Epithelia compartmentalize multicellular organisms and provide interfacing between the inside and outside. Apart from regulating the exchange of solutes, uptake of nutrients, and excretion of waste products, their major function is to prevent uncontrolled access of foreign material to immune-competent compartments. Progress in understanding this barrier function toward larger solutes and its possible defects, as can be seen in a variety of diseases, is largely hampered by a lack of methods to spatiotemporally resolve transepithelial passage of macromolecules. Using different cell culture epithelia, we applied biotinylated dextran tracers carrying an acceptor fluorophore. These bind to cell-adherent avidin carrying donor fluorophore at the basolateral membranes of single-layered epithelial sheets. Confocal fluorescence microscopy was applied to living epithelia in order to image apical-to-basolateral tracer passage as a Förster resonance energy transfer signal of the fluorescent dextran–avidin pair over time. Stimulated macromolecule passage using barrier-perturbing agents proved its effectiveness for the leak imaging method presented herein. Over hours of imaging, spontaneous leaks were rare, occurring transiently on the scale of minutes and for the most part associated with rearranging cell junctions. The discussed approach to leak imaging is expected to promote the understanding of epithelial barriers, particularly, the nature and dynamics of the epithelial cell leak pathway.

中文翻译：

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探索泄漏途径：大分子通过上皮细胞的活细胞成像

上皮细胞将多细胞生物分隔开来，并提供内部和外部之间的接口。除了调节溶质交换、营养物质吸收和废物排泄外，它们的主要功能是防止外来物质不受控制地进入免疫室。由于缺乏时空解决大分子跨上皮通道的方法，在很大程度上阻碍了理解这种针对较大溶质的屏障功能及其可能缺陷的进展，正如在各种疾病中所见。使用不同的细胞培养上皮细胞，我们应用了携带受体荧光团的生物素化葡聚糖示踪剂。它们与单层上皮层基底外侧膜上携带供体荧光团的细胞粘附亲和素结合。将共聚焦荧光显微镜应用于活体上皮细胞，以便将顶端到基底外侧的示踪剂通道成像为荧光葡聚糖-抗生物素蛋白对随时间变化的 Förster 共振能量转移信号。使用障碍扰动剂的受激大分子通道证明了其对本文介绍的泄漏成像方法的有效性。在数小时的成像过程中，自发性泄漏很少见，仅在几分钟内短暂发生，并且大部分与重新排列细胞连接有关。所讨论的泄漏成像方法有望促进对上皮屏障的理解，特别是上皮细胞泄漏通路的性质和动力学。使用障碍扰动剂的受激大分子通道证明了其对本文介绍的泄漏成像方法的有效性。在数小时的成像过程中，自发性泄漏很少见，仅在几分钟内短暂发生，并且大部分与重新排列细胞连接有关。所讨论的泄漏成像方法有望促进对上皮屏障的理解，特别是上皮细胞泄漏通路的性质和动力学。使用障碍扰动剂的受激大分子通道证明了其对本文介绍的泄漏成像方法的有效性。在数小时的成像过程中，自发性泄漏很少见，仅在几分钟内短暂发生，并且大部分与重新排列细胞连接有关。所讨论的泄漏成像方法有望促进对上皮屏障的理解，特别是上皮细胞泄漏通路的性质和动力学。