Quantification of epithelial permeability using zinc-based ultrasensitive membrane assay in MDCK cells

Abstract

Epithelium creates seals between different compartments, lining most organs in human body. The cell-cell junction responsible for the barrier formation is the tight junction (TJ) that is formed by serval different proteins, such as the transmembrane proteins claudins and occludin as well as different adaptor proteins like zonula occludens (ZOs). TJs meditate the paracellular molecular transport. Claudins form pores for ions to pass though, generating the pore pathway. Larger molecules are transported via leak pathway, which seems to be regulated by occludin, ZO-1 and claudin strands. In the past TJs have been thought to be static diffusion barriers, but increasing evidence shows that TJs are highly dynamic structures. Therefore, developing methods following permeability changes between individual cell-cell junctions gives us more insight into the dynamic nature of epithelial permeability. In this master’s thesis, my first goal was to set up an imaging assay for detecting permeability changes between MDCK II cells, called zinc-based ultrasensitive membrane assay (ZnUMBA). This method was originally developed by Stephenson et al. in 2019 for Xenopus laevis embryos and later adapted for MDCK II claudin-2 knockout (KO) cells by Higashi et al. in 2023. In ZnUMBA, medium with zinc ions is added to the one side of the epithelium and medium with zinc indicator is added to the opposite side, and a confocal microscope is used to image the epithelium. In cell culture experiments, the cells were grown on the bottom of a semipermeable cell culture inserts. The whole insert was then placed to an imaging chamber, allowing high resolution imaging of the epithelium with an inverted confocal microscope. When there is a breach in TJ, it is detected as increased intensity at the junction. The second goal of the thesis was to study the epithelial transport and permeability by using the ZnUMBA approach. Overall, the ZO-1 KO cells appeared to have higher baseline junction intensity compared to WT or ZO-2 KO cells. Permeability changes, termed leaks, were detected in WT MDCK, ZO-1 KO and ZO-2 KO cells. The leak duration varied between cell lines, WT cells having the longest median leak duration and ZO-1 KO cells having the shortest. There was small variation in median leak intensities between the cell lines, WT cells having higher leak intensity median, and ZO-1 KO cell the lowest. Calculated leak frequencies for 100 cell-cell junctions for 1-minute period had no statistically significant difference. However, when calculating the percentage of junctions that had a leak from all detected junctions, WT cells again had the highest percentage and ZO-1 KO cells the lowest. The higher baseline junctional intensity in ZO-1 KO cell line affects the leak detection. In addition, it is likely that the leaks are affected by actomyosin dynamics, and as ZO-1 KO cells lack one of the major connections between TJ transmembrane proteins and the actin cytoskeleton, this can cause the lower number of leaks. In addition, the leak duration detected across all cell lines ranged between few seconds to few minutes, suggesting that a fast mechanism for leak repair and detection is present in cells. We speculate that the leaks could be repaired by different mechanisms and that actomyosin dynamics play a major role in the leak formation.