Exact interaction mechanism between Bax and Bcl-XL, two key Bcl-2 family proteins, is an interesting and controversial issue. Partial acceptor photobleaching-based quantitative fluorescence resonance energy transfer (FRET) measurement, PbFRET, is a widely used FRET quantification method in living cells. In this report, we implemented pixel-to-pixel PbFRET imaging on a wide-field microscope to map the FRET e±ciency (ET images of single living HepG2 cells co-expressing CFP-Bax and YFP-Bcl-XL. The E value between CFP-Bax and YFP-Bcl-XL was 4.59% in cytosol and 11.31% on mitochondria, conclusively indicating the direct interaction of the two proteins, and the interaction of the two proteins was strong on mitochondria and modest in cytosol.

光敏剂N-aspartyl chlorin e6 (NPe6)能特异性定位于溶酶体, 溶酶体光损伤光能触发线粒体凋亡通路从而诱导细胞凋亡。Bax 是Bcl-2 家族一员, 是调控细胞凋亡的关键因子之一。静息态下, Bax 定位于细胞质中; 而在细胞凋亡过程中, Bax 会从细胞质转位到线粒体, 损伤线粒体, 从而启动细胞凋亡。在本研究中, 我们在活细胞内实时监控溶酶体光损伤诱导细胞凋亡过程中Bax亚细胞定位的动态变化。结果表明, 溶酶体光损伤后约170 min, Bax开始转位到线粒体, 在30 min之内便大量聚集在线粒体上。该研究结果实时动态地展示了细胞凋亡过程中Bax的时空变化过程。

PUMA (p53 up-regulated modulator of apoptosis, also called Bbc3) was first identified as a BH3-only Bcl-2 family protein that is transcriptionally up-regulated by p53 and activated upon p53-dependent apoptotic stimuli, such as treatment with DNA-damaging drugs or UV irradiation. Recently, studies have shown that PUMA is also up-regulated in response to certain p53-independent apoptotic stimuli, such as growth factor deprivation or treatment with glucocorticoids or STS (staurosporine). However, the molecular mechanisms of PUMA up-regulation and how PUMA functions in response to p53-independent apoptotic stimuli remain poorly understood. In this study, based on real-time single cell analysis, flow cytometry, and western blotting technique, we investigated the function of PUMA in living human lung adenocarcinoma cells (ASTC-a-1) after STS treatment. Our results show that FOXO3a was activated by STS stimulation and then translocated from cytosol to nucleus. The expression of PUMA was up-regulated via a FOXO3a-dependent manner after STS treatment, while p53 had little function in this process. Moreover, cell apoptosis and Bax activation induced by STS were not blocked by Pifithrin- α (p53 inhibitor), which indicated that p53 was not involved in this signaling pathway. Taken together, these results suggest that PUMA promoted Bax activation in a FOXO3a-dependent pathway during STS-induced apoptosis, while p53 was dispensable in this process.

Apoptosis is an important cellular event that plays a key role in the therapy of many diseases. The mechanism of the initiation and regulation of photodynamic therapy (PDT)–induced apoptosis is complex. Our previous study found that Photofrin was localized primarily in mitochondria, the primary targets of Photofrin-PDT. The key role of Bax in the mitochondria-mediated apoptosis has been demonstrated in many systems. In order to determine the role of Bax in the mitochondrion-mediated apoptosis induced by Photofrin-PDT, we used the GFP-Bax plasmid to monitor the dynamics of Bax activation after PDT treatment. With laser scanning confocal microscopy, we found that Bax did not translocate from the cytosol to mitochondria when the mitochondrial membrane potential (ΔΨm) disappeared, measured by TMRM. Thus, for Photofrin-PDT, the commitment to cell death is independent of Bax activation.

紫外辐射诱导的细胞凋亡是一个包括DNA损伤、死亡受体启动、线粒体破裂等一系列反应的复杂过程。有研究报道,由紫外辐射引发的DNA损伤可以激活肿瘤抑制因子p53,从而触发细胞凋亡级联反应,其中一个重要的事件就是上调促凋亡蛋白(p53 up-regulated modulator of apoptosis,PUMA)的表达。但是,PUMA 在紫外辐射诱导凋亡过程中的作用机制尚不清楚。 利用荧光共振能量转移技术(Fluorescence resonance energy transfer,FRET),在人类肺腺癌细胞(ASTC-α-1)内实时研究了紫外辐射诱导凋亡过程中PUMA,Bax,Bcl-Xl三者之间的相互作用关系,从而揭示PUMA的作用机制。 用120 mJ/cm2的紫外辐射剂量诱导细胞凋亡,通过共转染荧光探针GFP-PUMA和YFP-Bcl-Xl或者CFP-Bcl-Xl和YFP-Bax,在激光共聚焦扫描显微镜下实时检测PUMA和Bcl-Xl或者Bcl-Xl和Bax之间的相互作用。 结果显示,紫外辐射后GFP-PUMA与YFP-Bcl-Xl之间的FRET现象逐渐增强,说明PUMA与Bcl-Xl的作用越来越强;CFP-Bcl-Xl与YFP-Bax之间的FRET现象逐渐减弱,说明Bcl-Xl与Bax的作用越来越弱,此过程伴随有YFP-Bax在线粒体的明显定位。该实验结果暗示PUMA是竞争结合了Bcl-Xl,解除了Bcl-Xl对Bax的抑制作用,进而活化了Bax。

细胞凋亡是机体生命活动中重要的细胞学事件,在许多疾病的治疗中也起着关键性的作用.在多种凋亡因子刺激下,Bax的构象发生改变,寡聚化,插入线粒体外膜上.虽然关于Bax蛋白的研究已经取得了很大进展,但是Bax蛋白是如何转位到线粒体以及如何引起细胞色C释放等许多问题尚未十分清楚.为了进一步对Bax蛋白的生物学行为进行研究,特别是在无损伤、活细胞生理条件下,本实验采用了荧光蛋白标记和荧光成像技术对PDT作用凋亡过程中Bax蛋白在活细胞内分布的动态过程进行了初步研究.结果表明:在没有PDT作用时,Bax蛋白比较均匀地分布在整个细胞内,而PDT处理15分钟后,Bax蛋白开始不均匀分布在整个细胞,定位在线粒体上.该研究为今后使用荧光蛋白标记的方法在无损伤、活细胞生理条件下研究Bax蛋白定位机理以及如何诱导细胞色素C释放等问题打下了坚实的基础.

Bax(Bcl-2-associated X protein)是bcl-2家族中的一个很重要的促凋亡蛋白,在正常的细胞生理状态下,主要分布在细胞质,在UV等凋亡刺激因子的作用下,则从细胞质转位到线粒体从而诱导细胞色素C的释放,最终引起细胞凋亡.缺失Bax的细胞对UV诱导的细胞凋亡有抗性,因此研究Bax的特性有助于深入了解UV诱导的细胞凋亡信号转导通路的分子机制.以前的研究大都采用生化的手段,无法在活细胞上实时动态的观察Bax蛋白的变化过程.运用激光共聚焦扫描显微成像技术单细胞实时检测了UV诱导的细胞凋亡过程中Bax的动态变化过程.研究结果表明:UV诱导的细胞凋亡过程中Bax的转位大约起始于UV照射后5 h～6 h,整个转位过程持续20 min～30 min.