CELL BIOLOGY OF GAP JUNCTIONS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。我们一直在研究与先天性白内障相关的Cx50突变体。这种突变体在与内质网、高尔基溶酶体不同的隔室中积累。突变体不与转铁蛋白受体（用作内吞途径的标记物）共定位。我们建议研究突变蛋白的运输、积累的形成机制和时间过程。我们可以用四胱氨酸基序构建Cx50突变体并将其转染到细胞中。将转染的细胞与FLASH或ReASH孵育，可以跟踪这些聚积形成的时间过程和路径。用FLASH孵育，然后用ReASH孵育（反之亦然），可以深入了解哪一种蛋白质（旧的和/或新的）参与了积累的形成。此外，我们可以利用ReASH试剂的光氧化特性，这将允许通过电子显微镜观察突变蛋白所在的隔室，并与获得的活细胞图像直接相关。

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. We have been studying a mutant Cx50 associated to congenital cataracts. This mutant accumulates in the transfected cells in a compartment different from the ER, Golgi orlysosomes. The mutant does not co-localize with the transferrin receptor (used as a marker of the endocytic pathway). We propose to study the trafficking of the mutant protein and the mechanism and time course of formation of the accumulations. We can generate a construct of the Cx50 mutant with the tetracysteine motif and transfect it into cells. The incubation of the transfected cells with FLASH or ReASH would allow to follow the time course and path of formation of these accumulations. Incubation with FLASH and, subsequently, with ReASH (or vice versa) might give insights as to which pool of protein (old and/or new) participate in the formation of the accumulations. Moreover, we can take advantage of the photooxidative properties of the ReASH reagent which would allow to look at the compartments in which the mutant protein is located by electron microscopy and to make a direct correlation with the live cell images obtained.