de Bakker Bärbel I, de Lange Frank, Cambi Alessandra, Korterik Jeroen P, van Dijk Erik M H P, van Hulst Niek F, Figdor Carl G, Garcia-Parajo Maria F
Applied Optics Group, Faculty of Science & Technology, MESA+ Institute for Nanotechnology, University of Twente, The Netherlands.
Chemphyschem. 2007 Jul 16;8(10):1473-80. doi: 10.1002/cphc.200700169.
DC-SIGN, a C-type lectin exclusively expressed on dendritic cells (DCs), plays an important role in pathogen recognition by binding with high affinity to a large variety of microorganisms. Recent experimental evidence points to a direct relation between the function of DC-SIGN as a viral receptor and its spatial arrangement on the plasma membrane. We have investigated the nanoscale organization of fluorescently labeled DC-SIGN on intact isolated DCs by means of near-field scanning optical microscopy (NSOM) combined with single-molecule detection. Fluorescence spots of different intensity and size have been directly visualized by optical means with a spatial resolution of less than 100 nm. Intensity- and size-distribution histograms of the DC-SIGN fluorescent spots confirm that approximately 80 % of the receptors are organized in nanosized domains randomly distributed on the cell membrane. Intensity-size correlation analysis revealed remarkable heterogeneity in the molecular packing density of the domains. Furthermore, we have mapped the intermolecular organization within a dense cluster by means of sequential NSOM imaging combined with discrete single-molecule photobleaching. In this way we have determined the spatial coordinates of 13 different individual dyes, with a localization accuracy of 6 nm. Our experimental observations are all consistent with an arrangement of DC-SIGN designed to maximize its chances of binding to a wide range of microorganisms. Our data also illustrate the potential of NSOM as an ultrasensitive, high-resolution technique to probe nanometer-scale organization of molecules on the cell membrane.
DC-SIGN是一种仅在树突状细胞（DC）上表达的C型凝集素，通过与多种微生物高亲和力结合在病原体识别中发挥重要作用。最近的实验证据表明，DC-SIGN作为病毒受体的功能与其在质膜上的空间排列之间存在直接关系。我们通过近场扫描光学显微镜（NSOM）结合单分子检测，研究了完整分离的DC上荧光标记的DC-SIGN的纳米级组织。不同强度和大小的荧光斑点已通过光学手段直接可视化，空间分辨率小于100 nm。DC-SIGN荧光斑点的强度和大小分布直方图证实，约80%的受体组织成随机分布在细胞膜上的纳米级结构域。强度-大小相关性分析揭示了结构域分子堆积密度的显著异质性。此外，我们通过顺序NSOM成像结合离散单分子光漂白，绘制了密集簇内的分子间组织。通过这种方式，我们确定了13种不同单个染料的空间坐标，定位精度为6 nm。我们的实验观察结果均与DC-SIGN的一种排列方式一致，这种排列方式旨在最大限度地增加其与多种微生物结合的机会。我们的数据还说明了NSOM作为一种超灵敏、高分辨率技术在探测细胞膜上分子纳米级组织方面的潜力。
