Cell-cell junctions: the role of actin and viruses in modulating epithelial barrier function

Barriers are critical in the body, and dysregulation of barriers is a hallmark of many diseases, including irritable bowel disease, leaky cancer vasculature, and many viral infections. Epithelial paracellular permeability, what passes between cells, is regulated by the tight junction which is formed by specialized adhesive membrane proteins, adaptor proteins, and the actin cytoskeleton. Despite the tight junction’s critical physiological role, a molecular-level understanding of how tight junction assembly sets the permeability of epithelial tissue is lacking. In chapter 2, we identify a 28-amino acid sequence in the tight junction adaptor protein ZO-1 that is responsible for actin binding and show that this interaction is essential for tight junction barrier function. In contrast to the strong interactions with actin at the adherens junction, we find that the affinity between ZO-1 and actin is surprisingly weak, and we propose a model based on kinetic trapping to explain how affinity could affect tight junction assembly. Finally, by tuning the affinity of ZO-1 to actin, we demonstrate that epithelial monolayers can be engineered with a spectrum of permeabilities, which points to a new target for treating transport disorders, addressing viral breakdown of cell-cell barriers, and improving drug delivery.Viruses are known to dysregulate epithelial barriers by targeting tight junctions. The current SARS-CoV-2, the virus responsible for COVID-19, outbreak has created a devastating global health crisis that has been exacerbated by limited treatment options. Similarly, last year there were 39-56 million flu cases and 24,000-62,000 flu deaths in the USA. In chapter 3, we investigate the potential role of SARS-CoV-2 E protein and the influenza NS1 protein in one major symptom of both COVID-19 and influenza – breakdown of epithelial barrier function. Tight junctions are organized by proteins containing PDZ domains, which bind to PDZ binding motifs (PBM) at the C-terminus of other proteins. To test the importance of the E protein PBM and NS1 protein PBM on epithelial barrier function, we built ectopic expression cell culture models and measured tight junction protein localization and barrier function. We found that the PBM of NS1 from different strains of influenza had a variable effect on barrier function, with most PBMs significantly decreasing barrier function despite variable amino acid sequence. In contrast, we found that the PBM of the E protein of SARS-CoV-2 did not affect barrier function in our cell culture experiments, though expression of the full-length E protein did reduce barrier function in lung epithelial cells. Our work has revealed how actin binding at the tight junction influences barrier function, but there is a lack of molecular tools to modulate actin binding in live cells. In chapter 4, we present the development of a switchable actin binder that binds to actin only when a cell-permeable small molecule is administered. The switch comprises three fused protein domains – an actin-binding motif and two flanking domains that heterodimerize to block actin binding. For the actin-binding motif, we chose the actin binding site (ABS) of ZO-1 and sandwiched it between truncated Bcl-xL and the modified peptide, BH3, whose binding can be disrupted by the small molecule A-1155463. We show that the switchable actin binder colocalizes with actin in the presence of the small molecule and is tunable; with increasing concentration of the small molecule, there is increasing colocalization of the switchable actin binder with actin. To demonstrate functionality of the probe, we engineered ZO-1 with the switchable actin binder and found that inducing actin binding modulated barrier function. As a second demonstration of the switchable actin binder, we found that tethering the actin cortex to the plasma membrane altered macrophage phagocytosis and receptor enrichment. As indicated by these results, a switchable actin binder that controls when, where, and how much actin binding occurs in live cells has the potential to be a useful and versatile tool for investigating the role of actin networks in cells

A Weak Link with Actin Organizes Tight Junctions to Control Epithelial Permeability

In vertebrates, epithelial permeability is regulated by the tight junction (TJ) formed by specialized adhesive membrane proteins, adaptor proteins, and the actin cytoskeleton. Despite the TJ's critical physiological role, a molecular-level understanding of how TJ assembly sets the permeability of epithelial tissue is lacking. Here, we identify a 28-amino-acid sequence in the TJ adaptor protein ZO-1, which is responsible for actin binding, and show that this interaction is essential for TJ permeability. In contrast to the strong interactions at the adherens junction, we find that the affinity between ZO-1 and actin is surprisingly weak, and we propose a model based on kinetic trapping to explain how affinity could affect TJ assembly. Finally, by tuning the affinity of ZO-1 to actin, we demonstrate that epithelial monolayers can be engineered with a spectrum of permeabilities, which points to a promising target for treating transport disorders and improving drug delivery.Y

Azobenzene-based sinusoidal surface topography drives focal adhesion confinement and guides collective migration of epithelial cells

Surface topography is a key parameter in regulating the morphology and behavior of single cells. At multicellular level, coordinated cell displacements drive many biological events such as embryonic morphogenesis. However, the effect of surface topography on collective migration of epithelium has not been studied in detail. Mastering the connection between surface features and collective cellular behaviour is highly important for novel approaches in tissue engineering and repair. Herein, we used photopatterned microtopographies on azobenzene-containing materials and showed that smooth topographical cues with proper period and orientation can efficiently orchestrate cell alignment in growing epithelium. Furthermore, the experimental system allowed us to investigate how the orientation of the topographical features can alter the speed of wound closure in vitro. Our findings indicate that the extracellular microenvironment topography coordinates their focal adhesion distribution and alignment. These topographic cues are able to guide the collective migration of multicellular systems, even when cell–cell junctions are disrupted.publishedVersionPeer reviewe

