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Analyzing Mechanotransduction Through the LINC Complex in Isolated Nuclei

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1840)

Abstract

The mechanical properties of the cellular microenvironment can impact many aspects of cell behavior, including molecular processes in the nucleus. Recent studies indicate that the LINC complex and its associated nuclear envelope transmit and transduce mechanical stress into biochemical pathways that ultimately regulate nuclear structure or gene expression. Here we describe a method to apply tensional forces to the LINC complex of isolated nuclei. Using magnetic beads and magnets, this technique can be used to explore the biochemical pathways that are activated in response to tension applied to the surface of isolated nuclei.

Key words

Mechanotransduction Lamina Nucleus Mechanical tension Nesprin 
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Notes

Acknowledgments

We thank Keith Burridge for his continuous support. C.G. is supported by grants from the Agence Nationale de la Recherche (ANR-13-JSV1-0008) and from European Research Council (ERC) under European Union’s Horizon 2020 research and innovation program (ERC Starting Grant n°639300).

References

  1. 1.
    Pederson T (2011) The nucleus introduced. Cold Spring Harb Perspect Biol 3:a000521.  https://doi.org/10.1101/cshperspect.a000521CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Capco DG, Wan KM, Penman S (1982) The nuclear matrix: three-dimensional architecture and protein composition. Cell 29:847–858CrossRefPubMedGoogle Scholar
  3. 3.
    Lehto VP, Virtanen I, Kurki P (1978) Intermediate filaments anchor the nuclei in nuclear monolayers of cultured human fibroblasts. Nature 272:175–177CrossRefPubMedGoogle Scholar
  4. 4.
    Crisp M, Liu Q, Roux K, Rattner JB, Shanahan C, Burke B, Stahl PD, Hodzic D (2006) Coupling of the nucleus and cytoplasm: role of the LINC complex. J Cell Biol 172:41–53.  https://doi.org/10.1083/jcb.200509124CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Chang W, Worman HJ, Gundersen GG (2015) Accessorizing and anchoring the LINC complex for multifunctionality. J Cell Biol 208:11–22.  https://doi.org/10.1083/jcb.201409047CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Gundersen GG, Worman HJ (2013) Nuclear positioning. Cell 152:1376–1389.  https://doi.org/10.1016/j.cell.2013.02.031CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Maniotis AJ, Chen CS, Ingber DE (1997) Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure. Proc Natl Acad Sci U S A 94:849–854CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Aureille J, Belaadi N, Guilluy C (2017) Mechanotransduction via the nuclear envelope: a distant reflection of the cell surface. Curr Opin Cell Biol 44:59–67.  https://doi.org/10.1016/j.ceb.2016.10.003CrossRefPubMedGoogle Scholar
  9. 9.
    Guilluy C, Osborne LD, Van Landeghem L, Sharek L, Superfine R, Garcia-Mata R, Burridge K (2014) Isolated nuclei adapt to force and reveal a mechanotransduction pathway in the nucleus. Nat Cell Biol 16:376–381.  https://doi.org/10.1038/ncb2927CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Swift J, Ivanovska IL, Buxboim A, Harada T, Dingal PCDP, Pinter J, Pajerowski JD, Spinler KR, Shin J-W, Tewari M, Rehfeldt F, Speicher DW, Discher DE (2013) Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation. Science 341:1240104.  https://doi.org/10.1126/science.1240104CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Enyedi B, Jelcic M, Niethammer P (2016) The cell nucleus serves as a mechanotransducer of tissue damage-induced inflammation. Cell 165:1160–1170.  https://doi.org/10.1016/j.cell.2016.04.016CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Kumar A, Mazzanti M, Mistrik M, Kosar M, Beznoussenko GV, Mironov AA, Garrè M, Parazzoli D, Shivashankar GV, Scita G, Bartek J, Foiani M (2014) ATR mediates a checkpoint at the nuclear envelope in response to mechanical stress. Cell 158:633–646.  https://doi.org/10.1016/j.cell.2014.05.046CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Marjoram RJ, Guilluy C, Burridge K (2016) Using magnets and magnetic beads to dissect signaling pathways activated by mechanical tension applied to cells. Methods San Diego Calif 94:19–26.  https://doi.org/10.1016/j.ymeth.2015.09.025CrossRefGoogle Scholar
  14. 14.
    Zhao X-H, Laschinger C, Arora P, Szászi K, Kapus A, McCulloch CA (2007) Force activates smooth muscle alpha-actin promoter activity through the Rho signaling pathway. J Cell Sci 120:1801–1809.  https://doi.org/10.1242/jcs.001586CrossRefPubMedGoogle Scholar
  15. 15.
    Guilluy C, Swaminathan V, Garcia-Mata R, O’Brien ET, Superfine R, Burridge K (2011) The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins. Nat Cell Biol 13:722–727.  https://doi.org/10.1038/ncb2254CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Millon-Frémillon A, Aureille J, Guilluy C (2017) Analyzing cell surface adhesion remodeling in response to mechanical tension using magnetic beads. J Vis Exp.  https://doi.org/10.3791/55330
  17. 17.
    Arsenovic PT, Ramachandran I, Bathula K, Zhu R, Narang JD, Noll NA, Lemmon CA, Gundersen GG, Conway DE (2016) Nesprin-2G, a component of the nuclear LINC complex, is subject to myosin-dependent tension. Biophys J 110:34–43.  https://doi.org/10.1016/j.bpj.2015.11.014CrossRefPubMedCentralPubMedGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.l’institut du thorax, Inserm, CNRS, Université de NantesNantesFrance
  2. 2.Institute for Advanced Biosciences, Centre de recherche UGA—INSERM U1209—CNRS UMR 5309GrenobleFrance

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