Institute of Complex Systems, ICS-7: Biomechanics, Forschungszentrum Jülich, 52425 Jülich, Germany.
Proc Natl Acad Sci U S A. 2013 Nov 12;110(46):18513-8. doi: 10.1073/pnas.1313491110. Epub 2013 Oct 28.
Keratins are major components of the epithelial cytoskeleton and are believed to play a vital role for mechanical integrity at the cellular and tissue level. Keratinocytes as the main cell type of the epidermis express a differentiation-specific set of type I and type II keratins forming a stable network and are major contributors of keratinocyte mechanical properties. However, owing to compensatory keratin expression, the overall contribution of keratins to cell mechanics was difficult to examine in vivo on deletion of single keratin genes. To overcome this problem, we used keratinocytes lacking all keratins. The mechanical properties of these cells were analyzed by atomic force microscopy (AFM) and magnetic tweezers experiments. We found a strong and highly significant softening of keratin-deficient keratinocytes when analyzed by AFM on the cell body and above the nucleus. Magnetic tweezers experiments fully confirmed these results showing, in addition, high viscous contributions to magnetic bead displacement in keratin-lacking cells. Keratin loss neither affected actin or microtubule networks nor their overall protein concentration. Furthermore, depolymerization of actin preserves cell softening in the absence of keratin. On reexpression of the sole basal epidermal keratin pair K5/14, the keratin filament network was reestablished, and mechanical properties were restored almost to WT levels in both experimental setups. The data presented here demonstrate the importance of keratin filaments for mechanical resilience of keratinocytes and indicate that expression of a single keratin pair is sufficient for almost complete reconstitution of their mechanical properties.
角蛋白是上皮细胞骨架的主要成分，被认为在细胞和组织水平上对机械完整性起着至关重要的作用。角蛋白细胞是表皮的主要细胞类型，表达一组特定于分化的 I 型和 II 型角蛋白，形成稳定的网络，是角蛋白细胞力学特性的主要贡献者。然而，由于角蛋白的代偿性表达，在单个角蛋白基因缺失的情况下，角蛋白对细胞力学的总体贡献难以在体内进行检查。为了解决这个问题，我们使用了缺乏所有角蛋白的角蛋白细胞。通过原子力显微镜（AFM）和磁镊实验分析这些细胞的力学特性。我们发现，在用 AFM 分析细胞体和细胞核上方时，缺乏角蛋白的角蛋白细胞表现出强烈且高度显著的软化。磁镊实验完全证实了这些结果，此外，还显示了缺乏角蛋白的细胞中磁珠位移的高粘性贡献。角蛋白缺失既不影响肌动蛋白或微管网络，也不影响它们的总蛋白浓度。此外，肌动蛋白解聚在缺乏角蛋白的情况下可保持细胞软化。在重新表达唯一的基底表皮角蛋白对 K5/14 时，角蛋白丝网络得以重建，并且在两种实验设置中，力学特性几乎恢复到 WT 水平。这里呈现的数据表明角蛋白丝对于角蛋白细胞的机械弹性的重要性，并表明表达单个角蛋白对几乎完全重建它们的机械特性是足够的。
