Dual-color dual-focus line-scanning FCS for quantitative analysis of receptor-ligand interactions in living specimens.
Dual-color dual-focus line-scanning FCS for quantitative analysis of receptor-ligand interactions in living specimens.
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DOI：
10.1038/srep10149
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发表时间：
2015-05-07
影响因子：
4.6
通讯作者：
Nienhaus GU
中科院分区：
文献类型：
--
作者：
Dörlich RM;Chen Q;Niklas Hedde P;Schuster V;Hippler M;Wesslowski J;Davidson G;Nienhaus GU
多细胞生物体中的细胞通讯在很大程度上由结合特异性配体的大量细胞表面受体介导。深入了解细胞信号网络需要定量信息的配体受体相互作用在生活系统。原则上，基于荧光相关光谱（FCS）的方法可以提供这样的数据，但活细胞应用已被证明极具挑战性。在这里，我们已经开发出一种集成的双色双焦点线扫描荧光相关光谱（2c 2f lsFCS）技术，大大方便了活细胞和组织实验。配体和受体的绝对浓度及其在细胞膜内的扩散系数可以定量，而无需进行额外的校准实验。我们还确定了在同一实验中的细胞外的介质中扩散的配体的浓度，通过使用光栅图像相关光谱（RICS）为基础的分析。我们应用这种强大的技术来研究两种Wnt拮抗剂Dickkopf 1和Dickkopf 2（Dkk 1/2）与其同源受体低密度脂蛋白受体相关蛋白6（LRP 6）在活HEK 293 T细胞质膜中的相互作用。我们获得了显着低于亲和力比以前报道的使用体外研究，强调需要测量活细胞或组织的数据。
Cellular communication in multi-cellular organisms is mediated to a large extent by a multitude of cell-surface receptors that bind specific ligands. An in-depth understanding of cell signaling networks requires quantitative information on ligand-receptor interactions within living systems. In principle, fluorescence correlation spectroscopy (FCS) based methods can provide such data, but live-cell applications have proven extremely challenging. Here, we have developed an integrated dual-color dual-focus line-scanning fluorescence correlation spectroscopy (2c2f lsFCS) technique that greatly facilitates live-cell and tissue experiments. Absolute ligand and receptor concentrations and their diffusion coefficients within the cell membrane can be quantified without the need to perform additional calibration experiments. We also determine the concentration of ligands diffusing in the medium outside the cell within the same experiment by using a raster image correlation spectroscopy (RICS) based analysis. We have applied this robust technique to study the interactions of two Wnt antagonists, Dickkopf1 and Dickkopf2 (Dkk1/2), to their cognate receptor, low-density-lipoprotein-receptor related protein 6 (LRP6), in the plasma membrane of living HEK293T cells. We obtained significantly lower affinities than previously reported using in vitro studies, underscoring the need to measure such data on living cells or tissues.
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