Human Gut-On-A-Chip Supports Polarized Infection of Coxsackie B1 Virus In Vitro

Analysis of enterovirus infection is difficult in animals because they express different virus receptors than humans, and static cell culture systems do not reproduce the physical complexity of the human intestinal epithelium. Here, using coxsackievirus B1 (CVB1) as a prototype enterovirus strain, we demonstrate that human enterovirus infection, replication and infectious virus production can be analyzed in vitro in a human Gut-on-a-Chip microfluidic device that supports culture of highly differentiated human villus intestinal epithelium under conditions of fluid flow and peristalsis-like motions. When CVB1 was introduced into the epithelium-lined intestinal lumen of the device, virions entered the epithelium, replicated inside the cells producing detectable cytopathic effects (CPEs), and both infectious virions and inflammatory cytokines were released in a polarized manner from the cell apex, as they could be detected in the effluent from the epithelial microchannel. When the virus was introduced via a basal route of infection (by inoculating virus into fluid flowing through a parallel lower ‘vascular’ channel separated from the epithelial channel by a porous membrane), significantly lower viral titers, decreased CPEs, and delayed caspase-3 activation were observed; however, cytokines continued to be secreted apically. The presence of continuous fluid flow through the epithelial lumen also resulted in production of a gradient of CPEs consistent with the flow direction. Thus, the human Gut-on-a-Chip may provide a suitable in vitro model for enteric virus infection and for investigating mechanisms of enterovirus pathogenesis

Electrical simulations and experimental results of 4-electrode AC transepithelial electrical resistance measurements in gut-on-a-chip

Transepithelial electrical resistance (TEER) measurements are often used in organs-on-chips to monitor the barrier tightness of e.g. gut epithelium. Here, we present a chip with four integrated electrodes and a four-terminal alternating current (AC) measurement protocol to perform TEER measurements. The resulting impedance spectra are interpreted using electrical simulations, which include the chip with microfluidic channels, the four electrodes, the AC measurement protocol and the intestinal barrier cultured inside the device, which is modelled as a flat monolayer or as tissue with villi. Eventually, these simulations will be used to quantify TEER in Ohms*cm^2 to enable comparison among different platforms

Cytokine secretion and caspase-3 expression in infected gut chips.

<p>IP-10 (<b>A</b>) and IL-8 (<b>B</b>) were quantified in effluents from the apical (gray bars) and basal (white bars) microchannels in gut chips that were either uninfected or infected apically or basally (n = 4 chips/condition; *p < 0.05, **p < 0.001) at 24 hpi. (<b>C</b>) Epithelium lining gut chips that were uninfected (controls C1 and C2), or infected apically (A1, A2, A3) or basally (B1, B2, B3, B4), or infected apically or basally, were lysed and pro-caspase-3 and cleaved caspase-3 levels were visualized on the gel as indicated.</p

Non-invasive sensing of transepithelial barrier function and tissue differentiation in organs-on-chips using impedance spectroscopy

Here, we describe methods for combining impedance spectroscopy measurements with electrical simulation to reveal transepithelial barrier function and tissue structure of human intestinal epithelium cultured inside an organ-on-chip microfluidic culture device. When performing impedance spectroscopy measurements, electrical simulation enabled normalization of cell layer resistance of epithelium cultured statically in a gut-on-a-chip, which enabled determination of transepithelial electrical resistance (TEER) values that can be compared across device platforms. During culture under dynamic flow, the formation of intestinal villi was accompanied by characteristic changes in impedance spectra both measured experimentally and verified with simulation, and we demonstrate that changes in cell layer capacitance may serve as measures of villi differentiation. This method for combining impedance spectroscopy with simulation can be adapted to better monitor cell layer characteristics within any organ-on-chip in vitro and to enable direct quantitative TEER comparisons between organ-on-chip platforms which should help to advance research on organ function

CVB1 release is polarized towards the epithelial lumen.

<p>(<b>A</b>) Graph showing quantitation of viral loads in the effluents of the apical (epithelial) versus basal (vascular) microchannels after basal infection of the gut chips with CVB1 at an MOI of 0.2 (*p < 0.05 compared to 0–6 hpi). (<b>B</b>) Confocal fluorescence micrographs of apically and basally infected gut chips at 6 and 24 hpi, showing horizontal sections at the base, middle and top of the villi (left to right columns). Infected chips were stained for CVB1 (green) and nuclei (blue); bar, 100 μm.</p

Coxsackie virus B1 readily infects the human Gut-on-a-Chip.

<p>(<b>A</b>) Phase contrast micrographs of the villus epithelium within the Gut-on-a-Chip apically infected with CVB1 at 6, 24 and 48h post infection (hpi); bar, 100 μm. (<b>B</b>) Confocal immunofluorescence micrographs of uninfected and apically infected villus epithelium 24hpi that were stained for CVB1 (green), F-actin (red) and nuclei (blue). Note the destruction of villi in infected samples; bar, 100 μm. (<b>C</b>) Graph showing quantitation of viral loads in the effluents of the apical (epithelial) versus basal (vascular) microchannels after apical infection of the gut chips with CVB1 at an MOI of 0.2 (*p < 0.05 compared to 0–6 hpi).</p

Apical fluid flow generates a gradient of CPE.

<p>Phase contrast micrographs of apically and basally CVB1-infected Gut-on-a-Chips at 24hpi. The flow direction (from the inlet to the outlet) is from left to right (the arrow). Note the increasing CPE intensity towards the downstream outlet; bar, 100 μm.</p