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.

Isolated nuclei adapt to force and reveal a mechanotransduction pathway in the nucleus

Nature Cell Biology volume 16, pages 376–381 (2014)Cite this article

Subjects

Abstract

Mechanical forces influence many aspects of cell behaviour. Forces are detected and transduced into biochemical signals by force-bearing molecular elements located at the cell surface, in adhesion complexes or in cytoskeletal structures1. The nucleus is physically connected to the cell surface through the cytoskeleton and the linker of nucleoskeleton and cytoskeleton (LINC) complex, allowing rapid mechanical stress transmission from adhesions to the nucleus2. Although it has been demonstrated that nuclei experience force3, the direct effect of force on the nucleus is not known. Here we show that isolated nuclei are able to respond to force by adjusting their stiffness to resist the applied tension. Using magnetic tweezers, we found that applying force on nesprin-1 triggers nuclear stiffening that does not involve chromatin or nuclear actin, but requires an intact nuclear lamina and emerin, a protein of the inner nuclear membrane. Emerin becomes tyrosine phosphorylated in response to force and mediates the nuclear mechanical response to tension. Our results demonstrate that mechanotransduction is not restricted to cell surface receptors and adhesions but can occur in the nucleus.

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

Get just this article for as long as you need it

$39.95

Learn more

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

Figure 1: Isolated nuclei stiffen in response to force applied on nesprin-1.
Figure 2: The nucleoskeleton mediates nuclear stiffening in response to force.
Figure 3: Emerin phosphorylation on Tyr 74 and Tyr 95 mediates the mechanical adaptation of isolated nuclei to force.
Figure 4: Emerin phosphorylation on Tyr 74 and Tyr 95 affects stress fibre formation and SRF-dependent gene expression.

References

  1. Hoffman, B. D., Grashoff, C. & Schwartz, M. A. Dynamic molecular processes mediate cellular mechanotransduction. Nature 475, 316–323 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Wang, N., Tytell, J. D. & Ingber, D. E. Mechanotransduction at a distance: Mechanically coupling the extracellular matrix with the nucleus. Nat. Rev. Mol. Cell Biol. 10, 75–82 (2009).

    Article  CAS  PubMed  Google Scholar 

  3. Maniotis, A. J., Chen, C. S. & Ingber, D. E. Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure. Proc. Natl Acad. Sci. USA 94, 849–854 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Wang, N., Butler, J. P. & Ingber, D. E. Mechanotransduction across the cell surface and through the cytoskeleton. Science 260, 1124–1127 (1993).

    Article  CAS  PubMed  Google Scholar 

  5. Matthews, B. D., Overby, D. R., Mannix, R. & Ingber, D. E. Cellular adaptation to mechanical stress: Role of integrins, Rho, cytoskeletal tension and mechanosensitive ion channels. J. Cell Sci. 119, 508–518 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Choquet, D., Felsenfeld, D. P. & Sheetz, M. P. Extracellular matrix rigidity causes strengthening of integrin-cytoskeleton linkages. Cell 88, 39–48 (1997).

