Tight junctions (TJs) are composed of anastomosing strands between epithelial cells which regulate paracellular transport. Members of the claudin family of proteins reside within TJ strands and either seal the paracellular space or assemble into charge and size-selective ion channels. Claudin-15 is a pore-forming claudin that polymerizes to form Na+-selective channels in the intestinal tract. However, high resolution arrangement of claudin ion channels in TJ strands is yet to be observed. Our investigation sheds light on the arrangement, structure, and functional mechanism of claudin-15 pores. We generated EGFP claudin-15 and mCherry-ZO-1 expressing claudin-quintuple knockout (qKO) MDCK cells (Otani et al., JCB 2019) and performed cryo-electron tomography (Cryo-ET) studies to directly visualize TJ strands in situ. Cryo-ET preserves native ultrastructure, enabling us to observe intact claudin-15 pores in TJ strands. 3D tomograms revealed the first in situ visualization of claudin-15 TJ pore ultrastructure and spatial organization, showing a paracellular space of 4.3 ± 0.8 nm with membrane thickness of 4.5 ± 0.7 nm at TJ sites. In mCherry-ZO-1 expressing qKO cells, membranes were still closely apposed, but no pores were observed in the absence of claudin-15. Importantly, alignment of our claudin-15 computational strand model to tomograms validates our previously reported model and provides new structural insights into TJ pore formation and arrangement. This work informs future investigations of claudin strands in native TJ ultrastructure.
Demchenko et al. (Sun,) studied this question.