Force-dependent vinculin binding of α-catenin accelerates adherens junction formation

Adherens junctions (AJs) mediate cell-cell adhesion and mechanical coupling in epithelial tissues. During AJ formation, punctate AJs (punctum adherens; PA) initially appear and subsequently transition into linear AJs or zonula adherens (ZAs). The mechanosensitive interaction of α-catenin with its binding partners-actin filaments and vinculin-is thought to act as a key switch that stabilizes AJs under tension. However, the physiological role of α-catenin's force sensitivity during the early stages of AJ formation remains unclear. Here, we analyzed α-catenin mutants with altered force sensitivity: Insensitive mutant L344P lacking vinculin binding, and Hypersensitive mutant L378P binding vinculin constitutively. Using calcium-switch assays combined with fluorescence and electron microscopy, we found that cells expressing insensitive α-catenin exhibited persistent, elongated PA-like structures corresponding to lateral associations of cellular protrusions from opposing cells, accompanied by delayed ZA formation. In contrast, cells expressing the hypersensitive mutant rapidly formed ZAs, possibly bypassing the PA stage. Similar phenotypes were observed in vinculin-knockout cells, indicating that the defects in Insensitive mutants result from the lack of vinculin recruitment to α-catenin. Based on these findings, we propose a model in which clusters of the cadherin-catenin complex (CCC) along actin filaments on opposing protrusions serve as initial adhesion sites. As protrusions shorten through actomyosin contraction, CCC clusters move toward the protrusion tips along actin filaments, where stretched α-catenin recruits vinculin to reinforce the adhesion, leading to PA formation. Thus, α-catenin's force sensitivity is crucial for smooth and timely AJ assembly, ensuring proper epithelial morphogenesis by coupling intercellular adhesion with cytoskeletal tension.Key words: α-catenin, vinculin, adherens junction, actin filament, force sensitivity.