クライオ電子線トモグラフィーはタイトジャンクションにおける細胞間クローディン-15孔を明らかにする

Tight junctions (TJs) are composed of anastomosing strands between epithelial cells. Members of the claudin family of proteins reside within TJ strands and either seal the paracellular space or assemble into charge and size-selective pathways. Functional studies suggest that claudin-mediated conductance pathways resemble traditional ion channels. However, such postulated pores have not been directly visualized. Using a model claudin deficient epithelium where exogenously introduced EGFP-CLDN15 is the only claudin family member expressed, our investigation sheds light on the arrangement and structure of the postulated claudin pores. Following correlative light and electron microscopical identification of TJs and cryo-electron tomography, we identified series of linearly distributed electron lucent features that locate between two closely apposed plasma membranes of adjacent cells. At these sites, the median spacing between adjacent features is 2.25 nm (IQR = 1.83), with a median 1.66 nm (IQR = 0.92) diameter. In contrast, such features were not observed in claudin deficient model epithelium with exogenous mCherry-ZO-1 expression. These findings agree with the postulated and extensively modeled claudin pores formed within the simple columnar epithelium. This provides the first direct evidence of paracellular pore organization and paves way for future biophysical investigation.

By combining correlative fluorescence imaging, FIB milling, and cryo-ET within an epithelial system restricted to a single claudin isoform, we were able to visualize repetitive, low-density pore features within CLDN15-containing tight junctions (TJs), structures not previously resolved in intact epithelia. These features were absent in claudin-negative controls and displayed placement and geometry consistent with CLDN15 X-ray crystallography and molecular dynamics models. Quantitative measurements of pore diameter, paracellular gap width, and pore spacing further support their assignment as CLDN15 pores. These findings establish a structurally validated platform for defining claudin pore ultrastructure and provide a foundation for future efforts to compare pore-forming and barrier-forming claudins, understand disease-associated junction remodeling, and guide therapeutic modulation of epithelial barrier function.