Review

Dynamics and functions of tight junctions

Emily Steed, Maria S. Balda and Karl Matter
Department of Cell Biology, UCL Institute of Ophthalmology, University College London, Bath Street, London EC1V 9EL, UK

Tight junctions are intercellular adhesion complexes in vertebrates that are required for the formation of functional epithelial and endothelial barriers. Their morphological appearance and biochemical composition, that includes large multimeric protein complexes, have long fostered the belief that they are relatively rigid, nondynamic structures. Recent observations now suggest that at least some junctional elements and proteins can be very dynamic, and that such dynamic properties are important for different tight junction functions ranging from the regulation of paracellular permeability to junction-associated signalling mechanisms that guide cell behaviour. Combining such dynamic properties with existing tight junction models will help us to advance our understanding of the molecular mechanisms that underlie the functional properties of tight junctions. Introduction The tight junction, or zonula occludens, is an intercellular junctional complex found in epithelial and endothelial cells that is responsible for the formation of functional epithelial and endothelial barriers that regulate the passage of cells and solutes through the paracellular space (Figure 1A). In addition to tight junctions, the epithelial junctional complex contains adherens junctions and desmosomes [1]. These junctional complexes completely encircle the cells, resulting in a continuous junctional belt that interconnects neighbouring cells (Figure 1B). In epithelial cells, tight junctions are the most apical component of the junctional complex, whereas their localisation is more variable in endothelia [2,3]. Freeze-fracture electron microscopy reveals tight junctions as a series of anastomising membrane strands that reside at close contact sites between the outer leaets of the neighbouring plasma membranes, effectively occluding the intercellular space between adjacent cells (Figure 2A) (Refs [4,5]). Based upon these early morphological observations and on subsequent functional studies, tight junctions have been classically described either as creating a semi-permeable barrier that is capable of nely regulating the movement of ions, solutes and cells through the paracellular space and that differentiates on the basis of size and charge, or as a fence that participates in the establishment and maintenance of apico-basal polarity. In addition to the identication of multiple tight junction-associated protein complexes, their morphological appearance, that also gave rise to names such as terminal bar, has led to the perception of tight junctions as static, rigid structures in the plasma membrane, reecting their
Corresponding author: Matter, K. (k.matter@ucl.ac.uk).

function to facilitate such apparently passive processes. Indeed, the customary barrier and fence terminology we have come to accept as synonymous with the tight junction reinforces the image of these protein assemblies as solid, robust structures that determinedly prevent the passage of unwanted molecular trafc. Whereas the functions ascribed to tight junctions remain unchanged, emerging data suggests they could be more dynamic structures than has been previously thought. Here we review recently reported data concerning tight junction dynamics and discuss why such dynamic behaviour might be required and how it is achieved. We also begin to consider how current technical limitations might constrain progress towards understanding this aspect of tight junction biology and speculate upon how some of these can be overcome in the future. Structure of tight junctions The prevalent model of tight junction structure is of a stable multiprotein complex composed of integral and peripheral membrane proteins [5,6]. The two major types of integral membrane proteins are classied according to the number of transmembrane domains they contain: fourpass transmembrane proteins such as claudins, occludin and tricellulin, and single-span transmembrane proteins including the junctional adhesion molecule (JAM) and the Coxsackie and adenovirus-associated receptor (CAR) [7]. The identication of claudins as essential components of tight junction strands marked a major breakthrough in our understanding of tight junction structure [8,9]. Lateral association between claudin molecules within the plasma membrane, combined with homotypic adhesive interactions between claudin molecules on adjacent cells, is thought to underlie the characteristic structure of tight junction strands [5,10]. Because claudins are also believed to permit selective diffusion of ions across tight junctions, a speculative model of tight junction organisation emerged in which a series of claudin-based pores enable the passage of ions through an otherwise sealed membrane contact site (Box 1) (Refs [11,12]). However, the actual molecular architecture of such paracellular pores is still unknown. The association of other integral membrane proteins to the claudin-based strands provides additional complexity to tight junction structure. Occludin, the rst transmembrane component of tight junctions to be identied, also localises to the tight junction strands and has been implicated in regulating the permeability properties of tight junctions and, in particular, has been linked to the regulation of size-selective diffusion [1316]. JAMs and related proteins function as adhesion proteins, homotypically as

