CELL BIOLOGY OF GAP JUNCTIONS

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。 我们正在研究一种与遗传性先天性白内障相关的突变CX50。该突变体在88位氨基酸上有一个由脯氨酸到丝氨酸的替换，导致在转基因细胞中的蓄积不同于内质网、高尔基体或溶酶体。该突变体不与转铁蛋白受体(用作内吞途径的标志)共定位。我们正在研究突变蛋白的运输和积聚的形成机制和时间过程。我们构建了一个带有四半胱氨酸基序的CX50突变体，并将其导入细胞。将转基因细胞与闪光或ReAsH孵育，将允许跟踪这些积聚形成的时间进程和路径。与闪光以及随后与ReAsH(或反之亦然)的孵育提供了关于哪个蛋白质池(旧的和/或新的)参与积累的形成的洞察。此外，我们利用ReAsH试剂的光氧化特性，通过电子显微镜观察突变蛋白所在的隔间，并与获得的活细胞图像进行直接关联。

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.