Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast
Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast
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DOI：
10.3791/54587
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发表时间：
2016-10-01
影响因子：
1.2
通讯作者：
Wendland, Beverly
中科院分区：
文献类型：
--
作者：
Prosser, Derek C.;Wrasman, Kristie;Wendland, Beverly
绿色荧光蛋白（GFP）及其变体是广泛用于研究蛋白质定位和事件动力学的工具，例如活细胞中的细胞骨架重塑和囊泡运输。已经开发了使用嵌合GFP融合物的定量方法用于许多应用;然而，GFP在某种程度上对蛋白水解具有抗性，因此其荧光持续存在于溶酶体/液泡中，这可能阻碍内吞途径中货物运输的定量。用于定量内吞作用和内吞后运输事件的替代方法利用超脂质pHluorin，其是在酸性环境中淬灭的GFP的pH敏感性变体。pHluorin与跨膜货物蛋白质的胞质尾的嵌合融合导致在货物并入多泡体（MVB）中并递送至溶酶体/空泡腔时荧光减弱。因此，液泡荧光的淬灭有助于内吞作用和内吞途径中的早期事件的定量。本文描述了使用荧光素标记的货物通过荧光显微镜定量内吞作用的方法，以及使用流式细胞术的基于群体的测定。
Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.