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0962-8924/$ see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2009.12.002 Available online 12 January 2010

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Figure 1. Tight junctions and the epithelial junctional complex. (a) Schematic drawing of the epithelial junctional complex showing tight junctions as well as the two adhesive epithelial junctions: adherens junctions and desmosomes. The yellow arrows indicate the paracellular route for the diffusion of ions and hydrophilic solutes. Diffusion is not restricted until the level of the tight junction and this represents a regulated, semi-permeable diffusion barrier that is ion- and size-selective. Diffusion across the tight junction is a passive process that occurs along concentration gradients. (b) Immunofluorescence of junctional proteins. The figure shows a cultured human corneal epithelial cell surrounded by neighbouring cells that was stained for the tight junction protein occludin and the adherens junction protein b-catenin. Both proteins exhibit a continuous distribution along the contacts with neighbouring cells, indicating the formation of a junctional belt.

well as heterotypically, and regulate various processes such as leukocyte transmigration [17,18]. Underlying the membrane domain is the cytoplasmic plaque, a meshwork of densely packed peripheral proteins that connect the integral membrane proteins to the underlying actin cytoskeleton as well as to different types of signalling proteins. Prominent examples are the zona occludens proteins ZO-1, -2 and -3: each contains multiple protein-interaction domains, including three PDZ domains and an SH3 domain, through which they demonstrate afnity for a number of cytoskeletal proteins, signalling molecules and membrane proteins [19,20]. Many of these cytosolic adaptor proteins can associate with several other tight junction components and be part of different types of protein complexes [21]. Moreover, tight junction components can also engage in interactions with proteins of adherens junctions (e.g. ZO-1 with a-catenin and afadin/AF6), a type of complex formation that is observed in cells that possess only adherens junctions or, more transiently, during junction assembly [6,2224]. This complex network of protein interactions is thought to be necessary for the correct organisation of the integral membrane components of the junction, the regulation of junction assembly and function, as well as for the transmission of signals to the cell interior [20,25,26]. The need for tight junction dynamics The ultrastructural observations of tight junctions and the biochemical isolation of multimeric, junction-associated

protein complexes might convey an image of a stable plasma membrane structure. Nevertheless, ultrastructural alterations of intact tight junctions associated with paracellular glucose permeability were rst reported almost 20 years ago, suggesting that the tight junction is not an inexible structure [27]. Size-selective paracellular diffusion of such small hydrophilic molecules is likely to involve dynamic properties of tight junctions and the Rho-regulated actinomyosin cytoskeleton [25]. There are numerous other phenomena to consider that make a dynamic structure seem highly appropriate. During leukocyte transmigration, for example, endothelial and epithelial cells maintain their barrier properties while permitting neutrophil passage through the paracellular space. Many situations impact upon tight junctions because cells need to adapt to the behaviour of their neighbours to maintain their barrier function. Examples include the extrusion of cells following apoptosis as well as the movement of cell sheets and intercalation during development [28,29]. In some situations the maintenance of junction integrity is even linked to physical displacement of the tight junction along the lateral membrane, for example when dying cells are extruded from the intestinal epithelium or when maturing sperm migrate through the seminiferous epithelium [30,31]. At a biochemical level, several groups have begun to uncover evidence to suggest that the composition of junction-associated protein complexes is exible and is likely to depend on diverse factors including the state of junction
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Figure 2. Junctional intramembrane strands and paracellular permeability. (a) Visualisation of junctional intramembrane strands. The figure shows a freeze-fracture replica obtained from MDCK cells that were fully confluent. Based on reconstitution experiments in L-cells, claudin-based intramembrane stands could form a dynamic network of strands that promotes the diffusion of small hydrophilic tracers. (b) Model of the paracellular diffusion barrier formed by tight junctions. Tight junctions are shown as grey lines that represent the intramembrane strands. The strands contain ion-selective channels that are thought to be formed by claudins and can be in an open or a closed state, a switch that, at least for some claudins, seems to be regulated. Because claudins have characteristic ion-conductive properties, the types of claudin expressed determines the ion-selectivity of the paracellular pathway. The intramembrane strands are dynamic; hence, the network of strands is reshaping itself continuously, resulting in occasionally open network compartments that allow small solutes to enter and slowly diffuse across the junction in a manner similar to the way boats pass through river locks. The size selectivity is determined by the functional distance of the two neighbouring plasma membranes at the level of tight junctions. Size-selective paracellular diffusion is also regulated by various signalling pathways and could involve interactions with the actin cytoskeleton and additional strand components including occludin, a transmembrane protein known to regulate paracellular permeability. Because occludin is more mobile than claudin-1, strand association of occludin might be one way to regulate paracellular permeability [60]. In addition, because ion-conductivity and size-selective diffusion have been reported to be regulated in opposing directions, it is possible that increased strand dynamics might not be compatible with open ion-conductive pores.

