Applications of imaging techniques to studies of epithelial tight junctions

Advanced Drug Delivery Reviews 57 (2005) 111 – 121 www.elsevier.com/locate/addr

Applications of imaging techniques to studies of epithelial tight junctions Larry G. Johnson* Cystic Fibrosis Pulmonary Research and Treatment Center and the Departments of Medicine and Pharmacology, School of Medicine, CB# 7248, Rm. 7123A Thurston Bowles Bldg., The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7248, USA Received 19 April 2004; accepted 5 August 2004 Available online 30 September 2004

Abstract The intercellular junctional complex, which consists of the tight junction (TJ), adherens junction, and desmosomes, mediates cell–cell adhesion in epithelia and endothelia. The TJ forms the apical-most portion of this complex in epithelia, serving as a fence to lateral diffusion of apical and basolateral membrane components and as a semi-permeable barrier or gate to the flow of ions and solutes through the paracellular pathway. The TJ consists of a series of integral membrane and cytoplasmic plaque proteins with complex interactions. Included among the TJ proteins are the claudins, which play a major role in mediating the charge and solute selectivity of the junction. Yet, the profile of claudin and associated protein expression differs among epithelia and the function and regulation of many of the TJ proteins remain unknown. This review discusses the application of techniques to discern the function, localization, and regulation of epithelial TJs based on examples from published studies. D 2004 Elsevier B.V. All rights reserved. Keywords: Tight junctions; Epithelia; Immunofluorescence; Confocal microscopy; Permeability

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Introduction. . . . . . . . . . . . . . . . . . . Assessment of TJ function . . . . . . . . . . . 2.1. Assessment of fence function . . . . . . 2.2. Assessment of paracellular gate function 2.2.1. TER and dilution potentials . . 2.2.2. Imaging of ion permeability . . 2.2.3. Imaging of solute permeability. Imaging of tight junctions . . . . . . . . . . . 3.1. Ultrastructural analysis . . . . . . . . .

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* Tel.: +1 919 966 7052; fax: +1 919 966 5178. E-mail address: Larry_ [email protected]. 0169-409X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2004.08.004

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3.2. Fluorescence microscopy. . . . . . . . . 3.3. Dynamic imaging of fluorescent proteins 4. Conclusions . . . . . . . . . . . . . . . . . . . Acknowledgements. . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Epithelia promote organ homeostasis by restricting the flow of ions and solutes between cells across the epithelium. The areas of cell–cell contact in polarized epithelia form the intercellular junctional complex [1– 9]. This complex consists of the tight junction (TJ) or zonula occludens, the adherens junction or zonula adherens, and desmosomes or macular adherens (Fig. 1). The adherens junction and desmosomes, which serve as adhesive junctions imparting mechanical strength to the intercellular junction, are primarily composed of cadherins. E-cadherin, the major cell adhesion molecule in the adherens junction, has single transmembrane domains that form calcium-dependent homophilic interactions with the cadherins of the neighboring cells. The cytoplasmic domains of Ecadherins interact with the catenins, which promote interactions with cytoskeletal and cytoplasmic proteins through protein–protein interactions of PDZ and SH3 domains and phosphorylation. In addition to its

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structural role, h-catenin can be translocated into the nucleus and activate transcription of target genes after binding with a member of TCF/LEF transcription factors family [10]. Desmosomes are located below the adherens junction and are composed of members of the cadherin superfamily of transmembrane proteins, desmogleins and desmocollins, and the cytoplasmic catenin plaque proteins, desmoplakin, plakophilin, and plakoglobin. Interspersed within the intercellular junctional complex are gap junctions, communicating junctions formed by connexins assembled into channels that link the cytoplasm of adjacent cells. The TJ is located at the apical-most portion of the intercellular junction, separates the apical and basolateral compartments of epithelia and plays the key role in limiting paracellular permeability to ions and solutes. Tight junctions appear as multiple strands of fibrils forming a continuous circumferential seal around cells in freeze fracture replicas [11,12]. Four integral membrane proteins have been localized to the tight junction: occludin, claudins, junctional adhesion

Fig. 1. Schematic representation of the intercellular junctional complex.

