Lee W C, Lee Y C, Perng M D, Chen C M, Lai Y K
Institute of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
J Cell Biochem. 1993 Jul;52(3):253-65. doi: 10.1002/jcb.240520302.
Withangulatin A induced cell rounding up and the morphological alteration resulted from the reorganization of all of the major cytoskeletal components, i.e., vimentin, tubulin, and actin, as revealed by immunofluorescence techniques. When the withangulatin A-treated cells changed to a round-up morphology, vimentin intermediate filaments were found to be collapsed and clustered around the nucleus. The alteration was accompanied by characteristic changes of vimentin molecules, including augmentation of phosphorylation, retardation of electrophoretic mobility, and decrease in detergent extractability. The levels of vimentin phosphorylation were augmented by 2.5- and 1.8-fold in cells incubated with 50 microM withangulatin A for 1 and 3 h, respectively. The electrophoretic mobility of vimentin was partially retarded in cells treated with withangulatin A for 1 h at 10 microM and a completely upshift mobility was observed after 5 h treatment at 50 microM. In addition, vimentin molecules became less extractable by nonident P-40 after the cells were treated with withangulatin A and this effect was dose dependent. The decrease in solubility of vimentin was accompanied by the redistribution of HSP72 into the detergent nonextractable fraction and these two events were well correlated. Our results suggest that withangulatin A induced the modification of vimentin, which resulted in the alteration of cell morphology and redistribution of intracellular HSP72, an event that may play an important role in the induction of heat-shock response.
免疫荧光技术显示，Withangulatin A可诱导细胞变圆，且这种形态改变是由所有主要细胞骨架成分（即波形蛋白、微管蛋白和肌动蛋白）的重组引起的。当用Withangulatin A处理的细胞转变为圆球状形态时，发现波形蛋白中间丝塌陷并聚集在细胞核周围。这种改变伴随着波形蛋白分子的特征性变化，包括磷酸化增加、电泳迁移率减慢以及去污剂提取率降低。在用50μM Withangulatin A分别孵育1小时和3小时的细胞中，波形蛋白的磷酸化水平分别增加了2.5倍和1.8倍。在用10μM Withangulatin A处理1小时的细胞中，波形蛋白的电泳迁移率部分减慢，而在用50μM处理5小时后观察到完全的迁移率上移。此外，在用Withangulatin A处理细胞后，波形蛋白分子变得更难被非离子型P - 40提取，且这种效应呈剂量依赖性。波形蛋白溶解度的降低伴随着HSP72重新分布到去污剂不可提取部分，这两个事件密切相关。我们的结果表明，Withangulatin A诱导了波形蛋白的修饰，导致细胞形态改变和细胞内HSP72重新分布，这一事件可能在热休克反应的诱导中起重要作用。
