Cell responses to different substrate stiffness. (A) Phalloidin staining of the F-actin of fixed WT, Nespr-1ΔKASH and LMNAΔK32 myoblasts on fibronectin-coated glass and gel substrates of 700 kPa, 20 kPa, 12 kPa and 5 kPa. Nuclei are stained with DAPI. Scale bar: 40 µm. (B) Projected cell area as a function of substrate stiffness. Analysis was performed on glass and gel substrates of 700 kPa, 20 kPa, 12 kPa, and 5 kPa (each n > 50 cells). Values are means ± SEM; $p < 0.001 vs corresponding cell line value on glass; *p < 0.001 vs WT value at similar substrate rigidity.
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Increased contractility of Nespr-1ΔKASH and LMNAΔK32 myoblasts on matrix stiffness close to that of muscle
Contractile actin stress fibre accumulation in mutant cells
We next sought to investigate the contractile actin cytoskeleton organization in Nespr-1ΔKASH and LMNAΔK32 myoblasts cultured on matrix stiffness close to that of muscle, i.e., 12 kPa37. We found clear modifications in the organization of the actomyosin stress fibres in mutant cells compared with WT (Fig. 2A–D). As expected, WT myoblasts on 12 kPa displayed only few convex shaped contractile fibres at the cell periphery that resembled transverse arcs38. In contrast, both Nespr-1ΔKASH and LMNAΔK32 myoblasts had numerous thick actomyosin bundles, present both at the cell periphery and in the nuclear and perinuclear regions (Fig. 2B–D). These contractile stress fibres could extend throughout most of the cell length, thus resembling rg6 coaxial cable ventral stress fibres38. Importantly, they were present both at the basal and apical surfaces of the mutant cells (Fig. 2C), with a reduction in nuclear height in both Nespr-1ΔKASH and LMNAΔK32 compared with WT nuclei (Fig. 2E). The nuclear volume did not differ between WT and mutant nuclei (Suppl Fig. 1A), suggesting that different mechanisms controlled nuclear volume and nuclear thickness. In addition, the mRNA expression of MYH9, the gene encoding non-muscle myosin 2 A (NM-2A) was significantly up-regulated in Nespr-1ΔKASH and LMNAΔK32 compared with WT (Fig. 2F). These results show that Nespr-1ΔKASH and LMNAΔK32 myoblasts accumulate contractile stress fibres when plated in conditions close to their physiological stiffness.

Figure 2
Figure 2
Actin cytoskeleton on soft matrix (12 kPa). (A) Confocal images of WT, Nespr-1ΔKASH and LMNAΔK32 myoblasts on soft matrix (12 kPa) close to physiological muscle stiffness and stained for F-actin (phalloidin, red) and non-muscle myosin 2 A (NM-2A, green). Nuclei are stained with DAPI (blue). Scale bar: 10 µm. (B,C) Zoom-in of actin cytoskeleton at the cell periphery (B) and in the perinuclear regions (C). In C, confocal images are taken at the apical and basal surface of the cell. Scale bar: 5 µm. (D) Supranuclear actin rg6 coaxial cable number in WT, Nespr-1ΔKASH and LMNAΔK32 myoblasts. Values are means ± SEM; ***p < 0.001 compared with WT. (E) Nuclear thickness in WT, Nespr-1ΔKASH and LMNAΔK32 myoblasts. Values are means ± SEM; **p < 0.01 compared with WT. (F) mRNA expression of MYH9 gene expression coding for NM-2A in WT, Nespr-1ΔKASH and LMNAΔK32 myoblasts. Values are means ± SEM; **p < 0.01, *p < 0.05 compared with WT.