    Article  CAS  PubMed  Google Scholar 

  7. Guilluy, C. et al. The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins. Nat. Cell Biol. 13, 724–729 (2011).

    Article  CAS  Google Scholar 

  8. Hofmann, W. A. & de Lanerolle, P. Nuclear actin: To polymerize or not to polymerize. J. Cell Biol. 172, 495–496 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Dubash, A. D. et al. The small GTPase RhoA localizes to the nucleus and is activated by Net1 and DNA damage signals. PloS ONE 6, e17380 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Dahl, K. N. & Kalinowski, A. Nucleoskeleton mechanics at a glance. J. Cell Sci. 124, 675–678 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Pajerowski, J. D., Dahl, K. N., Zhong, F. L., Sammak, P. J. & Discher, D. E. Physical plasticity of the nucleus in stem cell differentiation. Proc. Natl Acad. Sci. USA 104, 15619–15624 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Lammerding, J. et al. Lamin A/C deficiency causes defective nuclear mechanics and mechanotransduction. J. Clin. Invest. 113, 370–378 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Sosa, B. A., Rothballer, A., Kutay, U. & Schwartz, T. U. LINC complexes form by binding of three KASH peptides to domain interfaces of trimeric SUN proteins. Cell 149, 1035–1047 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lei, K. et al. SUN1 and SUN2 play critical but partially redundant roles in anchoring nuclei in skeletal muscle cells in mice. Proc. Natl Acad. Sci. USA 106, 10207–10212 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Lammerding, J. et al. Abnormal nuclear shape and impaired mechanotransduction in emerin-deficient cells. J. Cell Biol. 170, 781–791 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Rowat, A. C., Lammerding, J. & Ipsen, J. H. Mechanical properties of the cell nucleus and the effect of emerin deficiency. Biophys. J. 91, 4649–4664 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Sawada, Y. et al. Force sensing by mechanical extension of the Src family kinase substrate p130Cas. Cell 127, 1015–1026 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Takahashi, A. et al. Nuclear localization of Src-family tyrosine kinases is required for growth factor-induced euchromatinization. Exp. Cell Res. 315, 1117–1141 (2009).

    Article  CAS  PubMed  Google Scholar 

  19. Chu, I. et al. p27 phosphorylation by Src regulates inhibition of cyclin E-Cdk2. Cell 128, 281–294 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Taagepera, S. et al. Nuclear-cytoplasmic shuttling of C-ABL tyrosine kinase. Proc. Natl Acad. Sci. USA 95, 7457–7462 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lim, S. T. et al. Nuclear FAK promotes cell proliferation and survival through FERM-enhanced p53 degradation. Mol. Cell 29, 9–22 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Tifft, K. E., Bradbury, K. A. & Wilson, K. L. Tyrosine phosphorylation of nuclear-membrane protein emerin by Src, Abl and other kinases. J. Cell Sci. 122, 3780–3790 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Ho, C. Y. & Lammerding, J. Lamins at a glance. J. Cell Sci. 125, 2087–2093 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Provenzano, P. P. & Keely, P. J. Mechanical signaling through the cytoskeleton regulates cell proliferation by coordinated focal adhesion and Rho GTPase signaling. J. Cell Sci. 124, 1195–1205 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Khatau, S. B. et al. A perinuclear actin cap regulates nuclear shape. Proc. Natl Acad. Sci. USA 106, 19017–19022 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Luxton, G. W., Gomes, E. R., Folker, E. S., Vintinner, E. & Gundersen, G. G. Linear arrays of nuclear envelope proteins harness retrograde actin flow for nuclear movement. Science 329, 956–959 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Folker, E. S., Ostlund, C., Luxton, G. W., Worman, H. J. & Gundersen, G. G. Lamin A variants that cause striated muscle disease are defective in anchoring transmembrane actin-associated nuclear lines for nuclear movement. Proc. Natl Acad. Sci. USA 108, 131–136 (2011).

    Article  CAS  PubMed  Google Scholar 

  28. Khatau, S. B. et al. The distinct roles of the nucleus and nucleus-cytoskeleton connections in three-dimensional cell migration. Sci. Rep. 2, 1–11 (2012).

    Article  Google Scholar 

  29. Ho, C. Y., Jaalouk, D. E., Vartiainen, M. K. & Lammerding, J. Lamin A/C and emerin regulate MKL1-SRF activity by modulating actin dynamics. Nature 497, 507–511 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Friedl, P., Wolf, K. & Lammerding, J. Nuclear mechanics during cell migration. Curr. Opin. Cell Biol. 23, 55–64 (2011).

    Article  CAS  PubMed  Google Scholar 

  31. Philip, J. T. & Dahl, K. N. Nuclear mechanotransduction: Response of the lamina to extracellular stress with implications in aging. J. Biomechanics 41, 3164–3170 (2008).

    Article  Google Scholar 

  32. Fisher, J. K. et al. Thin-foil magnetic force system for high-numerical-aperture microscopy. Rev. Sci. Instrum. 77, nihms8302 (2006).

