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

细胞凋亡过程中 Bcl-2 蛋白膜透化的实时可视化

• 批准号: 219396439
• 负责人: Dr. Tom Bender
• 金额: --
• 依托单位: Départment de biologie cellulaire
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/219396439?language=en
• 关键词: Real; time; visualization; membrane; permeabilization

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.

细胞凋亡是一种细胞自我毁灭程序，在外部死亡信号或内部刺激（如DNA损伤）后发生。线粒体外膜渗透（MOMP）发生在所谓的细胞凋亡内在途径中，导致细胞色素c等信号分子从线粒体膜间空间释放到细胞质中。这一过程受Bcl-2家族蛋白的亲凋亡和抗凋亡成员的控制。特别是，与BH3-only蛋白如tBid的相互作用导致促凋亡蛋白Bax靶向线粒体外膜，其同质寡聚，最终形成孔和外膜完整性的丧失。这些重排在体内伴随着线粒体的广泛裂变。有趣的是，融合或裂变中间体的形成加速了bax诱导的孔隙形成。生物物理模型表明，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。因此，这个项目的目标是阐明膜中间体和孔隙形成之间的联系。利用经过融合的巨大单层囊泡和荧光标记的重组蛋白，Bax低聚物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确定。此外，借助包裹在这些囊泡内并在膜渗透后释放的荧光右旋糖酐，将展示膜泄漏的空间和时间细节。这种体外MOMP的直接可视化将使我们全面了解Bcl-2蛋白在这一重要信号事件中的作用。

项目成果

Regulation of mitochondrial pyruvate uptake by alternative pyruvate carrier complexes

• DOI: 10.15252/embj.201490197
• 发表时间: 2015-04-01
• 期刊: EMBO JOURNAL
• 影响因子: 11.4
• 作者: Bender, Tom;Pena, Gabrielle;Martinou, Jean-Claude
• 通讯作者: Martinou, Jean-Claude

Mitochondrial pyruvate import and its effects on homeostasis.

• DOI: 10.1016/j.ceb.2014.10.008
• 发表时间: 2015-04
• 期刊: Current opinion in cell biology
• 影响因子: 7.5
• 作者: Benoît Vanderperre;Tom Bender;Edmund R. S. Kunji;J. Martinou

MPC1-like Is a Placental Mammal-specific Mitochondrial Pyruvate Carrier Subunit Expressed in Postmeiotic Male Germ Cells*

• DOI: 10.1074/jbc.m116.733840
• 发表时间: 2016-06
• 期刊: The Journal of Biological Chemistry
• 作者: Benoît Vanderperre;K. Cermakova;J. Escoffier;M. Kaba;Tom Bender;S. Nef;J. Martinou