Advertisement

Using Nesprin Tension Sensors to Measure Force on the LINC Complex

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

Abstract

Mechanotransduction, or the process by which mechanical forces regulate cellular functions, is increasingly studied in a variety of different physiological and pathological contexts. Although these forces are most often studied at cell-matrix and cell-cell adhesions, recent work has shown that the nuclear LINC complex is also subject to mechanical forces. Here we describe how to use a FRET-based biosensor, known as TSmod, in the LINC complex protein nesprin-2G. This approach allows for measurement of LINC complex forces in living cells with spatial-temporal resolution.

Key words

Mechanobiology FRET tension biosensors Nuclear LINC complex 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

This work was supported by NIH grant R35GM119617 and NSF CAREER CMMI1653299.

References

  1. 1.
    Harris AK, Wild P, Stopak D (1980) Silicone rubber substrata: a new wrinkle in the study of cell locomotion. Science 208:177–179CrossRefPubMedGoogle Scholar
  2. 2.
    Liu Z, Sniadecki NJ, Chen CS (2010) Mechanical forces in endothelial cells during firm adhesion and early transmigration of human monocytes. Cell Mol Bioeng 3:50–59CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Maruthamuthu V, Sabass B, Schwarz US, Gardel ML (2011) Cell-ECM traction force modulates endogenous tension at cell-cell contacts. Proc Natl Acad Sci U S A 108:4708–4713CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Ingber DE (2003) Tensegrity I. Cell structure and hierarchical systems biology. J Cell Sci 116:1157–1173CrossRefPubMedGoogle Scholar
  5. 5.
    Sims JR, Karp S, Ingber DE (1992) Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape. J Cell Sci 103(Pt 4):1215–1222PubMedGoogle Scholar
  6. 6.
    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
  7. 7.
    Hu S, Eberhard L, Chen J, Love JC, Butler JP, Fredberg JJ, Whitesides GM, Wang N (2004) Mechanical anisotropy of adherent cells probed by a three-dimensional magnetic twisting device. Am J Physiol Cell Physiol 287:C1184–C1191CrossRefPubMedGoogle Scholar
  8. 8.
    Hu S, Chen J, Fabry B, Numaguchi Y, Gouldstone A, Ingber DE, Fredberg JJ, Butler JP, Wang N (2003) Intracellular stress tomography reveals stress focusing and structural anisotropy in cytoskeleton of living cells. Am J Physiol Cell Physiol 285:C1082–C1090CrossRefPubMedGoogle Scholar
  9. 9.
    Alam S, Lovett DB, Dickinson RB, Roux KJ, Lele TP (2014) Nuclear forces and cell mechanosensing. Prog Mol Biol Transl Sci 126:205–215CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Wang N, Tytell JD, Ingber DE (2009) Mechanotransduction at a distance: mechanically coupling the extracellular matrix with the nucleus. Nat Rev Mol Cell Biol 10:75–82CrossRefPubMedGoogle Scholar
  11. 11.
    Lombardi ML, Jaalouk DE, Shanahan CM, Burke B, Roux KJ, Lammerding J (2011) The interaction between nesprins and sun proteins at the nuclear envelope is critical for force transmission between the nucleus and cytoskeleton. J Biol Chem 286:26743–26753CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Luxton GWG, Gomes ER, Folker ES, Vintinner E, Gundersen GG (2010) Linear arrays of nuclear envelope proteins harness retrograde actin flow for nuclear movement. Science 329:956–959CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Grashoff C, Hoffman BD, Brenner MD, Zhou R, Parsons M, Yang MT, McLean MA, Sligar SG, Chen CS, Ha T, Schwartz MA (2010) Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature 466:263–266CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Rothenberg KE, Neibart SS, LaCroix AS, Hoffman BD (2015) Controlling cell geometry affects the spatial distribution of load across Vinculin. Cell Mol Bioeng 8:364–382CrossRefGoogle Scholar
  15. 15.
