利用单分子荧光成像技术研究十字孢碱诱导细胞凋亡过程中糖原合成酶激酶-3β促进Bax转位

Using Single Molecule Fluorescence Imaging to Study Glycogen Synthase Kinase-3β Promotes Bax Translocation during Staurosporine-Induced Cell Apoptosis

糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)除了在抑制糖原合成中的重要作用外,越来越多的研究表明它是细胞凋亡过程中的一个关键信号调节蛋白。然而,在细胞凋亡过程中它调节的主要下游促凋亡蛋白依然不明确,尤其是Bcl-2家族的促凋亡蛋白(Bax是其中最重要的蛋白之一)。通过对GSK-3β和Bax两种蛋白进行荧光标记,在单分子水平上研究了十字孢碱(staurosporine,STS)诱导人肺腺癌细胞(ASTC-α-1)凋亡过程中,GSK-3β活化与Bax转位之间的关系。实验结果表明STS诱导ASTC-α-1凋亡过程中,共转染pCFP-Bax和pYFP-GSK-3β的细胞发生凋亡的时间明显早于单转染pYFP-Bax的细胞,并且Bax发生转位的时间也明显提前。这些结果显示在STS这种凋亡因素刺激下,GSK-3β可以通过促进Bax转位从而加速细胞凋亡。这是单分子荧光成像技术研究活细胞内分子事件的又一个重要应用。

Except for the inhibitive function on glycogen synthase, accumulated evidence indicates that glycogen synthase kinase-3β （ GSK-3β） is a critical signal-regulated factor in apoptosis. However, the main downstream pro-apoptotic proteins of GSK-3β regulation is unclear in the apoptotic process, especially for the pro-apoptotic proteins of Bcl-2 family, in which Bax is one of the most important member. Using fluorescently labeled GSK-3β and Bax, the present study is to investigate the relationship between GSK-3β activation and Bax translocation in staurosporine （STS）-induced ASTC-α-1 apoptosis at the single-molecular level. Results showed that eotransfection of pCFP-Bax and pYFP-GSK-3β accelerated apoptosis and Bax＇translocation in comparison with the cells expressed pYFP-Bax only during STS-induced ASTC-α-1 apoptosis, indicating that GSK-3β promoted apoptosis through accelerating Bax translocation. This is an important application of single molecule fluorescence imaging of the molecular events in living cells.