Characterizing Membrane Traffic in the Early Secretory Pathway Using the RUSH Retention System.
Characterizing Membrane Traffic in the Early Secretory Pathway Using the RUSH Retention System.
复制标题
使用 RUSH 保留系统表征早期分泌途径中的膜交通。
DOI：
10.1007/978-1-0716-2209-4_1
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Camus MD
中科院分区：
文献类型：
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作者：
Camus MD
早期分泌途径包括内质网（ER）和ER -高尔基中间室（ERGIC）细胞器。ERGIC现在被认为是一个复杂的货物分拣中心，涉及各种细胞和组织过程，然而进出ERGIC的交通途径仍然不清楚。经典的方法用于分析货物的旅程沿着分泌途径依赖于可逆交通阻塞导致货物积累在内质网。虽然这些方法是表征高尔基和后高尔基交通路线的关键，但它们对感兴趣的货物的特异性较差，时空分辨率有限，使得它们不足以精细表征早期分泌途径中的货物交通。在本章中，我们描述了一种使用保留选择性钩子（RUSH）系统研究早期分泌途径中货物蛋白交通的方案，这是一种具有高时空分辨率的高度特异性和敏感性的跟踪系统。以GLUT4和GLUT1分别作为非常规和常规分泌货物的例子，我们描述了在RUSH载体中克隆货物的步骤，并沿着早期分泌途径跟踪和量化它们的流量。这种RUSH方法也可用于研究早期分泌途径中其他货物蛋白的运输。
The early secretory pathway encompasses the endoplasmic reticulum (ER) and the ER–Golgi intermediate compartment (ERGIC) organelles. The ERGIC is now understood to be a complex cargo sorting hub involved in a variety of cellular and tissue processes, however the traffic pathways to and from the ERGIC are still unclear.Classical methods employed for the analysis of a cargo’s journey along the secretory pathway rely on reversible traffic blocks leading to cargo accumulation in the ER. Although these methods were key to characterize Golgi and post-Golgi traffic routes, their poor specificity to the cargo of interest and limited spatiotemporal resolution make them inadequate for the fine characterization of cargo traffic in the early secretory pathway.In this chapter, we describe a protocol to study the traffic of cargo proteins in the early secretory pathway using the Retention Using Selective Hook (RUSH) system, a highly specific and sensitive tracking system with a high spatiotemporal resolution. Taking GLUT4 and GLUT1 as examples of unconventionally and conventionally secreted cargo respectively, we describe the steps to clone the cargoes in the RUSH vector and follow and quantify their traffic along the early secretory pathway. This RUSH method can also be used to study the traffic of other cargo proteins in the early secretory pathway.