    Article  CAS  PubMed  Google Scholar 

  33. Guilluy, C. et al. The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins. Nat. Cell Biol. 13, 724–729 (2011).

    Article  CAS  Google Scholar 

  34. 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  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This study was supported by National Institutes of Health Grants numbers GM029860 (to K.B.), P41-EB002025-23A1 (R.S.) and R01-HL077546-03A2 (R.S.), and a grant from the University Cancer Research Fund from the Lineberger Comprehensive Cancer Center. C.G. is supported by a Marie Curie Outgoing International Fellowship from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 254747.

Author information

Author notes

  1. Christophe Guilluy & Laurianne Van Landeghem

    Present address: Present addresses: INSERM, UMR1087, L’Institut du Thorax, Nantes, F-44000, France (C.G.); INSERM, UMR913, Institut des Maladies de l’Appareil Digestif, Université de Nantes, Nantes, F-44093, France (L.V.L.).,

Authors and Affiliations

  1. Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA

    Christophe Guilluy, Laurianne Van Landeghem, Lisa Sharek, Rafael Garcia-Mata & Keith Burridge

  2. Department of Physics and Astronomy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA

    Lukas D. Osborne & Richard Superfine

  3. Lineberger Comprehensive Cancer Center, and UNC McAllister Heart Institute, University of North Carolina, Chapel Hill, North Carolina 27599, USA

    Keith Burridge

Authors
  1. Christophe Guilluy
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lukas D. Osborne
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Laurianne Van Landeghem
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lisa Sharek
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Richard Superfine
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Rafael Garcia-Mata
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Keith Burridge
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.G. designed and performed experiments. L.S., L.D.O., L.V.L., R.S. and R.G-M. helped with experimental design and procedures. C.G. and K.B. wrote the manuscript. K.B. directed the project. All authors provided detailed comments.

Corresponding authors

Correspondence to Christophe Guilluy or Keith Burridge.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Nuclear stiffening in response to force applied to nesprin-1.

Typical displacement plot for control sh (a—left panel), emerin sh2(b) and laminA/C sh1 (c). Displacement curve transformed into compliance with Jeffreys model fit (a—right panel) (Jeffreys model description in Supplementary Fig. 2a).

Supplementary Figure 2 Viscoelastic behavior of the nucleus.

To examine the stiffness of the nucleus in response to an applied force, the time-dependent compliance of the nucleus was calculated from the time-dependent displacement using:J(t) = 6πax(t)/F(t), where a is the bead radius. a, The viscoelastic response of the nucleus was characterized by fitting the compliance during force application to a Jeffreys model, a mechanical circuit model used to describe viscoelastic material. The Jeffreys model is formed by an elastic spring and viscous dashpot in parallel with a dashpot in series. b, Example bead displacement curve. c, Example bead displacement curve transformed into compliance with overlay of Jeffreys model fit.

Supplementary Figure 3 Characterization of the nuclear stiffening in response to force.

a, Nuclei isolated from Hela cells were incubated with anti-nesprin-1-coated magnetic beads and stimulated with a permanent magnet for different amounts of time. Active RhoA (RhoAGTP) was isolated with GSTRBD (Rho-binding domain) and analysed by western blotting. All results are representative of at least three independent experiments. b, Stable cell lines depleted for Lamin A/C, LAP2α, emerin, SUN1 or SUN2 were generated using shRNA. Efficiency of the knockdown was assessed by western blot. All results are representative of at least three independent experiments. c, Change in bead displacement between the first and the 6th pulse of force applied to beads coated with anti-nesprin-1 antibody bound to nuclei isolated from cells transfected with control siRNA (n = 12 beads) for MAN1 siRNA (n = 15 beads) and LBR siRNA (n = 17 beads). Displacements were calculated relative to the first pulse of force (error bars represent s.e.m., *P < 0.05, data were collected from 3 independent experiments and analysed by two-tailed unpaired t-test). d, Efficiency of siRNA knockdowns for MAN1 and LBR were assessed by w

Supplementary Figure 4 SFK(s) mediate the nuclear stiffening to force.

