CELL BIOLOGY OF GAP JUNCTIONS

这个子项目是许多研究子项目中的一个 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来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 研究中心，而研究中心不一定是研究者所在的机构。 我们正在研究与遗传性先天性白内障相关的突变型Cx 50。 该突变体在氨基酸88处具有脯氨酸至丝氨酸的取代，导致在转染细胞中不同于ER、高尔基体或溶酶体的区室中的积累。 突变体不与转铁蛋白受体（用作内吞途径的标记物）共定位。 我们正在研究突变蛋白的运输和积累的形成机制和时间过程。 我们产生了一个构建体的Cx 50突变体与四半胱氨酸基序，并将其转染到细胞中。 转染细胞与FLASH或ReAsH的孵育将允许跟踪这些积累形成的时间过程和路径。 用FLASH孵育，随后用ReAsH孵育（或反之亦然），可以了解哪些蛋白质池（旧的和/或新的）参与了积聚的形成。 此外，我们利用ReAsH试剂的光氧化性质，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 are studying a mutant Cx50 associated with hereditary congenital cataracts. This mutant, which has a proline to serine substitution at amino acid 88, results in accumulations in the transfected cells in a compartment different from the ER, Golgi or lysosomes. The mutant does not co-localize with the transferrin receptor (used as a marker of the endocytic pathway). We are studying the trafficking of the mutant protein and the mechanism and time course of formation of the accumulations. We generated a construct of the Cx50 mutant with the tetracysteine motif and transfected it into cells. The incubation of the transfected cells with FLASH or ReAsH would allow following the time course and path of formation of these accumulations. Incubation with FLASH and, subsequently, with ReAsH (or vice versa) provides insights as to which pool of protein (old and/or new) participate in the formation of the accumulations. Moreover, we take advantage of the photooxidative properties of the ReAsH reagent that would allow looking 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.