MECHANISMS OF APOPTOSIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 细胞色素c-GFP在细胞凋亡中的释放动力学约为5min。然而，绿色荧光蛋白的体积大约是细胞色素c的两倍，这引发了对这些发现的相关性的质疑。细胞色素c与四半胱氨酸基序融合形成一个融合蛋白，其大小与内源性细胞色素c的大小非常接近。这种融合蛋白的释放动力学与细胞色素c-GFP相似。这项研究的目的是利用NCMIR开发的相关显微技术来确定细胞色素c-四半胱氨酸在线粒体中的定位。具体地说，我们希望使用四半胱氨酸标签/双砷酸方法来可视化活细胞中光水平的融合和DAB的光转化，方法是直接激发双砷试剂ReAsH以高分辨率的电子显微镜(EM)显示细胞色素c-四半胱氨酸，然后进行断层重建。

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 kinetics of cytochrome c -GFP release in apoptosis was shown to be about 5 minutes. However, GFP is about twice as big as cytochrome c, raising the question as to the relevance of these findings. Cytochrome c fused to a tetracysteine motif results into a fusion protein whose size is much closer to that of the endogenous cytochrome c. This fusion has been shown to release with similar kinetics as cytochrome c-GFP. The purpose of this study is to determine the localization of cytochrome c-tetracysteine within mitochondria using correlated microscopy techniques developed at NCMIR. Specifically, we want to use the tetracysteine tag/biarsenical approach to visualize the fusion at the light level in living cells and the photoconversion of DAB through direct excitation of the biarsenical ReAsH to visualize cytochrome c-tetracysteine at high resolution by electron microscopy (EM) followed by tomographic reconstructions.