Blase Christopher, Becker Daniel, Kappel Sven, Bereiter-Hahn Jürgen
Institute for Cell Biology and Neuroscience, Center of Excellence Frankfurt: Macromolecular Complexes, JW Goethe University, Frankfurt/Main, Germany.
Eur J Cell Biol. 2009 Mar;88(3):131-9. doi: 10.1016/j.ejcb.2008.10.003. Epub 2008 Nov 25.
Cell volume is an important parameter in many physiological processes, and is closely regulated in many cell types. In those cells, swelling induced by hypotonic media is followed by an ion-driven regulatory volume decrease. In many cell types, this regulatory volume decrease requires an intact actin cytoskeleton. Therefore, we investigated the changes in the structure and polymerization state of the actin cytoskeleton in HaCaT keratinocytes during cell swelling and regulatory volume decrease. Disruption of the actin cytoskeleton by 2microM cytochalasin D inhibits regulatory volume decrease in HaCaT cells. Cells swollen in the presence of low concentrations of cytochalasin D (0.8microM, 305-250mosM) keep the elevated volume even after cytochalasin D removal. A further decrease of tonicity (250-200mosM) is again counteracted by regulatory volume decrease reaching the volume, which has been established at 250mosM. In contrast, no visible changes occurred in actin cytoskeleton morphology of EGFP-actin-transfected HaCaT cells during swelling or regulatory volume decrease. However, biochemical analysis showed an increase in total F-actin levels 90s after the onset of hypotonicity. The ratio of Triton-soluble to -insoluble actin also increased after hypotonic shock, suggesting that the measured increase in F-actin is primarily due to de novo polymerization and formation of short actin filaments, i.e., actin oligomers. These results show that a rapid reorganization of the actin cytoskeleton takes place after hypotonic treatment. This reorganization can influence signaling in response to hypotonicity either indirectly by means of sequestering or releasing actin-associated proteins, or directly by the interaction of short actin filaments with plasma membrane ion channels, and may be involved in determining a new volume set point.
细胞体积是许多生理过程中的一个重要参数，并且在许多细胞类型中受到严格调控。在这些细胞中，低渗介质诱导的肿胀之后会出现离子驱动的调节性体积减小。在许多细胞类型中，这种调节性体积减小需要完整的肌动蛋白细胞骨架。因此，我们研究了HaCaT角质形成细胞在细胞肿胀和调节性体积减小过程中肌动蛋白细胞骨架的结构和聚合状态的变化。用2微摩尔的细胞松弛素D破坏肌动蛋白细胞骨架会抑制HaCaT细胞的调节性体积减小。在低浓度细胞松弛素D（0.8微摩尔，305 - 250毫渗量）存在下肿胀的细胞，即使在去除细胞松弛素D后仍保持升高的体积。进一步降低张力（250 - 200毫渗量）会再次被调节性体积减小所抵消，细胞体积达到在250毫渗量时所确立的体积。相比之下，在肿胀或调节性体积减小过程中，转染了EGFP - 肌动蛋白的HaCaT细胞的肌动蛋白细胞骨架形态没有明显变化。然而，生化分析表明，低渗开始90秒后总F - 肌动蛋白水平增加。低渗休克后，Triton可溶性肌动蛋白与不溶性肌动蛋白的比例也增加，这表明所测得的F - 肌动蛋白增加主要是由于肌动蛋白的从头聚合和短肌动蛋白丝即肌动蛋白寡聚体的形成。这些结果表明，低渗处理后肌动蛋白细胞骨架会迅速重组。这种重组可能通过螯合或释放肌动蛋白相关蛋白间接影响对低渗的信号传导，或者通过短肌动蛋白丝与质膜离子通道的相互作用直接影响信号传导，并且可能参与确定新的体积设定点。
