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./nViruses 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./nOur 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.

Advisor: Fletcher, Daniel A.