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.
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