Nesprin‐2G knockout fibroblasts exhibit reduced migration, changes in focal adhesion composition, and reduced ability to generate traction forces

Abstract

The nuclear envelope protein nesprin‐2G is a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex and is responsible for mechanical and signaling crosstalk between the nucleus and cytoskeleton. A prior study has demonstrated that nesprin‐2G knockout mice show delayed wound healing. The goal was to elucidate the mechanism underlying the delayed wound closure in this mouse model. Primary fibroblasts from wild‐type and knockout neonatal mice were isolated. Knockout cells exhibited decreased focal adhesion (FA) size, number, and intensity. Consistent with this result, FA protein expression levels were decreased in knockout cells. Additionally, knockout fibroblasts displayed an abnormal actin cytoskeleton, as evidenced by loss of TAN line formation and both cytoplasmic and peri‐nuclear actin staining. Using collective and single cell motility assays, it was found that knockout cells exhibited a reduction in both speed and directed migration. Traction force microscopy revealed that knockout fibroblasts generated fewer traction forces compared with WT fibroblasts. In summary, the data indicated that changes in actin organization and defects in FAs result in a reduced ability of knockout fibroblasts to generate traction forces needed for efficient motility.

The LINC complex is required for endothelial cell adhesion and adaptation to shear stress and cyclic stretch

“…Because LINC complex disruption also reduced cell proliferation (Figure S2 nesprin-1 or nesprin-2 (King et al, 2014). Additional studies have shown delayed migration in LINC-disrupted fibroblasts (Luxton et al, 2010;Lombardi et al, 2011;Woychek and Jones, 2019). One notable observation in our movies of the wound healing assay was that the migratory behavior of DN-KASH expressing cells was more "chaotic" (Movie 4, Movie 5)…”

“…Of note, a prior study showed that inhibition of phosphorylation of Y1065 of vinculin leads to a uncontrolled exchange of vinculin and reduced cell adhesive forces (Küpper et al, 2010), suggesting that the change in phosphorylation state of FA proteins observed in endothelial cells expressing DN-KASH is indicative of impaired FA dynamics and reduced adhesivity. In addition two recent studies have shown that loss of nesprin-1 (Nguyen et al, 2019) or nesprin-2G (Woychek and Jones, 2019) negatively affect cell migration, further suggesting a relationship between the LINC complex and cell-matrix adhesion (Nguyen et al, 2019), potentially through direct cytoskeleton force transmission between these two regions of the cell. Notably, one of these studies showed that loss of nesprin-1 affected ERK and FAK signaling (Nguyen et al, 2019).…”

“…, 2010 ; Lombardi et al. , 2011 ; Woychek and Jones, 2019 ). One notable observation in our movies of the wound healing assay was that the migratory behavior of DN-KASH–expressing cells was more “chaotic” (Supplemental Movies 4 and 5), which may be indicative that disruption of the LINC complex causes a switch from collective cell migration to single cell migration.…”

ERK1/2 Phosphorylation of FHOD Connects Signaling and Nuclear Positioning Alternations in Cardiac Laminopathy

“…Movement in Cells Expressing a Muscle Disease-Associated Variant of Lamin A Nesprin-2 and FHOD1 knockdown or nesprin-2 knockout in fibroblasts inhibits nuclear movement (Kutscheidt et al, 2014;Luxton et al, 2010;Woychek and Jones, 2019). To test the impact of the phosphorylation of nesprin-2 or FHOD1 on nuclear movement, phosphomimetic and unphosphorylatable forms of these proteins were expressed in fibroblasts depleted of the endogenous proteins (Figures S4A and S4B).…”

Biomechanical analysis of the mechanical environment of the cell nucleus in serum starvation-induced vascular smooth muscle cell differentiation

“…Cyclic stretch stimulation induces phenotypic switching of VSMCs through the transcriptional regulators Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) signaling pathway (Wang et al, 2018). Although several studies have reported the biochemical or biomechanical factors involved in the stimulation of VSMC phenotypic transition, limited information is available about the effect on the mechanical environment of intracellular nucleus, including morphology and mechanical properties of the nucleus and intracellular forces exerted on the nucleus that are majorly involved in various cellular functions, such as cell migration (Woychek and Jones, 2019;Renkawitz et al, 2019) and proliferation (Versaevel, 2012; as well as the pathological conditions (Hoorntje et al, 2017).…”