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molecule (JAM), and the coxsackie and adenoviral serotype 2/5 receptor (CAR) [2,13–17]. JAM and CAR are members of the immunoglobulin gene superfamily containing single transmembrane spanning domains. They not only serve as adhesion molecules, but also as receptors for reovirus and adenovirus [18,19], respectively. Occludin and claudin have four transmembrane spanning domains that interact through a series of homophilic and heterophilic interactions to regulate the permselectivity of the junction. These integral membrane proteins are linked to a series of peripheral cytoplasmic plaque proteins (adaptors) located adjacent to the junction that not only anchor the integral membrane proteins to cytoplasmic and cytoskeletal elements, but also recruit regulatory and signaling molecules to the junction (Fig. 2). Included among these cytoplasmic adaptor proteins are the zonula occludens proteins (ZO-1, ZO-2, and ZO-3), cingulin, MAGI 1–3, PAR 3/6, Pal1, PATJ, MuPP1 and others (reviewed in Refs. [3,20,21]). Occludin has been associated with the regulation of diffusion of small hydrophilic tracers [22] and neutrophil transmigration [23,24], whereas JAM has been associated with mononuclear cell transmigration in vascular endothelium [25,26]. The role of CAR in epithelial TJs remains poorly delineated. Whereas only a few isoforms of JAM, CAR, and occludin exist—two

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splice variants of occludin [23,24], three isoforms of JAM in humans [27], and one isoform of CAR [17,28], more than 23 species of claudins have been identified, each with distinct tissue distributions [7,8,29,30]. This suggests that the profile of claudins within epithelia from different sites may determine the permeability properties of their tight junctions to ions and solutes. Occludin and claudins have both been localized to the TJ strand by immunolabeling of freeze fracture replicas [2,31]. Occludin was initially thought to be the major component of the tight junction fibril [13], but studies in occludin-deficient mice demonstrated the lack of a significant phenotype and normal tight junction fibrils ultrastructurally, suggesting that occludin was not required for the formation of tight junctions [13,32,33]. Accordingly, claudins were assumed to be the major protein constituting the TJ fibril and the predominant molecule regulating TJ permeability. Several studies have established a role for claudins in mediating the charge and solute selectivity of the junction. Over-expression of claudins in MDCK cells has been shown to increase (claudin-2) and to decrease (claudins 4 and 8) cation permeability [34–36]. Expression of molecular chimeras of claudins 2 and 4 and mutations of amino acids adjacent to conserved regions of the first extracellular loop of claudin-15 from acidic to basic residues has reversed the paracellular

Fig. 2. Schematic representation of the tight junction. Depicted are the integral membrane proteins and cytoplasmic proteins, which include adaptors with membrane associated guanylate kinase and PDZ domains, and regulatory and signaling molecules. For illustrative purposes, only a partial list of cytoplasmic proteins has been included (see reviews [1–4,7–9,29,30,77,78]).

