CELL BIOLOGY OF GAP JUNCTIONS

间隙连接的细胞生物学

• 批准号: 7601029
• 负责人: ERIC C BEYER
• 金额: $ 1.09万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7601029
• 关键词: Cells; Cellular biology; Computer Retrieval of Information on Scientific Projects Database; Electron Microscopy; Funding; Gap Junctions; Golgi Apparatus; Grant; Institution; Life; Localized; Pathway interactions; Property; Proteins; Reagent; Research; Research Personnel; Resources; Source; Time; Transferrin Receptor; United States National Institutes of Health; cellular imaging; congenital cataract; insight; mutant; trafficking

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.

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。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们一直在研究与先天性白内障相关的突变型Cx 50。 该突变体在转染细胞中积累在不同于ER、高尔基体或溶酶体的隔室中。 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和积累的机制和形成的时间过程。 我们可以产生一个具有四半胱氨酸基序的Cx 50突变体的构建体，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，然后用ReAsH孵育（反之亦然），可以了解哪些蛋白质池（旧的和/或新的）参与积累的形成。 此外，我们可以利用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。