Confocal FRET microscopy to measure clustering of ligand-receptor complexes in endocytic membranes
Confocal FRET microscopy to measure clustering of ligand-receptor complexes in endocytic membranes
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DOI：
10.1016/s0006-3495(03)74500-7
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发表时间：
2003-07-01
影响因子：
3.4
通讯作者：
Barroso, M
中科院分区：
文献类型：
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作者：
Wallrabe, H;Elangovan, M;Barroso, M
蛋白质在内吞膜中的分布动力学与许多细胞过程有关，如蛋白质分选、细胞器和膜微域生物发生、蛋白质-蛋白质相互作用、受体功能和信号转导。我们开发了一种基于荧光共振能量显微镜（FRET）和新的数学模型的检测方法，根据FRET强度对供体和受体浓度的依赖来区分荧光团结合分子的集群分布和随机分布。这些模型适合于扩展的簇，它可能被紧密包裹，并解释了膜结合蛋白之间的几何排斥效应。两个主要标准被用来证明标记的聚合物iga配体-受体复合物在极化MDCK细胞的顶胞膜内呈簇状组织：1)，能量传递效率（E%）水平与受体水平无关；2)随着未淬供体：受体比的增大，E%减小。簇密度的定量分析表明，供体标记的配体-受体复合物在其邻近区域应该有2.5-3个标记的复合物，并且簇可能发生在有限数量的离散膜位置和/或需要一种可以饱和的特定蛋白质。在此，我们提出了一种新的基于fret的方法来量化极化细胞顶端内噬膜中配体-受体复合物的共定位和分布。
The dynamics of protein distribution in endocytic membranes are relevant for many cellular processes, such as protein sorting, organelle and membrane microdomain biogenesis, protein-protein interactions, receptor function, and signal transduction. We have developed an assay based on Fluorescence Resonance Energy Microscopy (FRET) and novel mathematical models to differentiate between clustered and random distributions of fluorophore-bound molecules on the basis of the dependence of FRET intensity on donor and acceptor concentrations. The models are tailored to extended clusters, which may be tightly packed, and account for geometric exclusion effects between membrane-bound proteins. Two main criteria are used to show that labeled polymeric IgA-ligand-receptor complexes are organized in clusters within apical endocytic membranes of polarized MDCK cells: 1), energy transfer efficiency (E%) levels are independent of acceptor levels; and 2), with increasing unquenched donor: acceptor ratio, E% decreases. A quantitative analysis of cluster density indicates that a donor-labeled ligand-receptor complex should have 2.5-3 labeled complexes in its immediate neighborhood and that clustering may occur at a limited number of discrete membrane locations and/or require a specific protein that can be saturated. Here, we present a new sensitive FRET-based method to quantify the co-localization and distribution of ligand-receptor complexes in apical endocytic membranes of polarized cells.