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charge selectivity from cationic to anionic [37,38]. The deaths of claudin-1 deficient mice within the first 24 h of life and transgenic mice over-expressing claudin-6 within the first 48 h of life due to dehydration arising from a lack of barrier function against insensible water loss in the epidermis is consistent with regulation of TJ permeability by claudins [39,40]. In humans, naturally occurring mutations in claudin-16, which is expressed in the thick ascending limb of the loop of Henle, leads to an inability to absorb magnesium ions across the paracellular path of the tubules leading to renal Mg2+ wasting and severe hypomagnesemia [41]. Mutations in claudin-14 leading to deafness and in claudin-5 leading to velocardiofacial syndrome have also been reported [42,43]. However, the exact function of these claudins remains elusive, although claudin-14 may play a role in potassium homeostasis and claudin-5 may form polymers that increase solute permeability in airway cells [44,45]. A number of kinases, small GTPases, inflammatory mediators, and pharmacological agents appear to regulate permeability through the paracellular path. Protein kinase A and classical (c) and atypical (a) Protein Kinase C have each been linked to tight junctional assembly and disassembly (reviewed in Ref. [3]). Moreover, aPKCs form a complex with the PAR3/PAR6 signaling complex that localizes near the TJ. Cdc42 binds to PAR6 [46] and is a member of the Rho family of GTPases (Rac, Rho, and Cd42) that are important in the maintenance of epithelial cell polarity through effects on tight junctional and actin cytoskeletal components. Recently, guanine nucleotide exchange factor (GEF-H1/Lfc) has been shown to activate Rho and to regulate TJ permeability in MDCK cells [47]. Alterations in TJ permeability induced by inflammatory mediators have also been linked to pathogenesis of inflammatory bowel disease. Tumor necrosis factor alpha (TNFa), interferon gamma (IFNg), hepatocyte growth factor (HGF), interleukin-4 (IL-4) and IL-13 have each been reported to decrease barrier function in intestinal epithelia (reviewed in Ref. [48]). TNFa and IFNg are elevated in inflammatory bowel disease [49–51] and are also elevated (along with IL-8 and IL1h) in cystic fibrosis airways disease [52–54]. Thus, understanding the effects of inflammation on the TJ in health and disease is important.

Regulating paracellular permeability to enhance drug and peptide delivery has been a long-term interest of the pharmaceutical industry. A variety of agents have been identified that modulate paracellular permeability including cytochalasins, proteases, oxidants, hormones, calcium chelators, surfactants, fatty acids, toxins, and antibodies [55]. Most of these agents are nonspecific and some have significant toxicity. To better understand the consequences of changes in paracellular permeability, a variety of methods have been developed. This review will discuss the application of various techniques for studying the localization and function of epithelial tight junctions based on examples taken largely from airway and renal epithelia. However, the concepts are generally applicable to TJs of gut, skin, and endothelia.

2. Assessment of TJ function TJs perform both fence and gate functions in epithelia. The junctional fence prevents the lateral diffusion of lipids and proteins between the apical and basolateral domains of plasma membrane, whereas the gate restricts paracellular diffusion of ions and solutes in a charge and size selective manner. Agents or peptides that alter paracellular permeability may disturb both gate and fence functions, either singly or in combination [22,55–58]. 2.1. Assessment of fence function To address the issue, Balda et al. [22] developed methods for visualization of lipid diffusion from the apical to basolateral domain. The outer leaflet of control MDCK cells and MDCK cells expressing a cytoplasmic domain deletion mutant of occludin were labeled with the fluorescent lipid BODIPY FL C5sphingomyelin and laser scanning confocal microscopy in the XZ plane was performed. Lateral diffusion of fluorescent lipid was detected in cells expressing the cytoplasmic domain deletion mutant of occludin, but fluorescence was restricted to the apical plasma membrane of control cells. Surprisingly, the ultrastructure of the TJ was preserved by electron microscopy and freeze fracture replica analysis, consistent with a loss of fence, but not gate, function. Quantitative fluorometric analysis of diffusion to the

