Tissue barriers that restrict passage of liquids, ions, and larger solutes are essential for the development of multicellular organisms. In simple organisms this allows distinct cell types to interface with the external environment. In more complex species, the diversity of cell types capable of forming barriers increases dramatically. Although the plasma membranes of these barrier-forming cells prevent flux of most hydrophilic solutes, the paracellular, or shunt, pathway between cells must also be sealed. This function is accomplished in vertebrates by the zonula occludens, or tight junction. The tight junction barrier is not absolute but is selectively permeable and is able to discriminate between solutes on the basis of size and charge. Many tight junction components have been identified over the past 20 years, and recent progress has provided new insights into the proteins and interactions that regulate structure and function. This review presents these data in a historical context and proposes an integrated model in which dynamic regulation of tight junction protein interactions determines barrier function.

Binding of either ligand or agonistic antibodies to the death receptor CD95 (APO-1/Fas) induces the formation of the death-inducing signaling complex (DISC). We now show that signal initiation of CD95 in type I cells can be further separated into at least four distinct steps. (i) The first step is ligand-induced formation of CD95 microaggregates at the cell surface. (ii) The second step is recruitment of FADD to form a DISC. This step is dependent on actin filaments. (iii) The third step involves formation of large CD95 surface clusters. This event is positively regulated by DISC-generated caspase 8. (iv) The fourth step is internalization of activated CD95 through an endosomal pathway. The latter step is again dependent on the presence of actin filaments. The data indicate that the signal initiation by CD95 is a complex process actively regulated at various levels, providing a number of new drug targets to specifically modulate CD95 signaling.CD95 (APO-1/Fas) is the best-studied member of the death receptor family (26). We previously demonstrated that CD95 oligomerizes upon triggering, forming sodium dodecyl sulfate (SDS)-stable microaggregates on SDS-polyacrylamide gel electrophoresis (PAGE) (11). This activated receptor recruits the adapter molecule FADD and the initiator caspase 8 to form the death-inducing signaling complex (DISC) (11). Recently, Siegel et al. (37) refined this model by showing that unstimulated CD95 exists as preassociated complexes, and they and others (10, 24) confirmed the initial observation of the formation of SDS-stable aggregates by stimulated CD95 (11). In addition, CD95 has been reported to form clusters at the cell surface in a ligand-dependent fashion either late (43) or, in two other reports, very early (6, 9) after receptor triggering. The relationship between or the kinetic order of all these eventspreassociation, formation of SDS-stable microaggregates, formation of the DISC, and the appearance of higher-order receptor clusters, as seen by immunofluorescence microscopy-is unknown.We have previously described two different CD95 apoptosis pathways (32). In type I cells, caspase 8 is recruited to the DISC, resulting in release of active caspase 8 in quantities sufficient to directly activate caspase 3 (40). However, in type II cells, despite similar expression levels of surface CD95 and signaling molecules, formation of the DISC is so inefficient that only very small quantities of caspase 8 are generated at the cell surface. This amount of caspase 8 is insufficient to process caspase 3, but sufficient to cleave the BH3-only protein Bid (13,16,19), resulting in the apoptogenic activation of mitochondria. Therefore, the execution of apoptosis can be inhibited by overexpression of Bcl-2 or Bcl-x L only in type II cells (32). Recently, a number of transgenic and knockout studies have provided evidence for the existence of the two pathways in vivo (14,17,30,41,48,49). In all cases, CD95 apoptosis execution of thymocytes and peripheral T cells was independent of mitochondria, identi...

The tight junction defines epithelial organization. Structurally, the tight junction is comprised of transmembrane and membrane-associated proteins that are thought to assemble into stable complexes to determine function. In this study, we measure tight junction protein dynamics in live confluent Madin–Darby canine kidney monolayers using fluorescence recovery after photobleaching and related methods. Mathematical modeling shows that the majority of claudin-1 (76 ± 5%) is stably localized at the tight junction. In contrast, the majority of occludin (71 ± 3%) diffuses rapidly within the tight junction with a diffusion constant of 0.011 μm2s−1. Zonula occludens-1 molecules are also highly dynamic in this region, but, rather than diffusing within the plane of the membrane, 69 ± 5% exchange between membrane and intracellular pools in an energy-dependent manner. These data demonstrate that the tight junction undergoes constant remodeling and suggest that this dynamic behavior may contribute to tight junction assembly and regulation.

Background & Aims-Inflammatory bowel disease (IBD) is a multifactorial disease thought to be caused by alterations in epithelial function, innate and adaptive immunity, and luminal microbiota. The specific role of epithelial barrier function remains undefined, although increased activity of intestinal epithelial myosin light chain kinase (MLCK), which is the primary mechanism of tumor necrosis factor (TNF)-induced barrier dysfunction, occurs in human IBD. We aimed to determine whether in an intact epithelium, primary dysregulation of the intestinal epithelial barrier by pathophysiologically relevant mechanisms can contribute to development of colitis.

A critical function of the intestinal mucosa is to form a barrier that separates luminal contents from the interstitium. This intestinal barrier is compromised in a number of intestinal diseases, most notably inflammatory bowel disease. In vitro studies have demonstrated that cytokines elaborated by immune cells can cause the mucosal barrier to become leaky; these cytokines are known to be increased in intestinal mucosa involved in inflammatory bowel disease. Detailed information describing the mechanisms by which altered cytokine signaling occurs is not available, but recent data implicate the cytoskeleton within epithelial cells as a critical regulator of the mucosal barrier under physiological and pathophysiological conditions. Using available data, we describe a model of intestinal disease where an initial insult to the epithelial barrier may trigger a selfamplifying cycle of immune activation, cytokine release, and further barrier dysfunction. This model is supported by the observation that pharmacological abrogation of cytokine signaling corrects both barrier defects and clinical disease in animal models and human patients, although such therapy clearly has multiple mechanisms. Other therapeutic targets that represent strategies to prevent or reverse disease processes are also considered. The overarching hypothesis is that modulation of the mucosal epithelial barrier plays a critical role in the initiation and propogation of inflammatory intestinal diseases.