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

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.

The mammalian LINC complex regulates genome transcriptional responses to substrate rigidity

“…As myosin-generated cellular contractile forces can be exerted on the nuclear surface through the LINC complex141733, we asked whether non-muscle myosin II inhibition has similar effects on gene expression as with LINC complex disruption. Because myosin forces are small in cells on the soft substrate1734, we inhibited myosin activity on the stiff substrate with blebbistatin or with Y27632 (a Rho-kinase inhibitor, hereafter called Y27) in control KDEL cells (we have validated myosin inhibition with this approach in NIH 3T3 fibroblasts elsewhere1227; see also Supplementary Figure 3).…”

“…However, our identification of genes that are common to myosin inhibition and LINC disruption suggests a list of genes that likely depend directly on actomyosin tension exerted on the nucleus143356 for their mechanosensitivity. We have previously shown that SUN1L expression softens the integrated nucleus-cytoskeleton in fibroblasts25, which likely indicates altered forces on the nucleus.…”

Centrifugal Displacement of Nuclei Reveals Multiple LINC Complex Mechanisms for Homeostatic Nuclear Positioning

“…This conclusion is consistent with the findings of other recent studies. For example, a nesprin-2-based actin tension sensor revealed that static nuclei of mouse and human fibroblasts are under constant actomyosin force [45] and local displacement of nuclei by microneedles showed cytoskeletal dependent restoring forces [31, 32]. Also, in both the differentiation of mouse embryonic stem cells and early Drosophila development, nuclei are reported to be prestressed as their shape is altered by disrupting cytoskeletal elements [46].…”

The Work of Titin Protein Folding as a Major Driver in Muscle Contraction

“…These force sensors utilize two fluorescent proteins that are able to exchange energy through non-radiative transfer (FRET), separated with a short unstructured or structured peptide. These genetically encoded force sensors have been incorporated into spectrin proteins (115), vinculin and talin (116, 117), and nuclear nesprins (118), all of which are subject to tension from intracellular stress fibers. As the tension on the protein containing the force sensor increases, the FRET donor and acceptor separate and the FRET ratio decreases.…”