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basolateral membrane can also be performed by measuring fluorescence intensity of extracts obtained with defatted BSA applied to basolateral membranes and detergent extracts of labeled cells [22]. 2.2. Assessment of paracellular gate function The ion and size selectivity of the TJ gate differs across epithelia and is regulated by different physiological and pathological stimuli. Hence, a detailed analysis of the junctional gate function requires a combination of different types of assays that measure ion and solute permeability. 2.2.1. TER and dilution potentials Transepithelial resistance (TER) has been a frequently used tool for assessment of paracellular permeability. A major weakness of the use of TER to measure paracellular permeability is that it reflects the combined resistance of both cellular and shunt pathways. This measurement may be made with a commercially available hand-held electrometer and chopstick-like electrodes on filter-grown epithelial cells in culture dishes. Alternatively, polarized cultures may be mounted in modified Ussing chambers interfaced to an electrometer permitting continuous recording of transepithelial potential difference (V T) and TER. This latter configuration also permits measurement of the ion selectivity of the TJ under physiologic conditions by the measurement of dilution potentials. The dilution potential (corrected for the contribution of the filter) is determined as the change in V T upon switching the luminal bath from a high NaCl buffer to a low NaCl buffer of the same ionic strength [59]. The permeability of the epithelium to chloride relative to sodium ( P Cl/P Na) can then be calculated [59]. Accurate measurement of ion permeability across the TJ in low resistance epithelia may require blockade of transcellular ion transport across the apical membrane with specific inhibitors, although they may be of little value in high resistance epithelia. In a previous study, we used primary cystic fibrosis human airway epithelial (HAE) cells, which lack an apical membrane CFTR chloride conductance, and blocked luminal epithelial sodium channels (ENaC) with amiloride to overcome this limitation [57]. Although use of CFTR chloride

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channel blockers may be feasible in normal airway epithelia, highly effective blockers are not universally available. 2.2.2. Imaging of ion permeability Imaging of ion fluxes across the TJ enables both determination of the permeability of an individual TJ and analysis of the regulation of that permeability. Fluorescent indicator dyes, e.g. sodiumbinding benzofuran oxazole ammonium salt (SBFO) have been used to measure the ionic permeability of the TJ in MDCK II cells [60,61]. In this method, the lateral intercellular space (LIS) is filled or loaded with a solution containing SBFO. After washing, ratio imaging is performed to measure the rate of change in the LIS sodium concentration when buffer with a high sodium concentration is rapidly switched to one in which sodium has been replaced by lithium. The fluorescence emission intensity ratio with substitution of lithium for sodium reflects the decrease in the concentration of sodium in the LIS and the rate of decline in LIS sodium concentration is a measure of the relative sodium permeability of the TJ, which permits comparison to measurements in the presence of modulators of TJ permeability [60,61]. A similar experimental approach based on fluorescence ratio imaging of LIS may be feasible with other commercially available indicator dyes for cations and anions. As discussed above for electrophysiological methods, specific inhibitors of the transcellular pathways must be employed to limit measurement of ion flow to the paracellular path [35,57]. 2.2.3. Imaging of solute permeability Radiolabeled solutes of varying molecular size have been used to measure solute permeability through the paracellular path. Inulin, mannitol, and dextrans have been the most common radioisotopically labeled solutes used for this purpose. Radioactivity in serial samples from the basolateral bath following luminal application of tracer can be used to calculate unidirectional apparent permeability coefficients ( P app) to both large and small solutes [62]. The assumption here is that transcellular permeability to the tracers is limited, particularly to large solutes, whereas smaller solutes may permeate through cel-

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lular and shunt pathways. Nevertheless, radiotracer studies do not exclude measurement of transcellular flux of solutes. Use of commercially available fluorescent dyelabeled dextrans has improved the ability to not only measure permeability coefficients of paracellular tracers, but when combined with imaging techniques, also permits localization of tracer. Fluorescence of tracers can be measured fluorometrically in samples collected serially over time from the downstream reservoir after luminal application and permeability coefficients ( P app) calculated in a similar manner as performed with radiolabeled tracers [57,58]. To localize flow to either paracellular or transcellular routes, live cell imaging should be performed. In a prior study, Texas Red (TR)-labeled low molecular weight (LMW) and high molecular weight (HMW) dextrans were applied to the luminal bath of polarized primary human airway epithelial (HAE) cells treated with either vehicle or permeability enhancers [58]. No significant flow of label across vehicle-treated HAE cultures was detected by serial laser scanning confocal microscopy over 300 s in the XZ plane, whereas either addition of or luminal pretreatment of epithelial cultures with the sodium salt of the capric acid (C10) increased both paracellular and cellular permeability to HMW and LMW dextrans. Thus, live cell imaging by laser scanning confocal microscopy can be used to determine the relative contribution of transcellular flow to measurements of paracellular solute permeability. Spinning disk confocal microscopes, which have rotating arrays of microlenses and simultaneously rotating pinholes to generate confocality, have also been promoted for live cell imaging due to rapid, real time data acquisition. However, data acquisition in the XZ plane is not a feature of these microscopes [63,64].

