Regulation of cadherin adhesion at intercellular junctions

Adhesion proteins maintain cell-cell interactions, which are critical for tissue formation and the hierarchical organization of all multicellular organisms, and among them, cadherins are the major transmembrane cell-cell adhesion proteins in all vertebrate tissues. Regulation of cadherin mediated adhesion at cell-cell junctions is crucial to our understanding of development and disease. This thesis focuses on the regulation of cadherin adhesion, which can be influenced by its extracellular domain interactions, ligand or antibody binding, post translational modifications, or inside out signaling from cytoplasmic binding proteins. In this thesis, micropipette-based adhesion frequency measurements of cadherin-mediated, cell-cell binding kinetics identified a unique kinetic signature that appears to reflect both adhesive (trans) bonds between cadherins on opposing cells and lateral (cis) interactions between cadherins on the same cell. These kinetic measurements were used to assess the impact of confinement within narrow adhesion zones on the assembly of intercellular adhesions. Specifically, a unique kinetic signature suggested the formation of lateral interactions that were not detected in solution binding assays. Mutations postulated to disrupt lateral cadherin association altered the kinetic signature, but did not affect cadherin binding affinity. Perturbed kinetics further correlated with altered cadherin clustering at cell-cell junctions, wound healing dynamics, and paracellular permeability. Adhesion frequency measurements were used to demonstrate the allosteric regulation of cadherin adhesive function. In this thesis, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that activating anti-E-cadherin monoclonal antibodies or the dephosphorylation of a cytoplasmic binding partner, p120 catenin, increased the homophilic binding affinity of E-cadherin on Colo 205 cells. Further studies of Colo 205 cells demonstrated that four treatments, which similarly altered p120 catenin phosphorylation resulted in quantitatively similar enhancement in E-cadherin affinity. Using this approach, I further investigated the effect of N-linked and O-linked glycosylation on E-cadherin activity and function. Results revealed that, contrary to the influence of glycosylation on N-cadherin function, N-glycosylation of E-cadherin in the EC4 and EC5 domains negatively regulated cadherin adhesion, by altering binding kinetics and clustering at cell-cell junctions. This suggests the influence of N-glycosylation depends on its position in the cadherin ectodomain. In conclusion, this dissertation describes studies which elucidated different mechanisms regulating cadherin adhesive function. Results showed that cadherin binding is regulated by its ectodomain interactions at cell-cell junctions, by glycosylation, and by allosteric inside-out signaling. These findings were enabled by the adhesion frequency measurements, which enabled quantitative assessment of cadherin binding function, in the native context of the cell membrane and cytosolic binding partners

ZO-1 interactions with F-actin and occludin direct epithelial polarization and single lumen specification in 3D culture

Epithelia within tubular organs form and expand lumens. Failure of these processes can result in serious developmental anomalies. Although tight junction assembly is crucial to epithelial polarization, the contribution of specific tight junction proteins to lumenogenesis is undefined. Here, we show that ZO-1 (also known as TJP1) is necessary for the formation of single lumens. Epithelia lacking this tight junction scaffolding protein form cysts with multiple lumens and are defective in the earliest phases of polarization, both in two and three dimensions. Expression of ZO-1 domain-deletion mutants demonstrated that the actin-binding region and U5-GuK domain are crucial to single lumen development. For actin-binding region, but not U5-GuK domain, mutants, this could be overcome by strong polarization cues from the extracellular matrix. Analysis of the U5-GuK binding partners shroom2, α-catenin and occludin showed that only occludin deletion led to multi-lumen cysts. Like ZO-1-deficiency, occludin deletion led to mitotic spindle orientation defects. Single lumen formation required the occludin OCEL domain, which binds to ZO-1. We conclude that ZO-1–occludin interactions regulate multiple phases of epithelial polarization by providing cell-intrinsic signals that are required for single lumen formation

Paneth cell dysfunction in radiation injury and radio-mitigation by human α-defensin 5

IntroductionThe mechanism underlying radiation-induced gut microbiota dysbiosis is undefined. This study examined the effect of radiation on the intestinal Paneth cell α-defensin expression and its impact on microbiota composition and mucosal tissue injury and evaluated the radio-mitigative effect of human α-defensin 5 (HD5).MethodsAdult mice were subjected to total body irradiation, and Paneth cell α-defensin expression was evaluated by measuring α-defensin mRNA by RT-PCR and α-defensin peptide levels by mass spectrometry. Vascular-to-luminal flux of FITC-inulin was measured to evaluate intestinal mucosal permeability and endotoxemia by measuring plasma lipopolysaccharide. HD5 was administered in a liquid diet 24 hours before or after irradiation. Gut microbiota was analyzed by 16S rRNA sequencing. Intestinal epithelial junctions were analyzed by immunofluorescence confocal microscopy and mucosal inflammatory response by cytokine expression. Systemic inflammation was evaluated by measuring plasma cytokine levels.ResultsIonizing radiation reduced the Paneth cell α-defensin expression and depleted α-defensin peptides in the intestinal lumen. α-Defensin down-regulation was associated with the time-dependent alteration of gut microbiota composition, increased gut permeability, and endotoxemia. Administration of human α-defensin 5 (HD5) in the diet 24 hours before irradiation (prophylactic) significantly blocked radiation-induced gut microbiota dysbiosis, disruption of intestinal epithelial tight junction and adherens junction, mucosal barrier dysfunction, and mucosal inflammatory response. HD5, administered 24 hours after irradiation (treatment), reversed radiation-induced microbiota dysbiosis, tight junction and adherens junction disruption, and barrier dysfunction. Furthermore, HD5 treatment also prevents and reverses radiation-induced endotoxemia and systemic inflammation.ConclusionThese data demonstrate that radiation induces Paneth cell dysfunction in the intestine, and HD5 feeding prevents and mitigates radiation-induced intestinal mucosal injury, endotoxemia, and systemic inflammation

Tight junction channel regulation by interclaudin interference

Peer reviewe

Serine 408 phosphorylation is a molecular switch that regulates structure and function of the occludin α-helical bundle

Occludin is a tetramembrane-spanning tight junction protein. The long C-terminal cytoplasmic domain, which represents nearly half of occludin sequence, includes a distal bundle of three α-helices that mediates interactions with other tight junction components. A short unstructured region just proximal to the α-helical bundle is a phosphorylation hotspot within which S408 phosphorylation acts as molecular switch that modifies tight junction protein interactions and barrier function. Here, we used NMR to define the effects of S408 phosphorylation on intramolecular interactions between the unstructured region and the α-helical bundle. S408 pseudophosphorylation affected conformation at hinge sites between the three α-helices. Further studies using paramagnetic relaxation enhancement and microscale thermophoresis indicated that the unstructured region interacts with the α-helical bundle. These interactions between the unstructured domain are enhanced by S408 phosphorylation and allow the unstructured region to obstruct the binding site, thereby reducing affinity of the occludin tail for zonula occludens-1 (ZO-1). Conversely, S408 dephosphorylation attenuates intramolecular interactions, exposes the binding site, and increases the affinity of occludin binding to ZO-1. Consistent with an increase in binding to ZO-1, intravital imaging and fluorescence recovery after photobleaching (FRAP) analyses of transgenic mice demonstrated increased tight junction anchoring of enhanced green fluorescent protein (EGFP)-tagged nonphosphorylatable occludin relative to wild-type EGFP-occludin. Overall, these data define the mechanisms by which S408 phosphorylation modifies occludin tail conformation to regulate tight junction protein interactions and paracellular permeability