Synchronization of secretory protein traffic in populations of cells
Synchronization of secretory protein traffic in populations of cells
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DOI：
10.1038/nmeth.1928
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发表时间：
2012-05-01
期刊：
影响因子：
48
通讯作者：
Perez, Franck
中科院分区：
文献类型：
--
作者：
Boncompain, Gaelle;Divoux, Severine;Perez, Franck
为了剖析分泌交通，我们开发了使用选择性钩子（RUSH）系统的保留。 (RUSH) 是一种二态测定，基于与核心链霉亲和素融合并稳定锚定在供体室中的钩蛋白与与链霉亲和素结合肽 (SBP) 融合的目的报告蛋白的可逆相互作用。添加生物素会导致报告基因从钩子上同步释放。使用（RUSH）系统，我们分析了活细胞中生理温度下各种高尔基体和质膜报告基因的不同运输特性。使用两种货物的双色同步活细胞成像，我们观察到货物运输的高尔基内和高尔基后分离，与其他系统中的观察结果一致。我们初步表明（RUSH）系统可用于自动筛选。该系统应该有助于增加对运输机制的理解，并能够筛选扰乱病理性蛋白质运输的分子。
To dissect secretory traffic, we developed the retention using selective hooks (RUSH)) system. (RUSH) is a two-state assay based on the reversible interaction of a hook protein fused to core streptavidin and stably anchored in the donor compartment with a reporter protein of interest fused to streptavidin-binding peptide (SBP). Biotin addition causes a synchronous release of the reporter from the hook. Using the (RUSH) system, we analyzed different transport characteristics of various Golgi and plasma membrane reporters at physiological temperature in living cells. Using dual-color simultaneous live-cell imaging of two cargos, we observed intra- and post-Golgi segregation of cargo traffic, consistent with observation in other systems. We show preliminarily that the (RUSH) system is usable for automated screening. The system should help increase the understanding of the mechanisms of trafficking and enable screens for molecules that perturb pathological protein transport.