3. Imaging of tight junctions 3.1. Ultrastructural analysis Methods for ultrastructural characterization of the tight junction include electron microscopy (EM) and freeze fracture replica analysis. Both of these techniques may be combined with immunolabeling for specific localization of individual protein moieties.

On electron micrographs of ultrathin sections of epithelia, the TJ appears as discrete, punctate sites of close membrane contact, where the outer lipid leaflets of the adjacent plasma membrane appear to fuse with obliteration of the extracellular space [65]. Electron dense tracers, e.g. lanthanum, may be used to assess, in part, the function of the TJ at the ultrastructural level [66]. Development of specific antibodies to components of the TJ has permitted the localization with immunogold labeled secondary antibodies of TJ proteins to the junction, submembranous plaques, and even the nucleus. Freeze fracture techniques have been particularly useful for evaluating the nature of the TJ strand. This technique permits the fracturing of either fixed or unfixed frozen cells along the weak interior hydrophobic plane of cell membranes, which exposes integral transmembrane proteins. Examination under the electron microscope after fracturing reveals en face views of the TJ appearing as a continuous network of interconnected strands on the cytoplasmic leaflet of the plasma membrane or protoplasmic face (P face) and complementary empty grooves on the exoplasmic leaflet (E face) that encircles the apical zone of the lateral membranes of epithelial cells [65]. The combination of freeze fracture with immunogold labeling techniques has been used to localize occludin and claudins to the TJ strand [2,31]. In our laboratory, we have also used freeze fracture to evaluate the effects of inflammatory mediators on TJ strands [57]. 3.2. Fluorescence microscopy Fluorescence microscopy has been widely used to detect cytoplasmic and integral membrane proteins of the tight junction and cytoskeletal elements playing a role in tight junctional homeostasis. Specific fluorophore-conjugated antibodies and fluorophore-conjugated phalloidin, a peptide derived from Amanita phalloides mushroom that binds to polymerized actin filaments, have driven this field. Both wide field and laser scanning confocal microscopy can be potentially used for these studies. Wide field microscopy is limited by blurry images generated as a result of the collection of light from the entire depth of the specimen, rather than collection of light from a single plane of focus as occurs with confocal microscopy [67]. Deconvolution, the application of mathematical algorithms that can be

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used to either deblur an image within a single focal plane or to restore an image based on assumptions about the point spread function (blurring produced by a sub-resolution bead) from an objective, can improve the quality of images captured by widefield microscopy [64,67]. The need for deconvolution algorithms to obtain sharp high resolution images with widefield microscopy has made laser scanning confocal microscopy particularly attractive due to the optical sectioning

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capabilities and the use of pinholes to eliminate out of focus light (reviewed in Refs. [68–70]). Immunofluorescence staining of tight junctions appears as a honeycomb-like pattern when viewed parallel to the plane of the monolayers (XY plane) and as labeled wedges in the apical most portion of the lateral membrane in perpendicular (XZ plane) sections (Fig. 3). However, expression of TJ proteins is not restricted to the apical most portion of the intercellular junction. In polarized HAE cells, ZO-1 is expressed at the apical-most portion

Fig. 3. Localization of tight junction proteins by laser scanning confocal microscopy. (A) Upper panel, localization of ZO-1 (red) and JAM (green) in vehicle-treated polarized, well differentiated human airway epithelial cells. Lower panel, redistribution of ZO-1 and JAM following treatment of cells with TNFa and IFNg (reprinted from Molecular Biology of the Cell ([57]) with permission from the American Society of Cell Biology). (B) Localization of claudins 1, 3, and 7 in sections from excised human bronchus. Frozen sections were stained with commercially available anticlaudin antibodies and texas red secondary antibodies (adapted from Am. J. Physiol. Lung. Cell. Mol. Physiol. [44] with permission from the American Physiological Society).

