Hutten Saskia, Dormann Dorothee
Johannes Gutenberg Universität Mainz, Institute of Molecular Physiology, Mainz, Germany.
Institute of Molecular Biology (IMB) Mainz, Mainz, Germany.
Methods Mol Biol. 2022;2502:81-90. doi: 10.1007/978-1-0716-2337-4_5.
In the recent years, defective nuclear import has emerged as an important pathomechanism of neurodegenerative diseases, particularly in amyotrophic lateral sclerosis (ALS). Here, specific nuclear RNA binding proteins (RBPs) mislocalize and aggregate in the cytoplasm of neurons and glial cells in degenerating brain regions. Bona fide transport assays that measure nuclear import in a quantitative manner allow one to distinguish whether disease-linked RBP mutations that cause cytosolic RBP mislocalization directly result in reduced nuclear import or cause increased cytoplasmic localization of the RBP through other mechanisms. Here we describe the quantitative analysis of nuclear import rates of RBPs using a hormone-inducible system by live cell imaging.
近年来，核输入缺陷已成为神经退行性疾病的一种重要发病机制，尤其是在肌萎缩侧索硬化症（ALS）中。在此，特定的核RNA结合蛋白（RBPs）在退化脑区的神经元和胶质细胞胞质中发生错误定位并聚集。以定量方式测量核输入的真实转运分析能够让人区分导致胞质RBP错误定位的疾病相关RBP突变是直接导致核输入减少，还是通过其他机制导致RBP胞质定位增加。在此，我们描述了通过活细胞成像，利用激素诱导系统对RBPs核输入速率进行的定量分析。
