Measuring dynamics of caspase-8 activation in a single living HeLa cell during TNFα-induced apoptosis
Measuring dynamics of caspase-8 activation in a single living HeLa cell during TNFα-induced apoptosis
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DOI：
10.1016/s0006-291x(03)00559-x
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发表时间：
2003-05-02
影响因子：
3.1
通讯作者：
Chang, DC
中科院分区：
文献类型：
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作者：
Luo, KQ;Yu, VC;Chang, DC
在这项研究中，我们首次测量了caspase-8在单个活细胞中的激活动力学。这项测量是使用一种基于FRET(荧光共振能量转移)技术的特殊开发的分子传感器进行的。该传感器是通过将CFP(青色荧光蛋白)和YFP(黄色荧光蛋白)与含有串联caspase-8特异性切割位点的接头融合而成的。FRET比率在卵裂时的变化大于4倍。利用该传感器，我们发现，在肿瘤坏死因子α诱导的细胞凋亡过程中，caspase-8的激活过程比caspase-3慢，而且它的启动时间比caspase-3的激活早得多。抑制caspase-9延迟了caspase-3的完全激活，但不影响caspase-8的动力学。这些单细胞测量的结果表明，在肿瘤坏死因子α诱导HeLa细胞凋亡的过程中，caspase-3通过两条平行的途径被caspase-8激活。(C)2003年埃尔塞维尔科学公司(美国)。版权所有。
In this study, we reported the first measurement of the dynamics of activation of caspase-8 in a single living cell. This measurement was conducted using a specially developed molecular sensor based on the FRET (fluorescence resonance energy transfer) technique. This sensor was constructed by fusing a CFP (cyan fluorescent protein) and a YFP (yellow fluorescent protein) with a linker containing a tandem caspase-8-specific cleavage site. The change of the FRET ratio upon cleavage was larger than 4-fold. Using this sensor, we found that during TNFalpha-induced apoptosis, the activation of caspase-8 was a slower process than that of caspase-3, and it was initiated much earlier than the caspase-3 activation. Inhibition of caspase-9 delayed the full activation of caspase-3 but did not affect the dynamics of caspase-8. Results of these single-cell measurements suggested that caspase-3 was activated by caspase-8 through two parallel pathways during TNFalpha-induced apoptosis in HeLa cells. (C) 2003 Elsevier Science (USA). All rights reserved.