Plectin, a highly versatile cytolinker protein, provides tissues with mechanical stability through the integration of intermediate filaments (IFs) with cell junctions. Here, we hypothesize that plectin-controlled cytoarchitecture is a critical determinant of the intestinal barrier function and homeostasis. Mice lacking plectin in an intestinal epithelial cell (IEC; PleΔIEC) spontaneously developed colitis characterized by extensive detachment of IECs from the basement membrane (BM), increased intestinal permeability, and inflammatory lesions. Moreover, plectin expression was reduced in the colons of ulcerative colitis (UC) patients and negatively correlated with the severity of colitis. Mechanistically, plectin deficiency in IECs led to aberrant keratin filament (KF) network organization and the formation of dysfunctional hemidesmosomes (HDs) and intercellular junctions. In addition, the hemidesmosomal α6β4 integrin (Itg) receptor showed attenuated association with KFs, and protein profiling revealed prominent downregulation of junctional constituents. Consistent with the effects of plectin loss in the intestinal epithelium, plectin-deficient IECs exhibited remarkably reduced mechanical stability and limited adhesion capacity in vitro. Feeding mice with a low-residue liquid diet that reduced mechanical stress and antibiotic treatment successfully mitigated epithelial damage in the PleΔIEC colon.

Leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) is produced in a broad spectrum of mouse embryonic and adult tissues and its deficiency results in embryonal or perinatal lethality. The LGR4 function was mainly related to its potentiation of canonical Wnt signaling; however, several recent studies associate LGR4 with additional signaling pathways. To obtain a suitable tool for studying the signaling properties of Lgr4, we generated a tagged variant of the Lgr4 receptor using gene targeting in the mouse oocyte. The modified Lgr4 allele expresses the Lgr4 protein fused with a triple hemagglutinin (3HA) tag located at the extracellular part of the protein. The allele is fully functional, enabling tracking of Lgr4 expression in the mouse tissues. We also show that via surface labeling, the 3HA tag allows direct isolation and analysis of living Lgr4-positive cells obtained from the small intestinal crypts. Finally, the HA tag-specific antibody can be employed to characterize the biochemical features of Lgr4 and to identify possible biding partners of the protein in cells derived from various mouse tissues.Electronic supplementary materialThe online version of this article (doi:10.1007/s11248-017-0027-0) contains supplementary material, which is available to authorized users.

Plectin, a highly versatile cytolinker protein, provides tissues with mechanical stability through the integration of intermediate filaments (IFs) with cell junctions. Here, we hypothesize that plectin-controlled cytoarchitecture is a critical determinant of the intestinal barrier function and homeostasis. Mice lacking plectin in intestinal epithelial cells (IEC; PleΔIEC) spontaneously developed colitis characterized by extensive detachment of IECs from the basement membrane (BM), increased intestinal permeability, and inflammatory lesions. Moreover, plectin expression was reduced in colons of ulcerative colitis (UC) patients and negatively correlated with the severity of colitis. Mechanistically, plectin deficiency in IECs led to aberrant keratin filament (KF) network organization and formation of dysfunctional hemidesmosomes (HDs) and intercellular junctions. In addition, the hemidesmosomal α6β4 integrin (Itg) receptor showed attenuated association with KFs, and protein profiling revealed prominent downregulation of junctional constituents. Consistent with effects of plectin loss in the intestinal epithelium, plectin-deficient IECs exhibited remarkably reduced mechanical stability and limited adhesion capacity in vitro. Feeding mice with a low-residue liquid diet that reduced mechanical stress and antibiotic treatment successfully mitigated epithelial damage in the PleΔIEC colon.

No abstract