Real-time visualization of membrane permeabilization by Bcl-2 proteins during apoptosis

细胞凋亡是一种在外部死亡信号或内部刺激(如DNA损伤)后执行的细胞自毁程序。线粒体外膜通透性(MOMP)发生在所谓的细胞凋亡的内在途径中，导致细胞色素c等信号分子从线粒体的膜间隙释放到细胞质中。这一过程由Bcl2家族中的促凋亡和抗凋亡成员共同控制。特别是，与只有BH3的蛋白如TBID的相互作用导致促凋亡蛋白Bax靶向线粒体膜外膜，其同源寡聚，最终导致孔的形成和外膜完整性的丧失。在体内，这些重排伴随着线粒体的广泛分裂。有趣的是，融合或裂变中间体的形成加速了Bax诱导的孔的形成。正如生物物理模型所暗示的那样，这些中间体可能显示出本质上不稳定的膜区域，这些区域可能会被Bax低聚物永久破坏稳定。因此，该项目的目标是阐明膜中间体和孔形成之间的联系。利用进行融合的巨大单层囊泡和荧光标记的重组蛋白，Bax寡聚体相对于半融合茎的定位将通过高时间分辨率的共聚焦视频显微镜来确定。此外，借助被包裹在这些囊泡中并在膜渗透时释放的荧光葡聚糖，膜泄漏的空间和时间细节将被展示出来。这种在体外对MOMP的直接可视化将导致关于Bcl-2蛋白在这一重要信号事件中的作用的全面图像。

Apoptosis is a cellular self-destruction program that is executed either after external death signals or internal stimuli, e.g. DNA damage. Mitochondrial outer membrane permeabilization (MOMP) occurs during the so-called intrinsic pathway of apoptosis and leads to the release of signaling molecules like cytochrome c from the intermembrane space of mitochondria to the cytosol. This process is controlled by both pro- and anti-apoptotic members of the Bcl-2 family of proteins. In particular, interaction with BH3-only proteins like tBid leads to the targeting of the pro-apoptotic protein Bax to the outer mitochondrial membrane, its homo-oligomerization and, finally, to the formation of pores and loss of outer membrane integrity. These rearrangements are accompanied in vivo by extensive fission of mitochondria. Interestingly, the formation of fusion or fission intermediates accelerates Bax-induced pore formation. As biophysical models suggest, these intermediates might display intrinsically instable membrane areas that could become permanently destabilized by Bax oligomers. The goal of this project thus is to clarify the connection between membrane intermediates and pore formation. Using giant unilamellar vesicles undergoing fusion and fluorescently labeled recombinant proteins, the localization of Bax oligomers with respect to hemifusion stalks will be determined by confocal videomicroscopy with high temporal resolution. Moreover, with the help of fluorescent dextrans encapsulated inside these vesicles and released upon membrane permeabilization, the spatial and temporal details of membrane leaks will be demonstrated. This direct visualization of MOMP in vitro will lead to a comprehensive picture regarding the role of Bcl-2 proteins in this important signaling event.