Nanoscale segregation of channel and barrier claudins enables paracellular ion flux

The paracellular passage of ions and small molecules across epithelia is controlled by tight junctions
(TJs), complex meshworks of claudin polymers that form tight seals between neighboring cells. How
the nanoscale architecture of TJ meshworks enables paracellular passage of specific ions or small
molecules without compromising barrier function is unknown. Here we combine super-resolution
Stimulated Emission Depletion (STED) microscopy in live and fixed cells and tissues, multivariate
classification of STED images and Fluorescence Resonance Energy Transfer (FRET) to reveal the
nanoscale organization of TJs formed by mammalian claudins. We show that only a subset of
claudins can assemble into characteristic homotypic meshworks, whereas TJs formed by multiple
claudins display nanoscale organization principles of intermixing, integration, induction,
segregation, and exclusion of strand assemblies. Interestingly, channel-forming claudins are spatially
segregated from barrier-forming claudins via determinants mainly encoded in their extracellular
domains that also harbor mutations leading to human diseases. Electrophysiological analysis of
claudins in genome-engineered TJ-free epithelial cells suggests that nanoscale segregation of distinct
channel-forming claudins enables integration of barrier function and specific paracellular ion flux
across TJs via an “ion maze”.