The isotype-specific composition of the keratin cytoskeleton is important for strong adhesion, force resilience, and barrier function of the epidermis. However, the mechanisms by which keratins regulate these functions are still incompletely understood. In this study, the role and significance of the keratin network for mechanical integrity, force transmission, and barrier formation were analyzed in murine keratinocytes. Following the time-course of single-cell wound closure, wild-type (WT) cells slowly closed the gap in a collective fashion involving tightly connected neighboring cells. In contrast, the mechanical response of neighboring cells was compromised in keratin-deficient cells, causing an increased wound area initially and an inefficient overall wound closure. Furthermore, the loss of the keratin network led to impaired, fragmented cell–cell junctions, and triggered a profound change in the overall cellular actomyosin architecture. Electric cell-substrate impedance sensing of cell junctions revealed a dysfunctional barrier in knockout (Kty^−/−) cells compared to WT cells. These findings demonstrate that Kty^−/− cells display a novel phenotype characterized by loss of mechanocoupling and failure to form a functional barrier. Re-expression of K5/K14 rescued the barrier defect to a significant extent and reestablished the mechanocoupling with remaining discrepancies likely due to the low abundance of keratins in that setting. Our study reveals the major role of the keratin network for mechanical homeostasis and barrier functionality in keratinocyte layers.

角蛋白细胞骨架的同种型特异性组成对于表皮的强粘附力、力恢复力和屏障功能很重要。然而，角蛋白调节这些功能的机制仍不完全清楚。在这项研究中，分析了小鼠角质形成细胞中角蛋白网络对于机械完整性、力传递和屏障形成的作用和意义。随着单细胞伤口闭合的时间进程，野生型（WT）细胞以集体方式缓慢闭合间隙，涉及紧密连接的相邻细胞。相反，角蛋白缺陷细胞中邻近细胞的机械反应受到损害，导致最初的伤口面积增加和整体伤口闭合效率低下。此外，角蛋白网络的丧失导致细胞与细胞连接受损、破碎，并引发整个细胞肌动球蛋白结构的深刻变化。细胞连接处的电细胞-基质阻抗传感揭示了与 WT 细胞相比，敲除 (Kty ^−/− ) 细胞中存在功能障碍的屏障。这些发现表明，Kty ^-/−细胞表现出一种新的表型，其特征是机械耦合丧失和无法形成功能屏障。 K5/K14 的重新表达在很大程度上挽救了屏障缺陷，并重新建立了机械耦合，而剩余的差异可能是由于该环境中角蛋白的丰度较低所​​致。我们的研究揭示了角蛋白网络对于角质形成细胞层的机械稳态和屏障功能的主要作用。