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

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. Loss-of-function in Caco-2 BBE cells showed that Spef1 depletion induced invaginations of the apical domain at tricellular contacts, with a redistribution of the tricellular and tight junction components. These changes, which were paralleled by increased activity of NM2A across the apical surface, compromised intestinal 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.