ReviewConceptual barriers to understanding physical barriers☆
Introduction
Since their discovery in 1998 [1], claudin proteins have become a central focus of tight junction research. It has become clear that expression of members of this large family of tetra-membrane spanning proteins modulates paracellular, i.e. tight junction, permeability to ions and water in a size- and charge-selective manner [2], [3], [4], [5], [6], [7], [8]. Increases in paracellular conductance induced by specific claudins can be defined as either anion- or cation-selective [6], [9], [10], [11], [12], [13]. The conductance pathways that are enhanced by increased expression of pore-forming claudins are size-selective, and appear to only admit solutes and solvents with radii up to ∼3.5 Å [7], [8], [14], [15], [16]. These claudins are often referred to as “pore-forming” claudins. Other claudins have been described as “sealing” claudins [17], [18]. There is some evidence to support the idea that these claudins form paracellular seals, including the severe epidermal barrier defects in claudin-1-deficient mice [19] and the observation that expression of specific claudins reduces transepithelial ion conductance in cultured monolayers [20], [21]. However, while this is a convenient model, it may well be an oversimplification of a far more complex biology. In this review, we will explore the mechanisms by which claudins, other proteins, and lipids form and regulate the tight junction barrier, both at steady-state and in response to exogenous stimuli.
Section snippets
Claudins: tight junction components, organizers, or both?
The initial report that identified claudins showed that claudin-1 and claudin-2 co-localized with occludin by fluorescence microscopy and were present within tight junction strands seen by freeze-fracture electron microscopy [1]. This was rapidly followed by the observation that, when expressed in fibroblasts, which lack tight junctions, claudin proteins concentrated at cell contact sites and induced formation of tight junction like strands [22]. This, along with the beaded appearance of tight
Claudins as paracellular ion channels
Abundant data are available to support the conclusion that claudins form paracellular ion channels. Initial work demonstrated, for example, that the differences between MDCK cell lines characterized by high (MDCK I) and low (MDCK II) transepithelial electrical resistance (TER) were almost entirely explained by expression of claudin-2 in the latter, but not the former [16]. Specifically, claudin-2 expression in high resistance MDCK monolayers resulted in increased paracellular Na+ and K+
Functions of ‘sealing’ claudins
As noted above, expression of some claudins, e.g. claudin-4, can increase transepithelial electrical resistance, i.e. reduce paracellular ion conductance [20]. Thus, claudin-4 expression can enhance paracellular barrier function. It is not clear if this is due to a primary sealing property of claudin-4 or reflects the ability of claudin-4 to disrupt pores formed by other claudins. For example, claudin-4 expression in low resistance MDCK II monolayers, which express claudin-2, reduces
Regulation of paracellular macromolecular flux
In molecular terms, enhanced tight junction permeability to macromolecules, i.e. the leak pathway, has been most closely associated with occludin or ZO-1 knockdown in cultured monolayers in vitro [45], [48], [56], [57], [58]. Stimulus-induced occludin endocytosis causes similar increases in macromolecular paracellular flux, both in vitro [59], [60] and in vivo [61]. These changes are often linked to actomyosin cytoskeletal regulation and can, for example, be triggered by pro-inflammatory
Potential contributions of claudins to paracellular macromolecular flux
TNF has been variably reported to decrease or increase claudin-2 expression in vitro [55], [70], [71]. It is however notable that these studies treated monolayers with TNF or cocktails of IFNγ and TNF for 24–72 h, times which are more than sufficient to allow for compensatory changes in other tight junction proteins. In some cases, high TNF concentrations that also induced apoptosis were used, which further complicates analysis. Conversely, no claudin-2 upregulation was detectable when cultured
How do claudins contribute to tight junction barrier function?
The clearest example of the relationship between claudins and tight junction barrier function is provided by a landmark study of a mouse epithelial line (Eph4) engineered to be deficient in both ZO-1 and ZO-2 [78]. These cells failed to develop tight junctions by several measures. First, they did not traffic claudin-3, and presumably other claudins, as well as occludin and JAM to the tight junction. In addition, these monolayers displayed negligible transepithelial electrical resistance (TER)
Proteins, lipids, or both?
