Visualization and Quantitative Analysis of Reconstituted Tight Junctions Using Localization Microscopy
Visualization and Quantitative Analysis of Reconstituted Tight Junctions Using Localization Microscopy
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DOI：
10.1371/journal.pone.0031128
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发表时间：
2012-02-02
期刊：
影响因子：
3.7
通讯作者：
Cremer, Christoph
中科院分区：
文献类型：
--
作者：
Kaufmann, Rainer;Piontek, Joerg;Cremer, Christoph
紧密连接（TJ）调节组织屏障的细胞旁通透性。紧密连接蛋白（Cld）形成TJ链的骨架。成孔紧密连接蛋白决定离子的渗透性，而溶质和大分子的渗透性被认为受到链形态的关键限制（即，密度、分支和连续性）。为了研究TJ链形态的决定因素，我们建立了一种使用定位显微镜的新方法。通过用屏障形成Cld 3或Cld 5稳定转染HEK 293细胞来重建TJ链。通过光谱位置测定显微镜（SPDM）（一种使用标准荧光团的定位显微镜方法）在细胞-细胞接触处研究链。观察到Cld 3-YFP和Cld 5-YFP的扩展TJ-网络。对于每个网络，检测到200，000至1，100，000个单个分子，平均定位精度接近20 nm，平均结构分辨率接近50 nm。与传统的荧光显微镜相比，这大大提高了链网络的可视化，并使定量形态分析。两个椭圆形网格群（平均直径
Tight Junctions (TJ) regulate paracellular permeability of tissue barriers. Claudins (Cld) form the backbone of TJ-strands. Pore-forming claudins determine the permeability for ions, whereas that for solutes and macromolecules is assumed to be crucially restricted by the strand morphology (i.e., density, branching and continuity). To investigate determinants of the morphology of TJ-strands we established a novel approach using localization microscopy. TJ-strands were reconstituted by stable transfection of HEK293 cells with the barrier-forming Cld3 or Cld5. Strands were investigated at cell-cell contacts by Spectral Position Determination Microscopy (SPDM), a method of localization microscopy using standard fluorophores. Extended TJ-networks of Cld3-YFP and Cld5-YFP were observed. For each network, 200,000 to 1,100,000 individual molecules were detected with a mean localization accuracy of similar to 20 nm, yielding a mean structural resolution of similar to 50 nm. Compared to conventional fluorescence microscopy, this strongly improved the visualization of strand networks and enabled quantitative morphometric analysis. Two populations of elliptic meshes (mean diameter