Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jan 5;110(1):34-43.
doi: 10.1016/j.bpj.2015.11.014.

Nesprin-2G, a Component of the Nuclear LINC Complex, Is Subject to Myosin-Dependent Tension

Paul T Arsenovic  1 , Iswarya Ramachandran  1 , Kranthidhar Bathula  1 , Ruijun Zhu  2 , Jiten D Narang  1 , Natalie A Noll  1 , Christopher A Lemmon  1 , Gregg G Gundersen  2 , Daniel E Conway  3
Affiliations

Nesprin-2G, a Component of the Nuclear LINC Complex, Is Subject to Myosin-Dependent Tension

Paul T Arsenovic et al. Biophys J. .

Abstract

The nucleus of a cell has long been considered to be subject to mechanical force. Despite the observation that mechanical forces affect nuclear geometry and movement, how forces are applied onto the nucleus is not well understood. The nuclear LINC (linker of nucleoskeleton and cytoskeleton) complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton onto the nucleus. Previously used techniques for studying nuclear forces have been unable to resolve forces across individual proteins, making it difficult to clearly establish if the LINC complex experiences mechanical load. To directly measure forces across the LINC complex, we generated a fluorescence resonance energy transfer-based tension biosensor for nesprin-2G, a key structural protein in the LINC complex, which physically links this complex to the actin cytoskeleton. Using this sensor we show that nesprin-2G is subject to mechanical tension in adherent fibroblasts, with highest levels of force on the apical and equatorial planes of the nucleus. We also show that the forces across nesprin-2G are dependent on actomyosin contractility and cell elongation. Additionally, nesprin-2G tension is reduced in fibroblasts from Hutchinson-Gilford progeria syndrome patients. This report provides the first, to our knowledge, direct evidence that nesprin-2G, and by extension the LINC complex, is subject to mechanical force. We also present evidence that nesprin-2G localization to the nuclear membrane is altered under high-force conditions. Because forces across the LINC complex are altered by a variety of different conditions, mechanical forces across the LINC complex, as well as the nucleus in general, may represent an important mechanism for mediating mechanotransduction.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Nesprin tension biosensor. (A) Schematic of nesprin-2G tension sensor and headless control. (B) Nesprin-2G tension sensor and headless control localized to the nuclear membrane in NIH3T3 fibroblasts. The FRET at the nuclear envelope was reduced for the tension sensor as compared to the headless control. (C) The tension sensor had significantly reduced FRET at the nucleus as compared to the force-insensitive headless control, t-test, p < 0.01. Bar graphs represent FRET pixels from discrete intensity ranges collected across a minimum of 20 cells per condition. Similar results were obtained for three independent experiments. (D) Nesprin-2G tension sensor rescues centrosome orientation in nesprin-2G depleted cells subjected to scratch wounding and LPA treatment, Fisher’s exact test, ∗p < 0.05; ns, not significant: p > 0.05. (E) The tension sensor relative FRET index per cell at various cross sections of the nucleus (apical, equatorial, and basal). Significantly reduced FRET was observed for apical and equatorial planes compared to basal (ANOVA, Newman–Keuls post-hoc test, p < 0.01). (F) No significant differences were observed for the headless control at various cross sections of the nucleus (apical, equatorial, and basal). To see this figure in color, go online.
Figure 2
Figure 2
Role of myosin contractility on nesprin tension. (A) NIH3T3 cells expressing the nesprin tension sensor were treated with either 1 nM calyculin A or both 10 μM Y-27632 and 10 μM ML7 and were compared to unstimulated cells. (B) Statistical analysis of each condition showed that calyculin A treatment significantly reduced FRET, whereas Y27632 and ML7 treatment increased FRET, ANOVA Newman-Keuls post-hoc test, p < 0.01. Bar graphs represent FRET pixels from discrete intensity ranges collected across a minimum of 20 cells per condition. Similar results were obtained for two independent experiments. To see this figure in color, go online.
Figure 3
Figure 3
Effect of cell elongation on nesprin tension. (A) NIH3T3 cells expressing the nesprin tension sensor were grown overnight on 20 μm wide micropatterned lines of fibronectin or unpatterned fibronectin. (B) Statistical analysis of each condition showed that cells grown on lines had reduced FRET compared to cells grown on the unpatterned surface, t-test, p < 0.01. Bar graphs represent FRET pixels from discrete intensity ranges collected across a minimum of 15 cells per condition. Similar results were obtained for three independent experiments. (C) Untransfected cells grown on lines or unpatterned surfaces were fixed with paraformaldehyde and stained with nesprin-2 antibody. (D) Histogram analysis of nesprin-2 intensity at the nuclear membrane showed increased nuclear membrane nesprin-2 expression in cells on lines as compared to unpatterned surface (averaged across a minimum of four separate fields of view per condition). Histograms were normalized so that the area under each histogram sums to 1. To see this figure in color, go online.
Figure 4
Figure 4
Nesprin tension in HGPS cells. (A) The nesprin tension sensor was expressed in normal and HGPS primary fibroblasts. (B) There was a significant increase in FRET for HGPS cells as compared to normal cells, t-test, p < 0.01. Bar graphs represent FRET pixels from discrete intensity ranges collected across a minimum of 20 cells per condition. Similar results were obtained for two independent experiments. (C) Histogram analysis of fluorescent intensity of the nesprin sensor for normal and HGPS cells. Histograms were normalized so that the area under each histogram sums to 1. To see this figure in color, go online.
Figure 5
Figure 5
Model of nesprin-2 orientation in response to applied force. (A) Nesprin-2 is oriented radially for nonpolarized cells. (B) In elongated cells, in which actin stress fibers are oriented parallel to the longitudinal axis, there is more force applied to the LINC complex, as well as additional nesprin-2 molecules recruited to the nuclear membrane. Nesprin-2 forces are spatially homogenous around the periphery of the nucleus, suggesting that nesprin-2 orients in the longitudinal direction.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Isermann P., Lammerding J. Nuclear mechanics and mechanotransduction in health and disease. Curr. Biol. 2013;23:R1113–R1121. - PMC - PubMed
    1. Harris A.K., Wild P., Stopak D. Silicone rubber substrata: a new wrinkle in the study of cell locomotion. Science. 1980;208:177–179. - PubMed
    1. Liu Z., Sniadecki N.J., Chen C.S. Mechanical forces in endothelial cells during firm adhesion and early transmigration of human monocytes. Cell. Mol. Bioeng. 2010;3:50–59. - PMC - PubMed
    1. Maruthamuthu V., Sabass B., Gardel M.L. Cell-ECM traction force modulates endogenous tension at cell-cell contacts. Proc. Natl. Acad. Sci. USA. 2011;108:4708–4713. - PMC - PubMed
    1. Ingber D.E. Tensegrity I. Cell structure and hierarchical systems biology. J. Cell Sci. 2003;116:1157–1173. - PubMed

Publication types