    Conway DE, Breckenridge MT, Hinde E, Gratton E, Chen CS, Schwartz MA (2013) Fluid shear stress on endothelial cells modulates mechanical tension across VE-cadherin and PECAM-1. Curr Biol 23:1024–1030CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Kuriyama S, Theveneau E, Benedetto A, Parsons M, Tanaka M, Charras G, Kabla A, Mayor R (2014) In vivo collective cell migration requires an LPAR2-dependent increase in tissue fluidity. J Cell Biol 206:113–127CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Borghi N, Sorokina M, Shcherbakova OG, Weis WI, Pruitt BL, Nelson WJ, Dunn AR (2012) E-cadherin is under constitutive actomyosin-generated tension that is increased at cell-cell contacts upon externally applied stretch. Proc Natl Acad Sci U S A 109:12568–12573CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Cai D, Chen S-C, Prasad M, He L, Wang X, Choesmel-Cadamuro V, Sawyer JK, Danuser G, Montell DJ (2014) Mechanical feedback through E-cadherin promotes direction sensing during collective cell migration. Cell 157:1146–1159CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Austen K, Ringer P, Mehlich A, Chrostek-Grashoff A, Kluger C, Klingner C, Sabass B, Zent R, Rief M, Grashoff C (2015) Extracellular rigidity sensing by talin isoform-specific mechanical linkages. Nat Cell Biol 17:1597–1606CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Brenner MD, Zhou R, Conway DE, Lanzano L, Gratton E, Schwartz MA, Ha T (2016) Spider silk peptide is a compact, linear nanospring ideal for intracellular tension sensing. Nano Lett 16(3):2096–2102CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Ostlund C, Folker ES, Choi JC, Gomes ER, Gundersen GG, Worman HJ (2009) Dynamics and molecular interactions of linker of nucleoskeleton and cytoskeleton (LINC) complex proteins. J Cell Sci 122:4099–4108CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Arsenovic PT, Bathula K, Conway DE (2017) A protocol for using Förster resonance energy transfer (FRET)-force biosensors to measure mechanical forces across the nuclear LINC complex. J Vis Exp.  https://doi.org/10.3791/54902
  23. 23.
    Kolega J (2004) Phototoxicity and photoinactivation of blebbistatin in UV and visible light. Biochem Biophys Res Commun 320:1020–1025CrossRefPubMedGoogle Scholar
  24. 24.
    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 110:34–43CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Arsenovic PT, Mayer CR, Conway DE (2017) SensorFRET: a standardless approach to measuring pixel-based spectral bleed-through and FRET efficiency using spectral imaging. Sci Rep 7:15609CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Kutscheidt S, Zhu R, Antoku S, Luxton GWG, Stagljar I, Fackler OT, Gundersen GG (2014) FHOD1 interaction with nesprin-2G mediates TAN line formation and nuclear movement. Nat Cell Biol 16:708–715CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Chang W, Worman HJ, Gundersen GG (2015) Accessorizing and anchoring the LINC complex for multifunctionality. J Cell Biol 208:11–22CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Wilson MH, Holzbaur ELF (2015) Nesprins anchor kinesin-1 motors to the nucleus to drive nuclear distribution in muscle cells. Development 142:218–228CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Zhu R, Antoku S, Gundersen GG (2017) Centrifugal displacement of nuclei reveals multiple LINC complex mechanisms for homeostatic nuclear positioning. Curr Biol 27:3097–3110.e5CrossRefPubMedGoogle Scholar
  30. 30.
    Khatau SB, Hale CM, Stewart-Hutchinson PJ, Patel MS, Stewart CL, Searson PC, Hodzic D, Wirtz D (2009) A perinuclear actin cap regulates nuclear shape. Proc Natl Acad Sci U S A 106:19017–19022CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Li Y, Lovett D, Zhang Q, Neelam S, Kuchibhotla RA, Zhu R, Gundersen GG, Lele TP, Dickinson RB (2015) Moving cell boundaries drive nuclear shaping during cell spreading. Biophys J 109:670–686CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of Biomedical EngineeringVirginia Commonwealth UniversityRichmondUSA

Personalised recommendations

Cite protocol

Buy options