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。 我们一直在研究一种与先天性白内障有关的突变CX50。该突变体在转基因细胞中聚集在不同于ER、高尔基体或溶酶体的隔室中。该突变体不与转铁蛋白受体(用作内吞途径的标志)共定位。我们建议研究突变蛋白的运输和堆积的形成机制和时间过程。我们可以构建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Flash孵育，随后与ReAsH(或反之亦然)孵育，可能会深入了解哪个蛋白质池(旧的和/或新的)参与积累的形成。此外，我们还可以利用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 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 following 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 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.