CRB3 and NF2 orchestrate cytoskeletal dynamics to control epithelial barrier assembly

TL;DR

CRB3 and NF2 proteins help control the structure of intestinal barriers by regulating cell tension and junctions, which is important for maintaining gut health and responding to inflammation.

Contribution

The study reveals a novel CRB3-NF2 signaling pathway that regulates intestinal epithelial barrier function through RhoA/ROCK signaling and actomyosin contractility.

Findings

CRB3 deficiency leads to impaired intestinal barrier function and hypercontractile actomyosin networks.

CRB3 interacts with NF2 to regulate junctional tension and AJC assembly.

ROCK-II or myosin II inhibition can reverse the effects of CRB3 loss on junctional architecture.

Abstract

The gastrointestinal epithelium depends on the apical junctional complex (AJC), composed of tight and adherens junctions, to regulate barrier function. Here, we identify the apical polarity protein Crumbs homolog 3 (CRB3) as an important regulator of AJC assembly and barrier function in intestinal epithelium. Using primary murine colonic epithelial cells (colonoids) from inducible, conditional Crb3-knockout (Crb3ERΔIEC) and control (Crb3fl/fl) mice, we show that CRB3 deficiency compromised barrier function that was associated with a hypercontractile perijunctional actomyosin network and impaired AJC assembly. Loss of CRB3 exacerbated proinflammatory cytokine–induced AJC remodeling, leading to increased intestinal permeability. Crb3ERΔIEC cells exhibited increased RhoA activity and junctional tension, which could be reversed by ROCK-II or myosin II inhibition, restoring junctional architecture. Mechanistically, CRB3A interacts with the actin cytoskeletal linker protein, Merlin (NF2) via its FERM-binding domain, and NF2 knockdown phenocopied CRB3 loss, suggesting their cooperative role in AJC assembly. These findings establish CRB3 and NF2 signaling as key regulators of perijunctional actomyosin contractility and AJC organization during both de novo junctional assembly and inflammation-induced remodeling. This work defines a CRB3- and NF2-dependent pathway by which epithelial cells regulate mechanical tension to coordinate barrier assembly during homeostasis and junctional remodeling under inflammatory stress.

CRB3–NF2 mediated RhoA/ROCK signaling is key regulator of perijunctional actomyosin contractility and intestinal epithelial barrier function during both de novo junction assembly and inflammation induced remodeling.

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