CRB3 and NF2 orchestrate cytoskeletal dynamics to control epithelial barrier assembly
Shuling Fan, Saranyaraajan Varadarajan, Vicky Garcia-Hernandez, Sven Flemming, Arturo Raya-Sandino, Ben Margolis, Charles A. Parkos, Asma Nusrat
Shuling Fan, Saranyaraajan Varadarajan, Vicky Garcia-Hernandez, Sven Flemming, Arturo Raya-Sandino, Ben Margolis, Charles A. Parkos, Asma Nusrat
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Research Article Cell biology Gastroenterology Inflammation

CRB3 and NF2 orchestrate cytoskeletal dynamics to control epithelial barrier assembly

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.

Authors

Shuling Fan, Saranyaraajan Varadarajan, Vicky Garcia-Hernandez, Sven Flemming, Arturo Raya-Sandino, Ben Margolis, Charles A. Parkos, Asma Nusrat

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Figure 6

PDZ binding motif (PBM) and FERM binding domain (FBD) of CRB3A control assembly of the AJC and perijunctional F-actin ring in IECs.

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PDZ binding motif (PBM) and FERM binding domain (FBD) of CRB3A control a...
(A) Schematic of WT human CRB3A, CRB3A mutant with 3 amino acid point mutations in FBD (mFBD, blue arrowheads), and CRB3A mutant lacking PBM (ΔPBM). Amino acids in the FBD are shown in brown, mutations shown in blue, and the PBM in purple. (B) Left: Immunofluorescent labeling and confocal images of apical junctional protein, ZO-1 (red, during junction assembly in subconfluent colonoids derived from Crb3ERΔIEC mice infected with lentiviral CRB3 WT (pLLV Myc-Crb3a WT), ΔPBM motif (pLLV Myc-Crb3a ΔPBM), and mFBD (pLLV Myc-Crb3a FBD mut) constructs. Scale bar: 25 μm. n = 3 independent experiments, 3 technical replicates each. Right: Graph showing the relative intensity of ZO-1 at cell-cell junctions normalized to the background signal of every junction. Data are mean ± SEM. Each dot represents an independent junction. ****P < 0.0001 by Brown-Forsythe and Welch’s ANOVA test. (C) Costaining of apical junctional protein ZO-1 (red) and Myc-tag (anti-Myc, green) in subconfluent colonoids derived from Crb3ERΔIEC mice expressing plasmids described in Figure 4B. Cells stained positive for Myc (green) represent the Crb3-KO murine colonoids expressing the lentiviral Crb3a constructs described in Figure 4A. White box of the zoomed area shown on the right panel. Scale bars: 25 μm. n = 3 independent experiments, 2 technical replicates each. (D) Costaining of perijunctional F-actin ring (phalloidin, red) and Myc-tag (green) in colonoids derived from Crb3ERΔIEC mice described in Figure 4B. White box highlights the zoomed-in area shown in the right panel. Scale bars: 25 μm. n = 3 independent experiments, 2 technical replicates each.