Amanda M. Marchiando

Epithelial Barriers in Homeostasis and Disease

Epithelia form barriers that are essential to life. This is particularly true in the intestine, where the epithelial barrier supports nutrient and water transport while preventing microbial contamination of the interstitial tissues. Along with plasma membranes, the intercellular tight junction is the primary cellular determinant of epithelial barrier function. Disruption of tight junction structure, as a result of specific protein mutations or aberrant regulatory signals, can be both a cause and an effect of disease. Recent advances have provided new insights into the extracellular signals and intracellular mediators of tight junction regulation in disease states as well as into the interactions of intestinal barrier function with mucosal immune cells and luminal microbiota. In this review, we discuss the critical roles of the tight junction in health and explore the contributions of barrier dysfunction to disease pathogenesis.

Caveolin-1–dependent occludin endocytosis is required for TNF-induced tight junction regulation in vivo

Although tight junction morphology is not obviously affected by TNF, this proinflammatory cytokine promotes internalization of occludin, resulting in disrupted barrier function within the intestine.

Tight Junction–associated MARVEL Proteins MarvelD3, Tricellulin, and Occludin Have Distinct but Overlapping Functions

This study identifies and characterizes marvelD3, a novel tight junction protein that contains a conserved MARVEL domain. Analyses using phylogenetic, expression profiling, microscopic, and functional approaches show that marvelD3, occludin, and tricellulin are related and have distinct but overlapping functions at the tight junction.

Occludin S408 phosphorylation regulates tight junction protein interactions and barrier function

Occludin S408 phosphorylation regulates interactions between occludin, ZO-1, and select claudins to define tight junction molecular structure and barrier function.

The Epithelial Barrier Is Maintained by In Vivo Tight Junction Expansion During Pathologic Intestinal Epithelial Shedding

BACKGROUND & AIMS Tumor necrosis factor (TNF) increases intestinal epithelial cell shedding and apoptosis, potentially challenging the barrier between the gastrointestinal lumen and internal tissues. We investigated the mechanism of tight junction remodeling and barrier maintenance as well as the roles of cytoskeletal regulatory molecules during TNF-induced shedding. METHODS We studied wild-type and transgenic mice that express the fluorescent-tagged proteins enhanced green fluorescent protein-occludin or monomeric red fluorescent protein 1-ZO-1. After injection of high doses of TNF (7.5 μg intraperitoneally), laparotomies were performed and segments of small intestine were opened to visualize the mucosa by video confocal microscopy. Pharmacologic inhibitors and knockout mice were used to determine the roles of caspase activation, actomyosin, and microtubule remodeling and membrane trafficking in epithelial shedding. RESULTS Changes detected included redistribution of the tight junction proteins ZO-1 and occludin to lateral membranes of shedding cells. These proteins ultimately formed a funnel around the shedding cell that defined the site of barrier preservation. Claudins, E-cadherin, F-actin, myosin II, Rho-associated kinase (ROCK), and myosin light chain kinase (MLCK) were also recruited to lateral membranes. Caspase activity, myosin motor activity, and microtubules were required to initiate shedding, whereas completion of the process required microfilament remodeling and ROCK, MLCK, and dynamin II activities. CONCLUSIONS Maintenance of the epithelial barrier during TNF-induced cell shedding is a complex process that involves integration of microtubules, microfilaments, and membrane traffic to remove apoptotic cells. This process is accompanied by redistribution of apical junctional complex proteins to form intercellular barriers between lateral membranes and maintain mucosal function.

MLCK-dependent exchange and actin binding region-dependent anchoring of ZO-1 regulate tight junction barrier function

The perijunctional actomyosin ring contributes to myosin light chain kinase (MLCK)-dependent tight junction regulation. However, the specific protein interactions involved in this process are unknown. To test the hypothesis that molecular remodeling contributes to barrier regulation, tight junction protein dynamic behavior was assessed by fluorescence recovery after photobleaching (FRAP). MLCK inhibition increased barrier function and stabilized ZO-1 at the tight junction but did not affect claudin-1, occludin, or actin exchange in vitro. Pharmacologic MLCK inhibition also blocked in vivo ZO-1 exchange in wild-type, but not long MLCK−/−, mice. Conversely, ZO-1 exchange was accelerated in transgenic mice expressing constitutively active MLCK. In vitro, ZO-1 lacking the actin binding region (ABR) was not stabilized by MLCK inhibition, either in the presence or absence of endogenous ZO-1. Moreover, the free ABR interfered with full-length ZO-1 exchange and reduced basal barrier function. The free ABR also prevented increases in barrier function following MLCK inhibition in a manner that required endogenous ZO-1 expression. In silico modeling of the FRAP data suggests that tight junction-associated ZO-1 exists in three pools, two of which exchange with cytosolic ZO-1. Transport of the ABR-anchored exchangeable pool is regulated by MLCK. These data demonstrate a critical role for the ZO-1 ABR in barrier function and suggest that MLCK-dependent ZO-1 exchange is essential to this mechanism of barrier regulation.

