Visualization and Quantitative Analysis of Reconstituted Tight Junctions Using Localization Microscopy

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 ∼20 nm, yielding a mean structural resolution of ∼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 The morphometry and single molecule information provided advances the mechanistic analysis of paracellular barriers. Applying this novel method to different TJ-proteins is expected to significantly improve the understanding of TJ on the molecular level.紧密连接（Tight Junctions, TJ）可调控组织屏障的细胞旁通透性。紧密连接蛋白（Claudins, Cld）是紧密连接丝（TJ-strands）的骨架成分。成孔型紧密连接蛋白决定离子的通透性，而溶质与大分子的通透性则被认为主要受紧密连接丝的形态（即密度、分支性与连续性）严格限制。为探究紧密连接丝形态的决定因素，我们建立了一种基于定位显微镜（localization microscopy）的全新研究方法。通过将屏障型紧密连接蛋白3（Cld3）或5（Cld5）稳定转染至HEK293细胞，我们成功重构了紧密连接丝。借助光谱位置确定显微镜（Spectral Position Determination Microscopy, SPDM）——一种采用标准荧光染料的定位显微镜技术——我们在细胞间接触位点对紧密连接丝开展了成像观测。我们观测到Cld3-YFP与Cld5-YFP融合蛋白所形成的延伸型紧密连接网络。对每个网络，我们分别检测到200000至1100000个独立分子，平均定位精度约为20 nm，由此获得约50 nm的平均结构分辨率。相较于传统荧光显微镜，该方法显著优化了紧密连接丝网络的可视化效果，并实现了定量形态计量分析。我们发现了两类椭圆形网孔群体（平均直径[数据缺失]）。本研究提供的形态计量学与单分子信息，推动了细胞旁屏障的机制性分析。将该新型方法应用于不同的紧密连接蛋白，有望在分子水平上大幅加深我们对紧密连接的认知。