The data discussed above as well as other studies definitively demonstrate that claudins and other tight junction proteins, including the tight junction-associated marvel proteins (TAMPs) occludin, tricellulin, and marvelD3, as well scaffolding proteins, e.g. ZO-1, ZO-2, and cingulin, are important for correct tight junction assembly and function. It is also clear that claudin proteins form channels that mediate flux across the pore pathway, and that occludin, tricellulin, ZO-1, and ZO-2
A cooperative model that integrates tight junction structure and function
We propose a model wherein the interaction between tight junction proteins in combination with membrane lipids organized as inverted micelles results in the formation of a unique lipid–protein structure that allows for both paracellular permeability and barrier-forming properties of the tight junction (Fig. 3). Abundant data, including those presented above, are consistent with a cooperative model in which ZO-1 and ZO-2 recruit claudins to the nascent tight junction and, in turn, claudins begin
Is it time to reconsider membrane fusion as a component of tight junction assembly?
On the basis of the data above and other work it is tempting to revisit the idea that lipid structures are of critical importance to tight junction structure and barrier function [111]. Could the earliest observations interpreted as membrane fusion [97] and lipid-based strands [23], [24], [92], [112] have been correct? If so, one might anticipate that lipids, and even some proteins, could diffuse between cells at these sites. However, several reports showing that a bulky glycolipid, Forssman
A potential explanation for paracellular macromolecular flux
If claudins form channels that mediate flux across the pore pathway, what defines leak pathway flux, and how can this be interpreted in the context of a lipid strand model of the tight junction? One idea, is that breaks within and fusions between fibroblast strands result in transient, focal barrier loss that allows step-by-step flux of solutes through the anastomosing network [105]. This is a reasonable hypothesis that could also be consistent with occasional transfer of claudins between cells
Concluding thoughts
The discussion above is speculative and based on studies spanning 40 years. However, the model presented is consistent with data from morphological, functional, and structural analyses of tight junctions and tight junction proteins. Although the general hypothesis of lipidic tight junction strands is not novel, some of the refinements proposed here are. Nevertheless, it remains an untested model. A formal analysis of these ideas will be difficult without technological advances, including new
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Regulatory roles of claudin-1 in cell adhesion and microvilli formation
2021, Biochemical and Biophysical Research CommunicationsCitation Excerpt :A TJ segregates the apical membrane from the basolateral membrane to maintain cell polarity (fence function), regulates paracellular flux of small molecules at cell-cell contacts (barrier function), and forms a platform of signal transduction on the plasma membrane. Through those functions, TJs play crucial roles in blood-brain barrier, intestinal epithelial barrier, development and differentiation [1,2]. Claudin, a tetraspan transmembrane protein, is a major component of a TJ strand [3,4].
Tight Junction Structure and Function Revisited
2020, Trends in Cell BiologyTime-dependent alteration to the tight junction structure of distal intestinal epithelia in type 2 prediabetic mice
2019, Life SciencesCitation Excerpt :The junctional content of TJ proteins, assessed at the cell-to-cell contact by immunofluorescence, showed a decrease in Cld-1 (in the ileum and colon segments) and Cld-3 (ileum) and occludin (colon) in prediabetic mice. These junctional proteins are well known to interact with each other to form the TJ strands and regulate the epithelial barrier function to ions and molecules [57–59]. The analysis of Cld-2 (that regulates the paracellular permeability to cations/water [57,60]) showed a significant reduction in the total and junctional contents of this protein in the ileum, while an increase in its junctional content was seen in the colon of mice after 60d HFD exposure.
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We thank members of our laboratory whose data and discussion have stimulated development of the hypothesis presented. Work in the authors’ laboratory is supported by the NIDDK (R01DK61931, R01DK68271), The University of Chicago Cancer Center (P30CA14599), and The University of Chicago Institute for Translational Medicine (UL1RR024999).