Bolshakova Anastasia, Magnusson Karl-Eric, Pinaev George, Petukhova Olga
Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Ave., St. Petersburg, 194064, Russia.
Histochem Cell Biol. 2015 Sep;144(3):223-35. doi: 10.1007/s00418-015-1331-5. Epub 2015 May 20.
To evaluate the role of actin cytoskeleton in the regulation of NF-κB transcription factor, we analyzed its involvement in the intracellular transport and nuclear translocation of the NF-κB RelA/p65 subunit in A431 epithelial cells stimulated with fibronectin and EGF. Live cell imaging and confocal microscopy showed that EGF activated the movement of RelA/p65 in the cytoplasm. Upon cell adhesion to fibronectin, RelA/p65 concentrated onto stress fibers, and EGF stimulated its subsequent allocation to membrane ruffles, newly organized stress fibers, and discrete cytoplasmic actin-rich patches. These patches also contained α-actinin-1 and α-actinin-4, vinculin, paxillin, α-tubulin, and PI3-kinase. Cytochalasin D treatment resulted in RelA/p65 redistribution to actin-containing aggregates, with the number of cells with RelA/p65-containing clusters in the cytoplasm increasing under the effect of EGF. Furthermore, EGF proved to induce RelA/p65 accumulation in the nucleus after cell pretreatment with actin-stabilizing and actin-destabilizing agents, which was accompanied by changes in its DNA-binding activity after either EGF stimulation or cytochalasin D treatment. Thus, EGF treatment of A431 cells results in simultaneous nuclear RelA/p65 translocation and cytoplasmic redistribution, with part of RelA/p65 pool forming a very tight association with actin-rich structures. Apparently, nuclear transport is independent on drug stabilization or destabilization of the actin.
为了评估肌动蛋白细胞骨架在核因子-κB（NF-κB）转录因子调控中的作用，我们分析了其在纤连蛋白和表皮生长因子（EGF）刺激的A431上皮细胞中参与NF-κB RelA/p65亚基的细胞内转运和核转位的情况。活细胞成像和共聚焦显微镜显示，EGF激活了RelA/p65在细胞质中的移动。细胞黏附到纤连蛋白上后，RelA/p65聚集到应力纤维上，EGF刺激其随后分配到膜皱褶、新形成的应力纤维以及离散的富含肌动蛋白的细胞质斑块中。这些斑块还含有α-辅肌动蛋白-1和α-辅肌动蛋白-4、纽蛋白、桩蛋白、α-微管蛋白和磷脂酰肌醇-3激酶（PI3-激酶）。用细胞松弛素D处理导致RelA/p65重新分布到含肌动蛋白的聚集体中，在EGF的作用下，细胞质中含有RelA/p65簇的细胞数量增加。此外，在用肌动蛋白稳定剂和肌动蛋白去稳定剂预处理细胞后，EGF被证明可诱导RelA/p65在细胞核中积累，这伴随着在EGF刺激或细胞松弛素D处理后其DNA结合活性的变化。因此，用EGF处理A431细胞会导致RelA/p65同时发生核转位和细胞质重新分布，其中一部分RelA/p65库与富含肌动蛋白的结构形成非常紧密的结合。显然，核转运独立于肌动蛋白的药物稳定或去稳定作用。
