Distribution of a glycosylphosphatidylinositol-anchored protein at the apical surface of MDCK cells examined at a resolution of <100 A using imaging fluorescence resonance energy transfer.
Distribution of a glycosylphosphatidylinositol-anchored protein at the apical surface of MDCK cells examined at a resolution of <100 A using imaging fluorescence resonance energy transfer.
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DOI：
10.1083/jcb.142.1.69
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发表时间：
1998-07-13
期刊：
影响因子：
--
通讯作者：
Edidin M
中科院分区：
文献类型：
--
作者：
Kenworthy AK;Edidin M
富含糖基磷脂酰肌醇（GPI）锚定蛋白、鞘糖脂和胆固醇的膜微区（“脂筏”）参与了从膜运输到信号转导的各种事件。虽然有这样的膜微区的生化证据，他们还没有被可视化的光学或电子显微镜。为了探测完整细胞膜中富含GPI锚定蛋白的微区，我们使用了一种新形式的数字显微镜，成像荧光共振能量转移（FRET），它将荧光显微镜的分辨率扩展到分子水平（<100 nm）。我们检测到MDCK细胞顶膜处供体和受体标记的抗GPI锚定蛋白5 '核苷酸酶（5' NT）的抗体之间存在显着的能量转移。能量转移的效率与受体标记抗体的表面密度密切相关。的FRET数据符合随机分布的分子之间的二维FRET的理论预测，是不一致的模型，其中5′ NT是组成性集群。虽然我们不能完全排除某些5′ NT成簇的可能性，但数据表明大多数5′ NT分子随机分布在MDCK细胞的顶面。这些发现限制了目前的模型脂筏和膜组织的GPI锚定蛋白。
Membrane microdomains (“lipid rafts”) enriched in glycosylphosphatidylinositol (GPI)-anchored proteins, glycosphingolipids, and cholesterol have been implicated in events ranging from membrane trafficking to signal transduction. Although there is biochemical evidence for such membrane microdomains, they have not been visualized by light or electron microscopy. To probe for microdomains enriched in GPI- anchored proteins in intact cell membranes, we used a novel form of digital microscopy, imaging fluorescence resonance energy transfer (FRET), which extends the resolution of fluorescence microscopy to the molecular level (<100 Å). We detected significant energy transfer between donor- and acceptor-labeled antibodies against the GPI-anchored protein 5′ nucleotidase (5′ NT) at the apical membrane of MDCK cells. The efficiency of energy transfer correlated strongly with the surface density of the acceptor-labeled antibody. The FRET data conformed to theoretical predictions for two-dimensional FRET between randomly distributed molecules and were inconsistent with a model in which 5′ NT is constitutively clustered. Though we cannot completely exclude the possibility that some 5′ NT is in clusters, the data imply that most 5′ NT molecules are randomly distributed across the apical surface of MDCK cells. These findings constrain current models for lipid rafts and the membrane organization of GPI-anchored proteins.