assembly, the extracellular stimulus and the proliferation state [6,32]. It therefore seems likely that the junctionassociated protein complexes are sufciently exible to support a dynamic structure. Tight junction dynamics can be considered at two levels. The rst is the dynamic properties of morphological structures and the junctional membrane, such as the intramembrane strands; the second concerns the dynamic behaviour of individual proteins or classes of proteins. The two levels naturally overlap because morphological alterations are, at least in part, likely to be driven by the dynamic properties of individual junctional constituents. Membrane dynamics The remarkable exibility of tight junctions during developmental processes and the selective diffusion of small hydrophilic molecules suggest that the strand-based junctional membrane structure is dynamic. Paired tight junction strands are formed when individual intramembrane strands from two adjacent cells interact laterally in an association that causes the obliteration of the local intercellular space. How does such a structure adapt to changes
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in cell shape or permit selective diffusion? Whereas electron microscopy allows high-resolution imaging of xed samples, the high density of endogenous tight junction strands and their orientation parallel to the observation axis in standard cell cultures complicates the visualisation of their behaviour in live cells. In an effort to observe paired strands in live cells, Sasaki and colleagues took advantage of transfected L cells; upon expression of claudin cDNAs these cells form adhesive plains containing paired intramembrane strands [10]. Using GFP-tagged claudin-1, they followed the behaviour of the resulting intramembrane strands in real time. They reported 3 kinds of dynamic behaviour: breaking and end-to-end annealing of existing strands, end-to-side interactions resulting in apparently branched strands, and side-to-side interactions [33]. These observations thus suggest that claudin-based intramembrane strands not only have the dynamic properties required for network formation but also for constant remodelling of the network without loosing overall integrity. Such a dynamic network of intramembrane strands could hence provide the structural core of a exible junction that can easily adapt to cell shape changes.

Review
As the authors rightly noted, however, it remains to be conrmed whether in situ tight junction strands share this dynamic behaviour because the presence of GFP at the Ctermini of the claudin molecules could impair interactions between the strands and the submembrane cytoskeleton. Another more serious concern is that native junctions have a much more complex composition than the reconstituted intramembrane strands in L cells, and there are many known links between junctional components and the distinctive junction-associated actin cytoskeleton. The importance of the cellular environment is already illustrated by the observation that the intramembrane strands observed in L cells do not reach the extent of organisation as those observed in epithelial cells with native tight junctions [10]. It is therefore likely that the cytoskeleton and junctional components other than claudins contribute to strand organisation and dynamics. A model of tight junctions based on a exible network of strands would also help us to understand their barrier properties. If such dynamic breaking, resealing, and branching of strands indeed occurs in native junctions, small solutes would be able to pass through the paracellular pathway without a general breakdown of the barrier because the overall integrity of the strand network remains intact. Such a dynamic model could hence explain size-selective paracellular diffusion (Figure 2B). Because the paracellular diffusion pathway is size-selective, the functional distance between the two neighbouring plasma membranes within the junction would be the probable determinant of the upper size limit. Moreover, because this size limit in a native junction is small, this model is not only compatible with the fence function of tight junctions but also explains why the diffusion of lipids is restricted in the outer but not in the inner membrane leaet the hydrophilic head domain of lipids would prevent them from diffusing into the junction. A dynamic strand model would provide a molecular basis for the model of opening and closing of serial barriers as originally proposed on the basis of freeze-fracture and functional permeability data [4,34]. Size-selective paracellular diffusion is a regulated process; hence, the dynamic behaviour of the strands would need to be controlled to explain this regulation. An obvious regulatory mechanism would be to modulate the interactions between membrane proteins within the strands and the cytoplasmic plaque. For example, the C-terminal cytoplasmic domain of occludin, that also associates with intramembrane strands, binds to the submembrane cytoskeleton; expression of occludin mutants lacking this domain stimulates size-selective paracellular permeability [14]. Similarly, modulation of ZO-1 expression, a plaque protein that serves as an interface between the junctional transmembrane proteins and the cytoskeleton, also regulates size-selective paracellular permeability [35,36]. Another possibility is that strand dynamics, and hence diffusion, are regulated by the mechanical force generated by the actin cytoskeleton. Because actin dynamics and myosin-II activity can be regulated by multiple machanisms such as Rho signalling, this could provide a route through which physiological stimuli might regulate paracellular permeability. Indeed, the actinomyosin system and

