MECHANISMS OF APOPTOSIS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。线粒体外膜的透化作用使蛋白质从膜间隙释放到胞浆中可能是细胞凋亡过程中的关键事件。这些蛋白质包括细胞色素C、Smac/Diablo、核酸内切酶G、腺苷酸激酶、Omi和AIF。这些蛋白质释放的机制尚未建立，并且是激烈辩论的主题。在单细胞中的研究表明，细胞色素C的释放是突然的，快速的和完全的，并且它可以在线粒体膜电位的可检测变化之前进行。其他研究表明，Smac/DIABLO释放与细胞色素C释放一致。另一方面，许多研究表明膜间空间蛋白的差异释放。这些实验中的大多数是通过跟踪GFP或GFP同源物标记的分子的荧光来进行的，GFP或GFP同源物标记的分子可以是内源性分子的两倍或甚至3倍。我们正在利用原位荧光标记的一个小的四半胱氨酸标签，以遵循Smac，腺苷酸激酶和细胞色素C的释放。我们已经观察到，细胞凋亡诱导剂触发释放的所有这些蛋白在个别细胞线粒体去极化之前。此外，Smac和腺苷酸激酶从共表达这些蛋白质的组合的单个细胞中以与细胞色素C相同的动力学释放。我们正在对AIF和核酸内切酶G进行四半胱氨酸标记，以研究它们的释放，并通过电子显微镜检查所有这些蛋白质的线粒体内定位。对于这第二个目的，我们正在利用在NCMIR开发的技术，该技术使用由ReAsH激发产生的活性氧对DAB进行光氧化。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The permeabilization of the mitochondrial outer membrane to release proteins from the intermembrane space into the cytosol is likely to be the pivotal event in the process of apoptosis. These proteins include cytochrome C, Smac/Diablo, endonuclease G, adenylate kinase, Omi and AIF. The mechanism by which these proteins are released has yet to be established and is the subject of intense debate. Studies in single cells suggest that the release of cytochrome C is sudden, rapid and complete, and that it can proceed before detectable changes in mitochondrial membrane potential. Other studies indicate that Smac/DIABLO release coincides with cytochrome C release. On the other hand, many studies have shown a differential release of intermembrane space proteins. Most of these experiments have been performed by following the fluorescence of GFP or a GFP homolog-tagged molecule, which can be twice or even 3-times as large as the endogenous molecule. We are taking advantage of the in situ fluorescence labeling of a small tetracysteine tag in order to follow the release of Smac, adenylate kinase and cytochrome C. We have observed that apoptotic inducers triggered the release of all these proteins in individual cells prior to mitochondrial depolarization. Furthermore, Smac and adenylate kinase were released with the same kinetics as cytochrome C from individual cells coexpressing combinations of these proteins. We are in the process of tetracysteine tagging AIF and endonuclease G in order to study their release, as well as examine the intra-mitochondrial localization of all these proteins by electron microscopy. For this second purpose, we are taking advantage of the technique developed at NCMIR that uses the photooxidation of DAB by reactive oxygen generated by the excitation of ReAsH.