Cryo-electron tomography reveals paracellular claudin-15 pores at the tight junction

Tight junctions (TJs) within simple epithelia are composed of networks of anastomosing strands at the apical end of the intercellular space. 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, postulated pores have not been directly visualized. Using claudin-deficient epithelia in which exogenously introduced EGFP-CLDN15 is the principal claudin family member expressed, our investigation sheds light on the arrangement and structure of the putative claudin pores. Following correlative light and electron microscopy identification of TJs and cryo-electron tomography, we identified a series of linearly distributed electron-lucent features located between two closely apposed plasma membranes of adjacent cells. In contrast, such features were not observed in claudin-deficient epithelia with exogenous mCherry-ZO-1 expression. Morphometric analyses showed the median diameter of these features is 1.5 nm, with a median distance between adjacent features of 2.3 nm. These findings agree with the postulated and extensively modeled claudin pores formed within simple epithelia. This provides the first direct evidence of paracellular claudin pore organization and paves the way for future biophysical investigations.