title: Constitutive activation of Rho proteins by CNF-1 influences tight junction structure and epithelial barrier function.
abstract: The apical-most epithelial intercellular junction, referred to as the tight junction (TJ), regulates paracellular solute flux in diverse physiological and pathological states. TJ affiliations with the apical filamentous actin (F-actin) cytoskeleton are crucial in regulating TJ function. F-actin organization is influenced by the Rho GTPase family, which also controls TJ function. To explore the role of Rho GTPases in regulating TJ structure and function, we utilized Escherichia coli cytotoxic necrotizing factor-1 (CNF-1) as a tool to activate constitutively Rho, Rac and Cdc42 signaling in T84 polarized intestinal epithelial monolayers. The biological effects of the toxin were polarized to the basolateral membrane, and included profound reductions in TJ gate function, accompanied by displacement of the TJ proteins occludin and zonula occludens-1 (ZO-1), and reorganization of junction adhesion molecule-1 (JAM-1) away from the TJ membrane. Immunogold electron microscopy revealed occludin and caveolin-1 internalization in endosomal/caveolar-like structures in CNF-treated cells. Immunofluorescence/confocal microscopy suggested that a pool of internalized occludin went to caveolae, early endosomes and recycling endosomes, but not to late endosomes. This provides a novel mechanism potentially allowing occludin to evade a degradative pathway, perhaps allowing efficient recycling back to the TJ membrane. In contrast to the TJ, the characteristic ring structure of proteins in adherens junctions (AJs) was largely preserved despite CNF-1 treatment. CNF-1 also induced displacement of a TJ-associated pool of phosphorylated myosin light chain (p-MLC), which is normally also linked to the F-actin contractile machinery in epithelial cells. The apical perjunctional F-actin ring itself was maintained even after toxin exposure, but there was a striking effacement of microvillous F-actin and its binding protein, villin, from the same plane. However, basal F-actin stress fibers became prominent and cabled following basolateral CNF-1 treatment, and the focal adhesion protein paxillin was tyrosine phosphorylated. This indicates differences in Rho GTPase-mediated control of distinct F-actin pools in polarized cells. Functionally, CNF-1 profoundly impaired TJ/AJ assembly in calcium switch assays. Re-localization of occludin but not E-cadherin along the lateral membrane during junctional reassembly was severely impaired by the toxin. A balance between activity and quiescence of Rho GTPases appears crucial for both the generation and maintenance of optimal epithelial barrier function. Overactivation of Rho, Rac and Cdc42 with CNF-1 seems to mirror key barrier-function disruptions previously reported for inactivation of RhoA.
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University, Whitehead Biomedical Research Building, Atlanta, GA 30322, USA. ahopkin@emory.edu
authors.forename: Ann M
authors.initials: AM
authors.lastname: Hopkins
authors.forename: Shaun V
authors.initials: SV
authors.lastname: Walsh
authors.forename: Paul
authors.initials: P
authors.lastname: Verkade
authors.forename: Patrice
authors.initials: P
authors.lastname: Boquet
authors.forename: Asma
authors.initials: A
authors.lastname: Nusrat
mesh_terms: D000199:Actins / Q000187:drug effects / Q000378:metabolism; D022005:Adherens Junctions / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D001427:Bacterial Toxins / Q000494:pharmacology*; D015820:Cadherins / Q000187:drug effects / Q000378:metabolism; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism; D051242:Caveolin 1; D022461:Caveolins / Q000187:drug effects / Q000378:metabolism; D015815:Cell Adhesion Molecules; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D003598:Cytoskeletal Proteins / Q000187:drug effects / Q000378:metabolism; D003603:Cytotoxins / Q000494:pharmacology*; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D029968:Escherichia coli Proteins; D006801:Humans; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism; D008840:Microfilament Proteins / Q000187:drug effects / Q000378:metabolism; D008854:Microscopy, Electron; D062793:Occludin; D051419:Paxillin; D010750:Phosphoproteins / Q000187:drug effects / Q000378:metabolism; D011956:Receptors, Cell Surface; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D014407:Tumor Cells, Cultured; D062826:Zonula Occludens-1 Protein; D020764:cdc42 GTP-Binding Protein / Q000187:drug effects / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D000199:Actins; D001427:Bacterial Toxins; C495540:CAV1 protein, human; D015820:Cadherins; D002352:Carrier Proteins; D051242:Caveolin 1; D022461:Caveolins; D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D003603:Cytotoxins; D029968:Escherichia coli Proteins; C407243:F11R protein, human; D008565:Membrane Proteins; D008840:Microfilament Proteins; C568985:OCLN protein, human; D062793:Occludin; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D011956:Receptors, Cell Surface; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; C026615:villin; C060296:cytotoxic necrotizing factor type 1; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins