Pei Yihui, Xing Da, Gao Xuejuan, Liu Lei, Chen Tongsheng
MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University, Guangzhou 510631, China.
Apoptosis. 2007 Sep;12(9):1681-90. doi: 10.1007/s10495-007-0091-7.
Bid, a member of the pro-apoptotic Bcl-2 protein family, is activated through caspase-8-mediated cleavage into a truncated form (p15 tBid) during TNF-alpha(tumor necrosis factor alpha)-induced apoptosis. Activated tBid can induce Bax oligomerization and translocation to mitochondria, triggering the release of cytochrome c, caspase-3 activation and cell apoptosis. However, it is debatable that whether Bid and tBid can interact directly with Bax in living cells. In this study, we used confocal fluorescence microscope, combined with both FRET (fluorescence resonance energy transfer) and acceptor photobleaching techniques, to study the dynamic interaction between Bid and Bax during TNF-alpha-induced apoptosis in single living cell. In ASTC-a-1 cells, full length Bid induced Bax translocation to mitochondria by directly interacting with Bax transiently in response to TNF-alpha treatment before cell shrinkage. Next, we demonstrated that, in both ASTC-a-1 and HeLa cells, Bid was not cleaved before cell shrinkage even under the condition that caspase-8 had been activated, but in MCF-7 cells Bid was cleaved. In addition, in ASTC-a-1 cells, caspase-3 activation was a biphasic process and Bid was cleaved after the second activation of caspase-3. In summary, these findings indicate that, FL-Bid (full length-Bid) directly regulated the activation of Bax during TNF-alpha-induced apoptosis in ASTC-a-1 cells and that the cleavage of Bid occurred in advanced apoptosis.
Bid是促凋亡Bcl-2蛋白家族的成员，在肿瘤坏死因子α（TNF-α）诱导的细胞凋亡过程中，通过caspase-8介导的切割被激活，形成截短形式（p15 tBid）。活化的tBid可诱导Bax寡聚化并转位至线粒体，触发细胞色素c的释放、caspase-3的激活以及细胞凋亡。然而，Bid和tBid在活细胞中是否能直接与Bax相互作用仍存在争议。在本研究中，我们使用共聚焦荧光显微镜，结合荧光共振能量转移（FRET）和受体光漂白技术，研究了在TNF-α诱导的单个活细胞凋亡过程中Bid和Bax之间的动态相互作用。在ASTC-a-1细胞中，全长Bid在细胞收缩前通过在TNF-α处理后与Bax短暂直接相互作用，诱导Bax转位至线粒体。接下来，我们证明，在ASTC-a-1和HeLa细胞中，即使在caspase-8已被激活的情况下，Bid在细胞收缩前也未被切割，但在MCF-7细胞中Bid被切割。此外，在ASTC-a-1细胞中，caspase-3的激活是一个双相过程，Bid在caspase-3第二次激活后被切割。总之，这些发现表明，全长Bid（FL-Bid）在ASTC-a-1细胞中TNF-α诱导的细胞凋亡过程中直接调节Bax的激活，且Bid的切割发生在晚期凋亡阶段。
