Farías Ginny G, Britt Dylan J, Bonifacino Juan S
Cell Biology and Neurobiology Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20892, USA.
Cell Biology and Neurobiology Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 35A Convent Drive, Room 2F226, MSC 3758, Bethesda, MD, 20892, USA.
Methods Mol Biol. 2016;1496:13-30. doi: 10.1007/978-1-4939-6463-5_2.
The study of polarized protein trafficking in live neurons is critical for understanding neuronal structure and function. Given the complex anatomy of neurons and the numerous trafficking pathways that are active in them, however, visualization of specific vesicle populations leaving the Golgi complex presents unique challenges. Indeed, several approaches used in non-polarized cells, and even in polarized epithelial cells, have been less successful in neurons. Here, we describe an adaptation of the recently developed Retention Using Selective Hooks (RUSH) system (Boncompain et al., Nat Methods 9:493-498, 2012), previously used in non-polarized cells, to analyze the polarized sorting of proteins from the Golgi complex to dendrites and axons in live neurons. The RUSH system involves the retention of a fluorescently tagged cargo protein fused to the streptavidin-binding peptide (SBP) in the endoplasmic reticulum (ER) through the expression of an ER-hook protein fused to streptavidin. Upon D-biotin addition, the cargo protein is released and its traffic to dendrites and axons can be analyzed in live neurons.
研究活神经元中极化蛋白运输对于理解神经元结构和功能至关重要。然而，鉴于神经元复杂的解剖结构以及其中活跃的众多运输途径，可视化从高尔基体复合体离开的特定囊泡群体面临着独特的挑战。实际上，在非极化细胞甚至极化上皮细胞中使用的几种方法，在神经元中并不那么成功。在这里，我们描述了一种对最近开发的选择性钩子保留（RUSH）系统（Boncompain等人，《自然方法》9:493 - 498，2012）的改进，该系统先前用于非极化细胞，用于分析活神经元中从高尔基体复合体到树突和轴突的蛋白极化分选。RUSH系统涉及通过与链霉亲和素融合的内质网（ER）钩子蛋白的表达，将与链霉亲和素结合肽（SBP）融合的荧光标记货物蛋白保留在内质网中。添加D - 生物素后，货物蛋白被释放，其向树突和轴突的运输可在活神经元中进行分析。