Dynamic migration of γδ intraepithelial lymphocytes requires occludin

γδ intraepithelial lymphocytes (IELs) are located beneath or between adjacent intestinal epithelial cells and are thought to contribute to homeostasis and disease pathogenesis. Using in vivo microscopy to image jejunal mucosa of GFP γδ T-cell transgenic mice, we discovered that γδ IELs migrate actively within the intraepithelial compartment and into the lamina propria. As a result, each γδ IEL contacts multiple epithelial cells. Occludin is concentrated at sites of γδ IEL/epithelial interaction, where it forms a ring surrounding the γδ IEL. In vitro analyses showed that occludin is expressed by epithelial and γδ T cells and that occludin derived from both cell types contributes to these rings and to γδ IEL migration within epithelial monolayers. In vivo TNF administration, which results in epithelial occludin endocytosis, reduces γδ IEL migration. Further in vivo analyses demonstrated that occludin KO γδ T cells are defective in both initial accumulation and migration within the intraepithelial compartment. These data challenge the paradigm that γδ IELs are stationary in the intestinal epithelium and demonstrate that γδ IELs migrate dynamically to make extensive contacts with epithelial cells. The identification of occludin as an essential factor in γδ IEL migration provides insight into the molecular regulation of γδ IEL/epithelial interactions.

Redistribution of the tight junction protein ZO-1 during physiological shedding of mouse intestinal epithelial cells

We questioned how tight junctions contribute to intestinal barrier function during the cell shedding that is part of physiological cell renewal. Intravital confocal microscopy studied the jejunal villus epithelium of mice expressing a fluorescent zonula occludens 1 (ZO-1) fusion protein. Vital staining also visualized the cell nucleus (Hoechst staining) or local permeability to luminal constituents (Lucifer Yellow; LY). In a cell fated to be shed, ZO-1 redistributes from the tight junction toward the apical and then basolateral cell region. ZO-1 rearrangement occurs 15 ± 6 min (n = 28) before movement of the cell nucleus from the epithelial layer. During cell extrusion, permeation of luminal LY extends along the lateral intercellular spaces of the shedding cell only as far as the location of ZO-1. Within 3 min after detachment from the epithelial layer, nuclear chromatin condenses. After cell loss, a residual patch of ZO-1 remains in the space previously occupied by the departed cell, and the size of the patch shrinks to 14 ± 2% (n = 15) of the original cell space over 20 min. The duration of cell shedding measured by nucleus movement (14 ± 1 min) is much less than the total duration of ZO-1 redistribution at the same sites (45 ± 2 min). In about 15% of cell shedding cases, neighboring epithelial cells also undergo extrusion with a delay of 5-10 min. With the use of normal mice, ZO-1 immunofluorescent staining of fixed tissue confirmed ZO-1 redistribution and the presence of ZO-1 patches beneath shedding cells. Immunostaining also showed that redistribution of ZO-1 occurred without corresponding mixing of apical and basolateral membrane domains as marked by ezrin or E-cadherin. ZO-1 redistribution is the earliest cellular event yet identified as a herald of physiological cell shedding, and redistribution of tight junction function along the lateral plasma membrane sustains epithelial barrier during cell shedding.

No Static at All

Permeability of the intestinal epithelial barrier is regulated in response to physiological and pathophysiological stimuli. Recent work has characterized a critical role of acute tight junction regulation in diarrhea secondary to T cell activation and cytokine release. The intracellular mediators of the ensuing barrier dysfunction include myosin light chain kinase, which phosphorylates myosin II regulatory light chain and triggers structural tight junction reorganization. While the molecular intermediates in this reorganization are not defined, the new discovery that individual tight junction–associated proteins are highly dynamic at steady state may provide insight into the mechanisms of regulation.

396 The ZO-1 Actin Binding Region (ABR) Is Required for Cytoskeletal Tight Junction Regulation

Background & Aims: Microvillus inclusion disease is a rare and fatal congenital enteropathy, presenting with intractable secretory diarrhea shortly after birth. The complete inability to absorb nutrients from intestinal lumen demands total parenteral nutrition, and, eventually, transplantation of the small intestine. MID characteristics varies among patients and generally comprises of villous atrophy and crypt hyperplasia, and, at the cellular level, by the apical brush border atrophy, accumulation of apical proteins, lysosomes and microvilli-like inclusions in the apical cytoplasm of intestinal absorptive cells. Previously we have shown that MID enterocytes display abnormal expression of apical recycling endosomal markers, i.e. Rab11a, FIP-1 (RCP), FIP-5 (Rip11), resulting in a defective apical recycling system in MID. In this study, we aimed to identify the genetic cause and functional consequences that underlie the microvillus inclusion disease. Methods: We screened the genomic DNA of three patients diagnosed with microvillus inclusion disease, their siblings and parents. Biopsies of small intestine from MID and control patients were used to analyze the organization of organelles and localization of proteins involved in intracellular trafficking of brush border proteins. Results: In all MID patients together we have identified two substitutions, one deletion, and two protein truncating mutations in the myosin 5B gene. The MYO5B encodes for an actin filament-binding molecular motor protein that interacts with the small GTPase Rab11a, a marker of recycling endosomes, and thus facilitates the intracellular trafficking of apical proteins towards the apical membrane. We also found aberrant expression and subcellular distribution of myosin Vb protein and other key proteins that interact withmyosin Vb and/or control apical recycling endosome-mediated protein trafficking. Conclusions: The endosomal system that ensures the recycling of brush border proteins, with myosin Vb as a critical regulator, is required to develop and maintain functional apical cell surface in human enterocytes, and perturbations in this can be causally linked to microvillus inclusion disease. Mutations occurring at different positions of MYO5B gene and thus affecting different functional regions of MYO5B protein could explain the diversity of phenotypes present in MID patients. The identification of mutations in MYO5B as the cause for MID brings a major advance in setting the reliable diagnosis, enables the genetic counseling and prenatal screening, as well as paves the way for developing alternative therapeutic strategies.