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Abstract：
The epithelial cytoskeleton encompasses actin filaments, microtubules, and keratin intermediate filaments. They are interconnected and attached to the extracellular matrix via focal adhesions and hemidesmosomes. To study their interplay, we inhibited actin and tubulin polymerization in the human keratinocyte cell line HaCaT by latrunculin B and nocodazole, respectively. Using immunocytochemistry and time-lapse imaging of living cells, we found that inhibition of actin and tubulin polymerization alone or in combination induced keratin network re-organization albeit differently in each situation. Keratin filament network retraction towards the nucleus and formation of bundled and radial keratin filaments was most pronounced in latrunculin-B treated cells but less in doubly-treated cells and not detectable in the presence of nocodazole alone. Hemidesmosomal keratin filament anchorage was maintained in each instance, whereas focal adhesions were disassembled in the absence of actin filaments. Simultaneous inhibition of actin and tubulin polymerization, therefore, allowed us to dissect hemidesmosome-specific functions for keratin network properties. These included not only anchorage of keratin filament bundles but also nucleation of keratin filaments, which was also observed in migrating cells. The findings highlight the fundamental role of hemidesmosomal adhesion for keratin network formation and organization independent of other cytoskeletal filaments pointing to a unique mechanobiological function.
摘要：
上皮细胞骨架包括肌动蛋白丝，微管，和角蛋白中间丝。它们通过粘着斑和半染色体相互连接并附着于细胞外基质。为了研究它们的相互作用，我们通过latrunculinB和nocodazole抑制人角质形成细胞系HaCaT中的肌动蛋白和微管蛋白聚合，分别。使用免疫细胞化学和活细胞延时成像，我们发现，肌动蛋白和微管蛋白聚合的抑制单独或组合诱导角蛋白网络重组，尽管在每种情况下不同。在latrunculin-B处理的细胞中，角蛋白细丝网络向细胞核的回缩以及成束和放射状角蛋白细丝的形成最为明显，但在双重处理的细胞中则较少，并且在单独存在诺考达唑的情况下无法检测到。在每种情况下都保持了半负点角蛋白丝锚定，而在没有肌动蛋白丝的情况下，粘着斑被分解。同时抑制肌动蛋白和微管蛋白聚合，因此,使我们能够解剖半染色体特定的角蛋白网络特性功能。这些不仅包括角蛋白丝束的锚固，还包括角蛋白丝的成核，这也在迁移细胞中观察到。研究结果强调了半网粒粘附在角蛋白网络形成和组织中的基本作用，而与其他细胞骨架细丝无关，指向独特的机械生物学功能。
