内体
高尔基体
细胞生物学
细胞内
生物
细胞器
流式细胞术
转运蛋白
细胞膜
细胞
内质网
生物化学
分子生物学
作者
Pei Zhi Cheryl Chia,Yasmin M. Ramdzan,Fiona Houghton,Danny M. Hatters,Paul A. Gleeson
出处
期刊:Traffic
[Wiley]
日期:2014-03-11
卷期号:15 (5): 572-582
被引量:13
摘要
Abstract Current methods for the quantitation of membrane protein trafficking rely heavily on microscopy, which has limited quantitative capacity for analyses of cell populations and is cumbersome to perform. Here we describe a simple flow cytometry‐based method that circumvents these limitations. The method utilizes fluorescent pulse‐width measurements as a highly sensitive indicator to monitor the changes in intracellular distributions of a fluorescently labelled molecule in a cell. Pulse‐width analysis enabled us to discriminate cells with target proteins in different intracellular locations including Golgi, lyso‐endosomal network and the plasma membrane, as well as detecting morphological changes in organelles such as Golgi perturbation. The movement of endogenous and exogenous retrograde cargo was tracked from the plasma membrane‐to‐endosomes‐to‐Golgi, by decreasing pulse‐width values. A block in transport upon RNAi ‐mediated ablation of transport machinery was readily quantified, demonstrating the versatility of this technique to identify pathway inhibitors. We also showed that pulse‐width can be exploited to sort and recover cells based on different intracellular staining patterns, e.g. early endosomes and Golgi, opening up novel downstream applications. Overall, the method provides new capabilities for viewing membrane transport in thousands of cells per minute, unbiased analysis of the trafficking of cargo, and the potential for rapid screening of inhibitors of trafficking pathways.
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