Effects of Plectin Depletion on Keratin Network Dynamics and Organization

The keratin intermediate filament cytoskeleton protects epithelial cells against various types of stress and is involved in fundamental cellular processes such as signaling, differentiation and organelle trafficking. These functions rely on the cell type-specific arrangement and plasticity of the keratin system. It has been suggested that these properties are regulated by a complex cycle of assembly and disassembly. The exact mechanisms responsible for the underlying molecular processes, however, have not been clarified. Accumulating evidence implicates the cytolinker plectin in various aspects of the keratin cycle, i.e., by acting as a stabilizing anchor at hemidesmosomal adhesion sites and the nucleus, by affecting keratin bundling and branching and by linkage of keratins to actin filament and microtubule dynamics. In the present study we tested these hypotheses. To this end, plectin was downregulated by shRNA in vulvar carcinoma-derived A431 cells. As expected, integrin β4- and BPAG-1-positive hemidesmosomal structures were strongly reduced and cytosolic actin stress fibers were increased. In addition, integrins α3 and β1 were reduced. The experiments furthermore showed that loss of plectin led to a reduction in keratin filament branch length but did not alter overall mechanical properties as assessed by indentation analyses using atomic force microscopy and by displacement analyses of cytoplasmic superparamagnetic beads using magnetic tweezers. An increase in keratin movement was observed in plectin-depleted cells as was the case in control cells lacking hemidesmosome-like structures. Yet, keratin turnover was not significantly affected. We conclude that plectin alone is not needed for keratin assembly and disassembly and that other mechanisms exist to guarantee proper keratin cycling under steady state conditions in cultured single cells.

角蛋白中间丝细胞骨架（keratin intermediate filament cytoskeleton）可保护上皮细胞抵御各类应激刺激，并参与信号传导、细胞分化及细胞器运输等核心细胞生物学过程。此类功能的发挥依赖于角蛋白系统的细胞类型特异性排布与可塑性。已有研究表明，此类特性受复杂的组装与解组装循环调控。然而，支撑这些分子过程的确切机制尚未阐明。越来越多的证据表明，细胞连接蛋白桥粒斑蛋白（plectin）参与角蛋白循环的多个环节：包括作为半桥粒黏附位点与细胞核的稳定锚定蛋白、调控角蛋白束的形成与分支，以及介导角蛋白与肌动蛋白丝、微管动力学的相互连接。本研究针对上述假说展开验证。实验中，我们通过短发夹RNA（short hairpin RNA，shRNA）在源自外阴癌的A431细胞中下调桥粒斑蛋白的表达。正如预期，整合素β4（integrin β4）与BPAG-1阳性的半桥粒结构显著减少，胞质肌动蛋白应力纤维数量增多。此外，整合素α3与β1的表达水平也出现降低。进一步实验显示，桥粒斑蛋白缺失会导致角蛋白丝分支长度缩短，但未改变细胞整体机械特性——该结论通过原子力显微镜压痕分析，以及利用磁镊技术检测细胞质超顺磁磁珠位移的实验得以验证。在桥粒斑蛋白耗竭的细胞中，我们观察到角蛋白运动性增强，这一现象与缺乏类半桥粒结构的对照细胞一致。然而，角蛋白的更新速率并未受到显著影响。综上，我们认为仅桥粒斑蛋白并不足以调控角蛋白的组装与解组装；在培养的单细胞稳态条件下，存在其他机制以保障角蛋白循环的正常运行。