell adhesion in normal and cancer cells.
Fig. 4: Myosin and α-actinin dorsal stress fibre recruitment depend on Tpm1.8/1.9.
Fig. 5: Tpm1.8/1.9 suppress growth on soft surfaces via mechanosensing.

Similar content being viewed by others

Data availability

The authors declare that all the data supporting the findings of this study are available within the article and the Supplementary Information. Requests for resources including plasmids should be directed to and will be fulfilled by the corresponding author. The publicly available mRNA sequence of mouse Tpm1.8/1.9 used in this study has the access code NCBI M_001164253.1. Source data are provided with this paper.

References

  1. Gumbiner, B. M. Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell 84, 345–357 (1996).

    Article  CAS  Google Scholar 

  2. Zamir, E. & Geiger, B. Molecular complexity and dynamics of cell–matrix adhesions. J. Cell Sci. 114, 3583–3590 (2001).

    Article  CAS  Google Scholar 

  3. Takada, Y., Ye, X. & Simon, S. The integrins. Genome Biol. 8, 215 (2007).

    Article  Google Scholar 

  4. 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 

  5. Zaidel-Bar, R., Cohen, M., Addadi, L. & Geiger, B. Hierarchical assembly of cell–matrix adhesion complexes. Biochem. Soc. Trans. 32, 416–420 (2004).

    Article  CAS  Google Scholar 

  6. Wolfenson, H. et al. Tropomyosin controls sarcomere-like contractions for rigidity sensing and suppressing growth on soft matrices. Nat. Cell Biol. 18, 33–42 (2016).

    Article  CAS  Google Scholar 

  7. Meacci, G. et al. α-Actinin links extracellular matrix rigidity-sensing contractile units with periodic cell-edge retractions. Mol. Biol. Cell 27, 3471–3479 (2016).

    Article  CAS  Google Scholar 

  8. Ghassemi, S. et al. Cells test substrate rigidity by local contractions on submicrometer pillars. Proc. Natl Acad. Sci. USA 109, 5328–5333 (2012).

    Article  CAS  Google Scholar 

  9. Zaidel-Bar, R., Milo, R., Kam, Z. & Geiger, B. A paxillin tyrosine phosphorylation switch regulates the assembly and form of cell–matrix adhesions. J. Cell Sci. 120, 137–148 (2006).

    Article  Google Scholar 

  10. Nakamura, K. et al. Tyrosine phosphorylation of paxillin α is involved in temporospatial regulation of paxillin-containing focal adhesion formation and F-actin organization in motile cells. J. Biol. Chem. 275, 27155–27164 (2000).

    Article  CAS  Google Scholar 

  11. López-Colomé, A. M., Lee-Rivera, I., Benavides-Hidalgo, R. & López, E. Paxillin: a crossroad in pathological cell migration. J. Hematol. Oncol. https://doi.org/10.1186/s13045-017-0418-y (2017).

  12. Bershadsky, A., Kozlov, M. & Geiger, B. Adhesion-mediated mechanosensitivity: a time to experiment, and a time to theorize. Curr. Opin. Cell Biol. 18, 472–481 (2006).

    Article  CAS  Google Scholar 

  13. Pellegrin, S. P. & Mellor, H. Actin stress fibres. J. Cell Sci. 120, 3491–3499 (2007).

    Article  CAS  Google Scholar 

  14. Yang, B. et al. Stopping transformed cancer cell growth by rigidity sensing. Nat. Mater. 19, 239–250 (2020).

    Article  CAS  Google Scholar 

  15. Sheetz, M. A tale of two states: normal and transformed, with and without rigidity sensing. Annu. Rev. Cell Dev. Biol. 35, 169–190 (2019).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Swaney, K. F. & Li, R. Function and regulation of the Arp2/3 complex during cell migration in diverse environments. Curr. Opin. Cell Biol. 42, 63–72 (2016).

    Article  CAS  Google Scholar 

  18. Suraneni, P. et al. The Arp2/3 complex is required for lamellipodia extension and directional fibroblast cell migration. J. Cell Biol. 197, 239–251 (2012).

    Article  CAS  Google Scholar 

  19. Brayford, S. et al. Tropomyosin promotes lamellipodial persistence by collaborating with Arp2/3 at the leading edge. Curr. Biol. 26, 1312–1318 (2016).

    Article  CAS  Google Scholar 

  20. Bareja, I. et al. Dynamics of Tpm1.8 domains on actin filaments with single-molecule resolution. Mol. Biol. Cell 31, 2452–2462 (2020).

