Occludin knockdown is not sufficient to induce transepithelial macromolecule passage.
Occludin, a tight junction protein, has been reported to regulate barrier function - particularly the leak pathway for larger solutes - in epithelia. Therefore, we aimed to precisely define its role in macromolecule passage at single cell-cell junctions. A combination of varying occludin expression by transient and stable knockdown including systematic seeding strategies was employed to achieve a broad and defined pattern of variance in occludin expression over epithelia. This variance model enabled us to examine occludin function in the leak pathway using global and local analysis, i.e. to analyze macromolecule flux across epithelia and macromolecule passage at single-cell level. Macromolecular flux was found not to correlate with occludin expression in intestinal epithelial cells. In fact, by spatially resolving macromolecular permeation sites using a recently developed method we uncovered leaky cell junctions at the edge of Transwells resulting in increased passage. This demonstrates that rare leaks can determine net flux of macromolecules across epithelia while the vast majority of cellular junctions do not contribute significantly. Hence, concomitant local analysis of macromolecule passage across epithelial barriers is indispensable for interpretation of global flux data. By combining this new approach with cell culture models of the leak pathway, we can present evidence that lack of occludin is not sufficient to stimulate the epithelial leak pathway.
['Richter|Jan F|JF|', 'Hildner|Markus|M|', 'Schmauder|Ralf|R|', 'Turner|Jerrold R|JR|', 'Schumann|Michael|M|', 'Reiche|Juliane|J|']
D018929:Cell Culture Techniques D006801:Humans D046911:Macromolecular Substances D062793:Occludin
occludin macromolecule passage tight junctions Leak pathway epithelial barrier