Claudin-4 polymerizes after the incorporation of just two extracellular claudin-3 residues

Tight junctions play a pivotal role in the functional integrity of the human body by forming barriers crucial for tissue compartmentalization and protecting the body from external threats. Essential components of tight junctions are the transmembrane claudin proteins, which can polymerize into tight junction strands and meshworks. This study delves into the structural determinants of claudin polymerization, utilizing the close homology yet strong difference in polymerization capacity between claudin-3 and claudin-4. Through a combination of sequence alignment and structural modeling, critical residues in the second extracellular segment are pinpointed. Molecular dynamics simulations provide insights into the interactions of and the conformational changes induced by the identified extracellular segment 2 residues, shedding light on the intricacies of claudin polymerization. Live-STED imaging demonstrates that introduction of these residues from claudin-3 into claudin-4 significantly enhances polymerization in non-epithelial cells. In tight junction-deficient epithelial cells, mutated claudin-4 not only influences tight junction morphology but also partially restores barrier function. Understanding the structural basis of claudin polymerization is of paramount importance, as it offers insights into the dynamic nature of tight junctions. This knowledge could be applied to targeted therapeutic interventions, offering insight to repair or prevent barrier defects associated with pathological conditions, or introduce temporary barrier openings during drug delivery. ### Competing Interest Statement The authors have declared no competing interest.