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Box 1. Tight junctions and the paracellular gate

Tight junctions form a semi-permeable paracellular diffusion barrier, or gate, that restricts the passive diffusion of molecules on the basis of size and charge. Transepithelial electrical resistance is an oftendetermined parameter that measures ion conductance across the epithelial sheet at a given moment in time. The ion conductance of tight junctions varies from tissue to tissue and can be experimentally manipulated by expressing or removing specific claudins [5,11]. It is hence thought that claudins form aqueous pores or channels within tight junctions that allow ion-selective diffusion along concentration gradients. The specificity of these pores is determined by the claudin composition and, more precisely, by the properties of their extracytoplasmic loops, such as electrostatic interaction sites [12,77]. Sizeselective diffusion of small hydrophilic tracers [e.g, small dextrans (4 kDa) and mannitol] also occurs along concentration gradients but is measured over a period of time (i.e. hours). The diffusion of small tracers can be measured in morphologically intact monolayers that have well-developed electrical resistance values and do not permit the paracellular diffusion of larger hydrophilic tracers such as large dextrans [78]. The molecular mechanisms that underlie size-selective paracellular diffusion are unclear. However, several studies reported a functional dissociation between transepithelial electrical resistance and size-selective paracellular diffusion upon specific modifications of either junctional components or signalling pathways (e.g. Rho signalling) that affect permeability [25]. Thus, the molecular bases of ion-selective and size-selective permeation seem to be distinct. Interestingly, under conditions of increased size-selective permeability, ion conductance has often been observed to be reduced [14,36,42]. Hence, activation of the mechanisms that promote sizeselective permeability might not be compatible with open and conductive paracellular ion channels. Under consideration of the dynamic properties of tight junctions discussed here, one could thus speculate that size-selective diffusion relies on dynamic junctional properties, such as moving claudin-based tight junction strands, whereas ion-conductance is mediated by pores formed by claudins within those strands that require more stabile, non-dynamic strands to be active.

