Spef1 is a microvillar component that limits apical actomyosin contractility and preserves intestinal barrier function.

Epithelial sheet integrity is established by adherent contacts that form between cells at the interface between their apical and basolateral domains. Although cell contacts are reinforced by actomyosin contractility, which generates tension that propagates across the apical surface, how epithelial cells tune tension to reinforce junctions without compromising their physical barrier properties remains unclear. Herein, we report that Sperm Flagellar 1 (Spef1) is a microvillar component enriched in the apical domain and terminal web of enterocytes that prevents actomyosin hypercontractility. The loss of function of Spef1 in Caco-2 BBE cells induced invaginations of the apical domain at tricellular contacts, with a redistribution of the tricellular and tight junction components. These changes were driven by an increase in the NM2A:NM2C heavy chain ratio, which elevated tension throughout the apical surface and ultimately compromised barrier function. These findings highlight Spef1 as a microvillar resident that tunes actomyosin contractility across the apical surface to a level appropriate for junctional reinforcement and maintenance of epithelial function.