a, Nuclei isolated from Hela cells were incubated with anti-nesprin-1-coated magnetic beads and stimulated with a permanent magnet for different amounts of time. Emerin was immunoprecipitated and its tyrosine phosphorylation was analysed by western blot. All results are representative of at least three independent experiments. b, Nuclei isolated from Hela cells were incubated with anti-nesprin-1-coated magnetic beads and pretreated 30 min with Gleevec (10 μM), SU66056 (2.5 μM) or FAK inhibitor (5 μM). After stimulation with a permanent magnet for 3 min, tyrosine phosphorylation of nuclear proteins was analysed by western blot. All results are representative of at least three independent experiments. c, Nuclei isolated from Hela cells were incubated with anti-nesprin-1-coated magnetic beads and stimulated with a permanent magnet for 3 min. Src expression, Src phosphorylation on Y416, FAK expression and FAK phosphorylation on Y397 were assessed by western blot. All results are representative of at least three independent experiments. d, Change in bead displacement between the first and 6th pulse of force applied to beads coated with anti-nesprin-1 antibody bound to nuclei incubated with no ATP (n = 14 beads) or with SU6656 (n = 22 beads) for 30 min (untreated control n = 13 beads). Displacements were calculated relative to the first pulse of force applied to untreated nuclei (Error bars represent s.e.m., *P < 0.05, data were collected from 3 independent experiments and analysed by two-tailed unpaired t-test).

Supplementary Figure 5 Tension induces emerin phosphorylation.

a, Emerin tyrosine phosphorylation was analysed after immunoprecipitation in MRC5 cells during adhesion to fibronectin or treated with blebbistatin. (‘total’ refers to the emerin level in nuclear lysates). All results are representative of at least three independent experiments. b, MRC5 cells were incubated with fibronectin-coated magnetic beads for 30 min. A permanent magnet was used to generate tensional force for different amounts of time. After cell lysis, emerin tyrosine phosphorylation was analysed. All results are representative of at least three independent experiments. c, Invasion of emerin knockdown Hela cells and emerin knockdown Hela cells re-expressing WT or 74-95FF emerin mutant was evaluated by Transwell migration assays. Cells were plated in the upper chamber of the filters and after 8 h cells that had migrated to the underside of the filters were fixed. Relative cell migration was determined by the number of cells that had migrated to the underside of the filter normalized to the total number of cells. A number of n = 24 fields were observed per condition. The value from control shRNA Hela cells was arbitrarily set at 100% (Error bars represent s.e.m., #P < 0.05 compared to control sh, *P < 0.05 compared to WT, data were collected from 4 independent experiments and analysed by one way ANOVA). d, Emerin knockdown MRC5 cells re-expressing WT or 74-95FF emerin mutant were grown on fibronectin-coated coverslips for 6 h, fixed, permeabilized and stained for YAP/TAZ and myc tagged emerin. To quantify YAP or TAZ nuclear localization (panel d) we calculated the percentage of cells with a predominant nuclear staining (delimited by DAPI staining) among the total cell number. n = 36 myc positive cells expressing WT emerin and n = 34 myc positive cells expressing 74-95FF were analysed (Error bars represent s.e.m., *P < 0.05, data were collected from 3 independent experiments and analysed by two-tailed unpaired t-test). Bar scale = 25 μm. e, Emerin knockdown MRC5 cells re-expressing WT or 74-95FF emerin mutant were incubated with fibronectin-coated magnetic beads for 20 min. A permanent magnet was used to generate tensional force for 90 min. IEX1 and GAPDH mRNA levels were analysed using real-time qPCR (error bars represent s.e.m., #P < 0.05 compared to WT control, data were collected from n = 3 independent experiments and analysed by two-tailed unpaired t-test).

Supplementary information

Supplementary Information

Supplementary Information (PDF 661 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Guilluy, C., Osborne, L., Van Landeghem, L. et al. Isolated nuclei adapt to force and reveal a mechanotransduction pathway in the nucleus. Nat Cell Biol 16, 376–381 (2014). https://doi.org/10.1038/ncb2927

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2927

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