upstream regulatory pathways involving RhoROCK and MLCK signalling can drive junction dissociation if stimulated strongly, whereas more subtle changes regulate size-selective diffusion without causing barrier breakdown [25,3747]. The activity of junction proteins and their interactions with each other could be regulated by other mechanisms such as protein phosphorylation that can also regulate permeability [48]. Important aims for the future will be to develop methods to analyze strand dynamics in actual tight junctions and to follow strand dynamics under conditions permitting interaction with the junctional cytoskeleton and with other proteins underlying the junctional membrane and other components of the intramembrane strands. It will be particularly important to determine how strand dynamics are affected by the manipulation of specic signalling pathways that alter permeability. It is also not yet known whether strand movements are active or passive are the strands exible in response to mechanical stress placed upon the cell? Or do strands move unless they are restricted? Protein dynamics Evidence is accumulating for the dynamic behaviour of both cytoplasmic and transmembrane proteins of tight junctions. The recruitment of dual localisation proteins to tight junctions is perhaps one of the most appreciated forms of tight junction dynamics (Figure 3) (Refs [49,50]).
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Figure 3. Dynamics of tight junction proteins. The figure shows a schematic drawing of the main proteins discussed in this review. Dynamic behaviour is represented by coloured arrows corresponding to individually coloured tight junction constituents. Claudin-1 (dark blue) assumes a relatively stable position in the tight junction. The majority of occludin (green) is thought be mobile and capable of lateral diffusion within the plasma membrane. Occludin might also be internalised away from the junctional complex under certain conditions. In addition to associating with different integral membrane proteins of the tight junction strands, a large fraction of ZO-1 (red) has been shown to exchange between distinct membrane and cytosolic pools. Another group of cytoplasmic tight junction-associated proteins includes adaptors and different types of signalling proteins that can be seen to localise in the cytoplasm or nucleus in addition to the tight junction.

These proteins can be found restricted to tight junctions but, under certain conditions, are also found distributed throughout the cytoplasm or in the nucleus. Generally, they seem to serve signalling functions and include junctional adaptor proteins such as ZO-2 and Par-6, the cell cycle kinase CDK4, as well as transcription and mRNA processing factors such as ZONAB, symplekin, and AP-1 (Refs [35,5159]). However, direct observations of how such proteins move in cells have not been performed. It has also recently come to light that the interactions between individual proteins are more dynamic than previously thought, allowing the tight junction to remodel rapidly under steady-state conditions. Shen and colleagues used transfected epithelial cells that form tight junctions to monitor the behaviour of GFP-tagged tight junction proteins by live cell imaging using uorescence recovery after photobleaching (FRAP) as well as photoactivatable GFP probes [60]. In keeping with an earlier report by Sasaki and colleagues in L-cells, the majority of claudin1 was seen to localise stably to the tight junction (Figure 3) (Ref. [33]). In contrast, occludin and ZO-1 demonstrated distinct dynamic behaviours [60]. 80% of occludin was mobile and diffused within the junctional membrane, whereas a large fraction of ZO-1 exchanged between the membrane and cytosolic pools. These dynamic patterns are unlikely to represent diffusion of an occludinZO-1 complex, but suggest that tight junction constituents are subject to a continuous cycle of rapid association and disassociation [60]. If the relative stability of claudin-1 is a feature common to other members of the claudin family, this would support the view that they indeed represent the main constituents and actual backbone of tight junction strands.
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Dynamics of additional strand-associated tight junction proteins might enable the properties of the strands to be ne-tuned, a role that could be played by proteins such as occludin or tricellulin. Tricellulin is enriched at tricellular corners but might possibly have some functional redundancy with occludin because it distributes more evenly along the junction in the absence of occludin or if over-expressed [6163]. Whereas little is known about the dynamic properties of tricellulin, occludin seems to be much more mobile than claudin-1 and diffuses in and out of the junction within the membrane [60]. This dynamic behaviour and the above-described functional properties suggest that occludin dynamics could indeed be a mechanism that regulates paracellular permeability because occludin can be internalised by caveolae and/or clathrin-mediated endocytosis under conditions of enhanced permeability [6466]. Clathrinmediated endocytosis of multiple junctional components including occludin was also observed after complete disruption of intercellular junctions by calcium removal [67]. Hence, intramembrane strands containing occludin might be more stable, or less dynamic, than those without, resulting in increased paracellular permeability when occludin is removed from the junction (Figure 2). Potentially, data from live cell experiments such as those carried out by Shen and colleagues [60] could also be helpful to determine which proteins form stable protein complexes with each other. The interpretation of such data is not trivial, however, because a given junctional protein can be part of a number of apparently distinct protein complexes [21]. Hence, a protein might be part of different complexes with distinct dynamic properties. Biochemical methods will therefore have to be used to complement live cell experiments to determine the frequency and stability of protein complexes. Moreover, mutant proteins that lack particular binding domains can be used to test the functional relevance of these interactions. For example, Shen and colleagues found that the PDZ-binding motif of claudin-1 was not required for immobilisation at the junction, indicating that PDZ domain-mediated interactions between claudin-1 and the junctional plaque are not an important determinant of claudin dynamics at the junction [60]. As with experiments focusing on strand dynamics, published studies focusing on single proteins have limitations. The two central problems are overexpression and possible effects of the GFP tag on protein behaviour. Although GFP-tagged junctional proteins that localise normally are available, it is difcult to rule out such problems entirely. Occludin, for example, has been tagged with GFP at its relatively short N-terminal cytoplasmic domain [60]. A much smaller N-terminal epitope tag, however, had previously been shown to affect the role of occludin in neutrophil transmigration and the junctional alignment of occludin [68]; hence, the N-terminal tag might alter occludins dynamic properties. The N-terminal domain of occludin binds to the ubiquitinprotein ligase itch [69]. Because protein ubiquitination is known to regulate protein trafcking and degradation, one could imagine that N-terminal modications of occludin could