    Article  CAS  Google Scholar 

  21. Tojkander, S. et al. A molecular pathway for myosin II recruitment to stress fibers. Curr. Biol. 21, 539–550 (2011).

    Article  CAS  Google Scholar 

  22. Bach, C. T. et al. Tropomyosin isoform expression regulates the transition of adhesions to determine cell speed and direction. Mol. Cell. Biol. 29, 1506–1514 (2009).

    Article  CAS  Google Scholar 

  23. Shin, H., Kim, D. & Helfman, D. M. Tropomyosin isoform Tpm2.1 regulates collective and amoeboid cell migration and cell aggregation in breast epithelial cells. Oncotarget 8, 95192–95205 (2017).

    Article  Google Scholar 

  24. Lees, J. G. et al. Tropomyosin regulates cell migration during skin wound healing. J. Invest. Dermatol. 133, 1330–1339 (2013).

    Article  CAS  Google Scholar 

  25. Koning, R. I., Koster, A. J. & Sharp, T. H. Advances in cryo-electron tomography for biology and medicine. Ann. Anat. 217, 82–96 (2018).

    Article  Google Scholar 

  26. Simpson, L. J., Reader, J. S. & Tzima, E. Mechanical forces and their effect on the ribosome and protein translation machinery. Cells https://doi.org/10.3390/cells9030650 (2020).

  27. Stahnke, S. et al. Loss of Hem1 disrupts macrophage function and impacts migration, phagocytosis, and integrin-mediated adhesion. Curr. Biol. https://doi.org/10.1016/j.cub.2021.02.043 (2021).

  28. Kim, D. H. & Wirtz, D. Focal adhesion size uniquely predicts cell migration. FASEB J. 27, 1351–1361 (2013).

    Article  CAS  Google Scholar 

  29. Meiring, J. C. M. et al. Co-polymers of actin and tropomyosin account for a major fraction of the human actin cytoskeleton. Curr. Biol. 28, 2331–2337.e2335 (2018).

    Article  CAS  Google Scholar 

  30. Prasad, G. L., Fuldner, R. A. & Cooper, H. L. Expression of transduced tropomyosin 1 cDNA suppresses neoplastic growth of cells transformed by the ras oncogene. Proc. Natl Acad. Sci. USA 90, 7039–7043 (1993).

    Article  CAS  Google Scholar 

  31. Cramer, L. P., Siebert, M. & Mitchison, T. J. Identification of novel graded polarity actin filament bundles in locomoting heart fibroblasts: implications for the generation of motile force. J. Cell Biol. 136, 1287–1305 (1997).

    Article  CAS  Google Scholar 

  32. Naumanen, P., Lappalainen, P. & Hotulainen, P. Mechanisms of actin stress fibre assembly. J. Microsc. 231, 446–454 (2008).

    Article  CAS  Google Scholar 

  33. Tojkander, S., Gateva, G. & Lappalainen, P. Actin stress fibers—assembly, dynamics and biological roles. J. Cell Sci. 125, 1855–1864 (2012).

    CAS  Google Scholar 

  34. Lazarides, E. & Burridge, K. α-Actinin: immunofluorescent localization of a muscle structural protein in nonmuscle cells. Cell 6, 289–298 (1975).

    Article  CAS  Google Scholar 

  35. Weber, K. & Groeschel-Stewart, U. Antibody to myosin: the specific visualization of myosin-containing filaments in nonmuscle cells. Proc. Natl Acad. Sci. USA 71, 4561–4564 (1974).

    Article  CAS  Google Scholar 

  36. Kemp, J. P. & Brieher, W. M. The actin filament bundling protein α-actinin-4 actually suppresses actin stress fibers by permitting actin turnover. J. Biol. Chem. 293, 14520–14533 (2018).

    Article  CAS  Google Scholar 

  37. Meiring, J. C. M. et al. Colocation of Tpm3.1 and myosin IIa heads defines a discrete subdomain in stress fibres. J. Cell Sci. https://doi.org/10.1242/jcs.228916 (2019).

  38. Elosegui-Artola, A. et al. Mechanical regulation of a molecular clutch defines force transmission and transduction in response to matrix rigidity. Nat. Cell Biol. 18, 540–548 (2016).

    Article  CAS  Google Scholar 

  39. Pollard, T. D. Reflections on a quarter century of research on contractile systems. Trends Biochem. Sci. 25, 607–611 (2000).

    Article  CAS  Google Scholar 

  40. Chitty, J. L. et al. The Mini‐Organo: a rapid high-throughput 3D coculture organotypic assay for oncology screening and drug development. Cancer Rep. https://doi.org/10.1002/cnr2.1209 (2020).

