Tight junctions regulate epithelial barrier function and have been shown to be influenced by multiple classes of proteins. Apical integrins have been identified as potential regulators of epithelial barrier function; however, only indirect approaches have been used to measure integrin regulation of the epithelial barrier. Here, we used polymeric nanowires conjugated with anti-integrin β1 antibodies to specifically target apically localized integrins in either their closed or open conformation. Barrier regulation by apical integrins was found to be conformation specific. Nanowires targeting integrins in the closed conformation increased epithelial permeability and caused zonula occludens-1 (ZO-1, also known as TJP1) to change from a linear to a ruffled morphology. Claudin-2 and claudin-4 colocalized with ZO-1 and were also ruffled; however, claudin-1 and claudin-7 remained linear. Ruffling was dependent on myosin light chain kinases (MLCKs) and Rho kinases (ROCKs). Conversely, targeting integrins in the open conformation decreased epithelial permeability and made junctions more linearized. Anti-integrin β1 nanowires differentially affected actin and talin (analyzed using pan-talin antibodies), depending on whether they contained activating or inhibitory antibodies. Thus, apical integrins can act as a conformation-sensitive switch that regulates epithelial barrier function.

Alcohol use disorder (AUD) is a significant risk factor for severe acute respiratory distress syndrome. We found that AUD causes a phenotypic shift in gene expression in human bronchial epithelial cells, enhancing expression of epidermal genes. AUD cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had higher levels of proinflammatory cytokine secretion and barrier dysfunction not present in infected non-AUD cells, consistent with increased early COVID-19 severity due to AUD.

Abstract Background Respiratory syncytial virus (RSV) is a leading viral respiratory pathogen in infants. The objective of this study was to generate RSV live-attenuated vaccine (LAV) candidates by removing the G-protein mucin domains to attenuate viral replication while retaining immunogenicity through deshielding of surface epitopes. Methods Two LAV candidates were generated from recombinant RSV A2-line19F by deletion of the G-protein mucin domains (A2-line19F-G155) or deletion of the G-protein mucin and transmembrane domains (A2-line19F-G155S). Vaccine attenuation was measured in BALB/c mouse lungs by fluorescent focus unit (FFU) assays and real-time polymerase chain reaction (RT-PCR). Immunogenicity was determined by measuring serum binding and neutralizing antibodies in mice following prime/boost on days 28 and 59. Efficacy was determined by measuring RSV lung viral loads on day 4 postchallenge. Results Both LAVs were undetectable in mouse lungs by FFU assay and elicited similar neutralizing antibody titers compared to A2-line19F on days 28 and 59. Following RSV challenge, vaccinated mice showed no detectable RSV in the lungs by FFU assay and a significant reduction in RSV RNA in the lungs by RT-PCR of 560-fold for A2-line19F-G155 and 604-fold for A2-line19F-G155S compared to RSV-challenged, unvaccinated mice. Conclusions Removal of the G-protein mucin domains produced RSV LAV candidates that were highly attenuated with retained immunogenicity.

Tight junctions are protein complexes that form at sites of cell contact that function to regulate paracellular flow of small molecules, water, and ions. Distinct protein components contribute to the barrier function of tight junctions including the claudin family of transmembrane proteins and scaffolding proteins such as Zonula Occludens-1(ZO-1). Changes in tight junction morphology from the established smooth cobblestone patterning to a spiked junction are associated with an increase in barrier permeability (leak). Tight junction spike formation is associated with changes in claudin-18 interacting partners, including ZO-1. This suggests that protein-claudin-18 interactions are essential for tight junction morphology and therefore function. Using rat alveolar epithelial cells (rAECs), we used BioID to investigate the proximal claudin-18 proteome. BioID uses a promiscuous biotinylating enzyme, BirA, which we fused to claudin-18 to biotinylate proximal and interacting proteins. To determine that BirA-claudin-18 localized to tight junctions, rAECs were transduced with adenovirus to express BirA-claudin-18 and stained with claudin-18 and streptavidin-Cy3. This showed that the assay was functional and our chimera trafficked to tight junctions and colocalized to proximal proteins. To identify biotinylated proteins, we used tandem mass spectrometry (MS/MS) and identified 3771 protein matches within the BirA-claudin-18 sample. Proteins of interest were vetted, and a list of protein candidates were compiled based on peptide spectrum matches (PSMs) and known protein localization. Proteins of interest include retinoic acid and palladin, which are of specific interest because they are associated with a spike like structure found in the testis, the tubulobulbar complex, which we hypothesize behaves as a signaling platform in the testis and is comparable to tight junction spikes found in the lung. These findings show that BioID is a valid method for identification of proteins to claudin-18, providing a discovery-based approach to identify novel proteins that play a role in barrier function and tight junction regulation.

Tight junctions between lung alveolar epithelial cells maintain an air-liquid barrier necessary for healthy lung function. Previously, we found that rearrangement of tight junctions from a linear, cortical orientation into perpendicular protrusions (tight junction spikes) is associated with a decrease in alveolar barrier function, especially in alcoholic lung syndrome. Using quantitative super-resolution microscopy, we found that spikes in control cells were enriched for claudin-18 as compared with alcohol-exposed cells. Moreover, using an in situ method to measure barrier function, tight junction spikes were not associated with localized increases in permeability. This suggests that tight junction spikes have a regulatory role as opposed to causing a physical weakening of the epithelial barrier. We found that tight junction spikes form at cell-cell junctions oriented away from pools of β-catenin associated with actin filaments, suggesting that adherens junctions determine the directionality of tight junction spikes. Dynamin-2 was associated with junctional claudin-18 and ZO-1, but showed little localization with β-catenin and tight junction spikes. Treatment with Dynasore decreased the number of tight junction spikes/cell, increased tight junction spike length, and stimulated actin to redistribute to cortical tight junctions. By contrast, Dynole 34-2 and MiTMAB altered β-catenin localization, and reduced tight junction spike length. These data suggest a novel role for dynamin-2 in tight junction spike formation by reorienting junction-associated actin. Moreover, the greater spatial separation of adherens and tight junctions in squamous alveolar epithelial cells as compared with columnar epithelial cells facilitates analysis of molecular regulation of the apical junctional complex.