Mapping receptor density on live cells by using fluorescence correlation spectroscopy.
Mapping receptor density on live cells by using fluorescence correlation spectroscopy.
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DOI：
10.1002/chem.200802305
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Tan W
中科院分区：
文献类型：
--
作者：
Chen Y;Munteanu AC;Huang YF;Phillips J;Zhu Z;Mavros M;Tan W
研究细胞膜上受体的密度、空间分布和分子相互作用提供了了解细胞行为和生物功能以及发现、设计和筛选新型治疗剂所需的知识。然而，以空间和时间有序的方式绘制活细胞上受体分布的图谱和监测配体-受体相互作用是具有挑战性的任务。在本文中，我们应用荧光相关光谱(FCS)通过引入荧光标记的适体分子来定位活细胞膜上的受体密度，这些适体分子特异性地结合到某些细胞表面受体上。FCS创造的飞升大小(0.4fl)的观察体积允许荧光适体检测到2个分子，似乎是研究活细胞上分子相互作用的理想和高灵敏度的生物物理工具。荧光团标记的适配子由于具有高的结合亲和力和特异性而被选择用于受体识别。FCS测定表明，适体sgc8与其靶分子膜受体人蛋白酪氨酸激酶-7(PTK7)具有较强的亲和力(解离常数Kd=790±150 pm)。然后我们构建了一个细胞模型，并应用这种适体-受体相互作用来估计细胞表面的受体密度和分布。用人白血病细胞系CCRF-CEM细胞(13 0 0±1 90 0受体μm−2)和HeLa宫颈癌细胞(5 5 0±90受体μm−2)研究PTK7的不同表达水平。与过量的非标记适配子的竞争研究和蛋白酶处理研究证明了密度估计方法的有效性。这种FCS密度估计方法具有直接测量、高灵敏度、快速分析和单细胞测量的固有优势，在未来的分子相互作用研究和亚细胞结构和膜受体的密度估计中具有潜在的应用前景。
Study of the density, spatial distribution, and molecular interactions of receptors on the cell membrane provides the knowledge required to understand cellular behavior and biological functions, as well as to discover, design, and screen novel therapeutic agents. However, the mapping of receptor distribution and the monitoring of ligand–receptor interactions on live cells in a spatially and temporally ordered manner are challenging tasks. In this paper, we apply fluorescence correlation spectroscopy (FCS) to map receptor densities on live cell membranes by introducing fluorescently marked aptamer molecules, which specifically bind to certain cell-surface receptors. The femtoliter-sized (0.4 fL) observation volume created by FCS allows fluorescent-aptamer detection down to 2 molecules and appears to be an ideal and highly sensitive biophysical tool for studying molecular interactions on live cells. Fluorophore-labeled aptamers were chosen for receptor recognition because of their high binding affinity and specificity. Aptamer sgc8, generated for specific cell recognition by a process called cell systematic evolution of ligands by exponential enrichment, was determined by FCS to have a binding affinity in the picomolar range (dissociation constant Kd = 790 ± 150 pM) with its target membrane receptor, human protein tyrosine kinase-7 (PTK7), a potential cancer biomarker. We then constructed a cellular model and applied this aptamer–receptor interaction to estimate receptor densities and distributions on the cell surface. Specifically, different expression levels of PTK7 were studied by using human leukemia CCRF-CEM cells (1300±190 receptors μm−2) and HeLa cervical cancer cells (550±90 receptors μm−2). Competition studies with excess nonlabeled aptamers and proteinase treatment studies proved the validity of the density-estimation approach. With its intrinsic advantages of direct measurement, high sensitivity, fast analysis, and single-cell measurement, this FCS density-estimation approach holds potential for future applications in molecular-interaction studies and density estimations for subcellular structures and membrane receptors.
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