Department of Biology, Temple University, Philadelphia, Pennsylvania.
Yale School of Medicine, Yale University, New Haven, Connecticut.
Curr Protoc. 2022 Sep;2(9):e520. doi: 10.1002/cpz1.520.
There is a significant current question regarding the viable copy numbers of nucleoporins required for the function of the nuclear pore complex (NPC) in eukaryotic cells. The NPC consists of approximately 30 different nucleoporins in an eight-fold symmetry, meaning that there are multiple duplicates of each nucleoporin present within the nuclear pore. We recently developed a method that combines auxin-inducible degrons and single-molecule super-resolution microscopy to evaluate the copy number of nuclear basket nucleoporins required for the successful function of the NPC. Here, we describe the theory behind this auxin-inducible degron and single-molecule super-resolution microscopy method, and we detail a step-by-step process to selectively degrade nucleoporins either completely or in a stepwise manner. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Degradation of target nucleoporins Basic Protocol 2: Quantification of nucleoporin copy number via narrow-field fluorescence microscopy.
目前有一个重要的问题是，真核细胞核孔复合体 (NPC) 的功能所需的核孔蛋白的可行拷贝数是多少。NPC 由大约 30 种不同的核孔蛋白以八重对称的方式组成，这意味着在核孔内存在每个核孔蛋白的多个重复。我们最近开发了一种结合了生长素诱导的降解结构域和单分子超分辨率显微镜的方法，以评估 NPC 成功功能所需的核篮核孔蛋白的拷贝数。在这里，我们描述了这种生长素诱导的降解结构域和单分子超分辨率显微镜方法的原理，并详细介绍了一个逐步的过程，以选择性地完全或逐步降解核孔蛋白。© 2022 威立出版公司。基本方案 1：靶核孔蛋白的降解基本方案 2：通过窄场荧光显微镜定量核孔蛋白拷贝数。