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affect occludin dynamics via itch. Indeed, occludin ubiquitination has recently been shown to induce occludin internalisation and increased paracellular permeability [66,70]. Overexpression is similarly difcult to control. The experimental systems used so far are based on expression of the studied proteins in cells that already express the endogenous protein. Overexpression of junctional proteins such as occludin, claudin-1 and ZO-1 can affect junctional properties as well as localisation, possibly because certain interactions can become saturated [14,15,35,71]. Hence, performing such dynamic experiments in null or knockdown backgrounds might be advantageous. Moreover, biochemical half-life determinations could be used to compare the turnover of transfected and endogenous proteins because enhanced endocytosis due to reduced stabilisation at the junction is likely to increase degradation. Conclusion and perspectives Dynamic properties of tight junctions have now been observed at different levels ranging from the ultrastructural analysis of the junctional membrane and altered distribution of junctional components in response to certain stimuli, to direct observations of dynamic behaviour and properties. Although the recent reports on the dynamic properties of junctional proteins and structures have already provided interesting new insights, the analysis of tight junction dynamics is at an early stage. Fuller understanding will require extension to other junctional proteins and technical innovations to permit visualisation of dynamic processes under more physiological conditions and at higher resolution. One important aspect will be to image a wider spectrum of tight junction proteins. For example, of the claudins, only claudin-1 has been investigated, but different family members might have different dynamic properties just as they have different effects on ion selectivity [11]. It will be equally important to determine how other components of tight junctions, both transmembrane and cytosolic, behave dynamically and how they affect the dynamic properties of junctional structures such as the intramembrane strands. To understand the relevance of such studies in a functional context will require that the constructs and conditions used are also analyzed for their effects on tight junction functions. Perhaps the bigger challenges to overcome are technical. Whereas it is feasible to study proteins at physiological expression levels, visualisation by light microscopy requires uorescent tags and these can interfere with protein function. Hence, it will be important to analyze the functional properties of uorescently tagged proteins carefully to understand imaging experiments. Moreover, it will be important to apply smaller tags that are less likely to interfere with protein function and that can also be used for direct localisation and ultrastructural studies by electron microscopy, such as the tetracysteine tags [72]. However, the imaging of native intramembrane strands within tight junctions still seems to be a long distance away due to the resolution required. This could be achieved in the future if methods that overcome the

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diffraction barrier of the light microscope can be adapted accordingly [73,74]. One of the older and still-debated questions in the tight junction eld concerns the contribution of lipids and nonconventional lipid structures to junction architecture and function [75,76]. Functional studies using different techniques to alter the lipid composition of cells have been performed, but these are difcult to interpret because the lipid environment can have indirect effects on the functional properties of transmembrane proteins as well as direct effects on tight junction structure. However, the combination of live cell imaging experiments with labelled lipids and proteins along with parallel functional and ultrastructural studies could eventually enable us to understand how these components work together to assemble functional tight junctions.
Acknowledgements
E.S. is supported by a studentship from the Medical Research Council. The work in our laboratories is supported by the Biotechnology and Biological Sciences Research Council, the Medical Research Council, the Biomedical Research Centre for Ophthalmology, Fight for Sight, and the Wellcome Trust.

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