  41. Bell, E., Ivarsson, B. & Merrill, C. Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc. Natl Acad. Sci. USA 76, 1274–1278 (1979).

    Article  CAS  Google Scholar 

  42. Vogel, V. & Sheetz, M. Local force and geometry sensing regulate cell functions. Nat. Rev. Mol. Cell Biol. 7, 265–275 (2006).

    Article  CAS  Google Scholar 

  43. Iskratsch, T., Wolfenson, H. & Sheetz, M. P. Appreciating force and shape—the rise of mechanotransduction in cell biology. Nat. Rev. Mol. Cell Biol. 15, 825–833 (2014).

    Article  CAS  Google Scholar 

  44. Hardeman, E. C. & Gunning, P. W. Life and death agendas of actin filaments. Nat. Mater. 19, 135–136 (2020).

    Article  CAS  Google Scholar 

  45. Molinie, N. & Gautreau, A. The Arp2/3 regulatory system and its deregulation in cancer. Physiol. Rev. 98, 215–238 (2018).

    Article  CAS  Google Scholar 

  46. Hendricks, M. & Weintraub, H. Tropomyosin is decreased in transformed cells. Proc. Natl Acad. Sci. USA 78, 5633–5637 (1981).

    Article  CAS  Google Scholar 

  47. Coombes, J. D. et al. Ras transformation overrides a proliferation defect induced by Tpm3.1 knockout. Cell. Mol. Biol. Lett. 20, 626–646 (2015).

    Article  CAS  Google Scholar 

  48. Hahn, W. C. et al. Creation of human tumour cells with defined genetic elements. Nature 400, 464–468 (1999).

    Article  CAS  Google Scholar 

  49. Ponten, J. & Saksela, E. Two established in vitro cell lines from human mesenchymal tumours. Int. J. Cancer 2, 434–447 (1967).

    Article  CAS  Google Scholar 

  50. Schevzov, G., Whittaker, S. P., Fath, T., Lin, J. J. & Gunning, P. W. Tropomyosin isoforms and reagents. Bioarchitecture 1, 135–164 (2011).

    Article  Google Scholar 

Download references

Acknowledgements

We would like to thank the Katharina Gaus Light Microscopy Facility, the Flow Cytometry Facility and the Electron Microscopy Unit of the Mark Wainwright Analytical Centre at UNSW, and the Cryo Electron Microscopy Facility through the Victor Chang Innovation Centre for their valuable help. Thanks to J. Hook and Y. Yao for their technical assistance. P.W.G. and E.C.H. were supported by grants from the ARC (DP160101623), the Australian NHMRC (APP1100202, APP1079866) and The Kid’s Cancer Project. N.A. was supported by the Australian NHMRC (APP1102730).

Author information

Authors and Affiliations

  1. School of Medical Sciences, Faculty of Medicine and Health, UNSW Sydney, Sydney, New South Wales, Australia

    Maria Lastra Cagigas, Nicole S. Bryce, Nicholas Ariotti, Simon Brayford, Peter W. Gunning & Edna C. Hardeman

  2. Electron Microscope Unit, Mark Wainwright Analytical Centre, UNSW Sydney, Sydney, New South Wales, Australia

    Nicholas Ariotti

  3. Children’s Cancer Institute, Lowy Cancer Research Centre, UNSW Sydney, Sydney, New South Wales, Australia

    Simon Brayford

Authors
  1. Maria Lastra Cagigas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nicole S. Bryce
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nicholas Ariotti
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Simon Brayford
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peter W. Gunning
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Edna C. Hardeman
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.L.C. conceptualized and wrote the manuscript. M.L.C., N.A. and S.B. performed the data acquisition and analysis. P.W.G., E.C.H., N.S.B. and N.A. reviewed the manuscript, supervised the work and acquired funding.

Corresponding author

Correspondence to Peter W. Gunning.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Materials thanks the anonymous reviewers for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–7 and Video 1.

Reporting Summary

41563_2021_1087_MOESM3_ESM.mp4

Supplementary Video 1 Cryo-tomogram of a nascent adhesion site. Related to Fig. 2. Notice the increased cellular density at the filaments/adhesion Z-plane compared to the cytoplasm above

Source data

Source Data Fig. 4

Source Data including uncropped blots has been provided as a pdf via the uploading link

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Cagigas, M.L., Bryce, N.S., Ariotti, N. et al. Correlative cryo-ET identifies actin/tropomyosin filaments that mediate cell–substrate adhesion in cancer cells and mechanosensitivity of cell proliferation. Nat. Mater. 21, 120–128 (2022). https://doi.org/10.1038/s41563-021-01087-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41563-021-01087-z

This article is cited by

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