Tight Junction Tortuosity Reflects the In-plane Apical Constriction in Madin-Darby Canine Kidney Cells

Po-Kai Wang^1*, Keng-hui Lin^1,2,3

¹Institute of Physics, Academia Sinica, Taipei, Taiwan
²Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan
³Genome and Systems Biology Degree Program, National Taiwan University, Taipei, Taiwan

* Presenter:Po-Kai Wang, email:kaicanfly@as.edu.tw

The tight junction outlines the apicolateral border of epithelial cells like a belt, sealing the paracellular space when cells form epithelial sheets. The permeability and morphology of the tight junction are regulated by actomyosin contractility, which has conventionally been attributed to the purse-string-like circumferential actomyosin belt along the tight junction. Spatially, the tight junction is located close to the apical actin network, which may exert inward contractions orthogonal to the tight junction. Given the observation of tortuous tight junctions when MDCK cells are under high tension, we propose that actomyosin networks beneath the apical surface—such as the terminal web—rather than the circumferential actomyosin belt, generate in-plane tension across the apical surface. This tension exerts inward contractions orthogonal to the tight junction, thereby inducing its wavy morphology. To test the contribution of apical constriction in driving tight junction curvature, we perturbed actomyosin activity in polarized Madin-Darby Canine Kidney (MDCK) epithelial cells using the ROCK inhibitor Y-27632, which reduced tight junction tortuosity. Consistent with these findings, laser ablation of specific spots on the apical surface of MDCK cells severed the apical cytoskeletal network, reducing in-plane tension, increasing the apical surface area, and resulting in a less tortuous tight junction morphology. To further investigate this mechanism, we are studying genes that regulate the assembly of the terminal web, with the goal of downregulating these genes to assess their effects on tight junction tortuosity. However, visualizing the apical actin network is challenging due to the dense microvilli covering the surface when using fluorescence-based microscopy. Therefore, cryo-electron tomography (cryo-ET) will be employed to visualize the structure of the terminal web in both wild-type and gene knockout cells, enabling us to explore the relationship between the terminal web, tight junction morphology, and epithelial cell shape.

Keywords: Actomyosin contractility, Tight junction morphology, Apical in-plane tension, Terminal web