MECHANISMS OF APOPTOSIS

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。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。列出的机构是 中心，不一定是研究者的机构。 细胞凋亡中细胞色素c-GFP释放的动力学显示为约5分钟。 然而，绿色荧光蛋白是细胞色素c的两倍，提出了这些发现的相关性的问题。 细胞色素c与四半胱氨酸基序融合产生的融合蛋白的大小更接近内源性细胞色素c的大小。 这种融合已经显示出以与细胞色素c-GFP类似的动力学释放。本研究的目的是确定线粒体内的细胞色素c-四半胱氨酸的本地化，使用相关的显微镜技术开发的NCMIR。 具体来说，我们希望使用四半胱氨酸标签/双砷方法来可视化活细胞中光水平下的融合以及DAB的光转化，通过直接激发双砷ReAsH以高分辨率可视化细胞色素c-四半胱氨酸电子显微镜（EM），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.