Rogerson Clare, Wotherspoon Duncan J, Tommasi Cristina, Button Robert W, O'Shaughnessy Ryan F L
Centre for Cell Biology and Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK.
Immunobiology and Dermatology, UCL Great Ormond Street Institute of Child Health, London, UK.
Cell Death Differ. 2021 Jun;28(6):1849-1864. doi: 10.1038/s41418-020-00712-9. Epub 2021 Jan 18.
Keratinocyte cornification and epidermal barrier formation are tightly controlled processes, which require complete degradation of intracellular organelles, including removal of keratinocyte nuclei. Keratinocyte nuclear destruction requires Akt1-dependent phosphorylation and degradation of the nuclear lamina protein, Lamin A/C, essential for nuclear integrity. However, the molecular mechanisms that result in complete nuclear removal and their regulation are not well defined. Post-confluent cultures of rat epidermal keratinocytes (REKs) undergo spontaneous and complete differentiation, allowing visualisation and perturbation of the differentiation process in vitro. We demonstrate that there is dispersal of phosphorylated Lamin A/C to structures throughout the cytoplasm in differentiating keratinocytes. We show that the dispersal of phosphorylated Lamin A/C is Akt1-dependent and these structures are specific for the removal of Lamin A/C from the nuclear lamina; nuclear contents and Lamin B were not present in these structures. Immunoprecipitation identified a group of functionally related Akt1 target proteins involved in Lamin A/C dispersal, including actin, which forms cytoskeletal microfilaments, Arp3, required for actin filament nucleation, and Myh9, a component of myosin IIa, a molecular motor that can translocate along actin filaments. Disruption of actin filament polymerisation, nucleation or myosin IIa activity prevented formation and dispersal of cytoplasmic Lamin A/C structures. Live imaging of keratinocytes expressing fluorescently tagged nuclear proteins showed a nuclear volume reduction step taking less than 40 min precedes final nuclear destruction. Preventing Akt1-dependent Lamin A/C phosphorylation and disrupting cytoskeletal Akt1-associated proteins prevented nuclear volume reduction. We propose keratinocyte nuclear destruction and differentiation requires myosin II activity and the actin cytoskeleton for two intermediate processes: Lamin A/C dispersal and rapid nuclear volume reduction.
角质形成细胞的角质化和表皮屏障形成是受到严格控制的过程，这需要细胞内细胞器的完全降解，包括角质形成细胞核的清除。角质形成细胞核的破坏需要Akt1依赖的核纤层蛋白Lamin A/C的磷酸化和降解，而Lamin A/C对于核完整性至关重要。然而，导致细胞核完全清除的分子机制及其调控尚未明确。大鼠表皮角质形成细胞（REKs）汇合后的培养物会经历自发且完全的分化，从而能够在体外观察和干扰分化过程。我们证明，在分化的角质形成细胞中，磷酸化的Lamin A/C会分散到整个细胞质中的结构上。我们表明，磷酸化的Lamin A/C的分散是Akt1依赖的，并且这些结构对于从核纤层中去除Lamin A/C具有特异性；这些结构中不存在核内容物和Lamin B。免疫沉淀鉴定出一组与Lamin A/C分散相关的功能相关的Akt1靶蛋白，包括形成细胞骨架微丝的肌动蛋白、肌动蛋白丝成核所需的Arp3以及肌球蛋白IIa的一个组成部分Myh9，肌球蛋白IIa是一种可以沿肌动蛋白丝转运的分子马达。肌动蛋白丝聚合、成核或肌球蛋白IIa活性的破坏会阻止细胞质中Lamin A/C结构的形成和分散。对表达荧光标记核蛋白的角质形成细胞进行实时成像显示，在最终核破坏之前，核体积减少步骤在不到40分钟内完成。阻止Akt1依赖的Lamin A/C磷酸化并破坏细胞骨架中与Akt1相关的蛋白会阻止核体积减少。我们提出，角质形成细胞核的破坏和分化需要肌球蛋白II活性和肌动蛋白细胞骨架参与两个中间过程：Lamin A/C的分散和快速的核体积减少。