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of the intercellular junction on XZ images, whereas expression of JAM extends from the apical-most portion to the lateral membrane (Fig. 3A). Similar patterns have evolved for individual claudin species. In excised human bronchus, claudin-1 localizes to the apical-most and lateral membranes of the intercellular junction and around basal cells, whereas claudin-3 is expressed exclusively at the apical-most portion of the junction, and claudin-7 is localized almost exclusively along the lateral membrane (Fig. 3B, [44]). The effects of agents regulating paracellular permeability on localization of TJ proteins can also be assessed. For example, basolateral treatment of polarized HAE cells with tumor necrosis alpha (TNFa) and interferon gamma (INFg) disrupts the localization of ZO-1 and JAM (Fig. 3A, [57,71]). In intestinal epithelia, these cytokines also disrupt claudins 1 and 4 [72]. Hence, confocal microscopy can provide a good indication of whether a protein is associated with tight junctions and the effects of regulatory agents on its localization. Immunofluorescent staining and fluorescence microscopy have traditionally been limited by the sensitivity of the antibodies for detection of proteins expressed at low levels and a lack of antibodies for newly discovered proteins. The application of multiple layering techniques, e.g. immunostaining with primary antibodies, biotinylated secondary antibodies, and avid or streptavidin-conjugated fluorophores, has reduced this limitation. Expression of tagged proteins for which highly sensitive antibodies are commercially available may overcome the lack of antibodies, but mandates proper folding and function of the tagged protein in the relevant cell systems. Immunofluorescence and confocal microscopy have often been applied to the co-localization of a number of integral membrane and cytoplasmic plaque proteins of the TJ (Fig. 3A, [58,71]). However, colocalization by immunofluorescence demonstrates that two proteins are in close proximity, rather than a physical interaction of co-localized proteins. Fluorescence resonance energy transfer (FRET) is a highresolution light microscopy method that may be useful for investigating protein-protein interactions of TJ components (reviewed in Refs. [73,74]). FRET occurs when two fluorophores are close enough together in an orientation that allows coupling between donor and acceptor fluorophores. Under these conditions, excitation of the acceptor occurs as a result of energy transfer

to the donor molecule, leading to a diminution in fluorescence emission by the donor coincident with an increase in fluorescence emission by the acceptor. The efficiency of FRET is inversely proportional to the sixth power of the distance separating the fluorophores making it a sensitive measure of the intermolecular separation. Limitations of FRET include the low efficiency with which hypothetical FRET pairs produce actual FRET in living cells [64,75]. Covalent linkage of donor and acceptor fluorophores may help overcome this limitation [75]. FRET has not been routinely applied to the study of protein–protein interactions relevant to tight junctions to date. 3.3. Dynamic imaging of fluorescent proteins The ability to evaluate function of genetically encoded fluorescent proteins has revolutionized live cell imaging. The spectral variants of green fluorescent (GFP) and red fluorescent protein have made it feasible to perform multicolor imaging of live cells. The field of tight junctions may benefit from the ability to study the spatial relationships of individual tight junctions, their rates of turnover, and the dynamic nature of the junction in living cells. In a recent study, evidence has arisen to support this notion. Matsuda et al. [76] expressed GFP in the amino terminus of claudin-3 in l-fibroblasts and evaluated the effects on claudin kinetics by time lapse microscopy. Surprisingly, the tightly apposed membranes of TJs were endocytosed together into one of the adjoining cells and during internalization, claudins segregated away from occludin, JAM, and ZO-1 to generate claudin-enriched vesicles lacking occludin, JAM, or ZO-1. Thus, GFP labeling of TJ proteins may permit dynamic imaging of regulation of TJ protein expression, kinetics, and fence and gate functions. One caveat to the expression of labeled TJ proteins is that tagged proteins must retain the targeting, folding, and function of the endogenous protein.

4. Conclusions TJs play a crucial role in maintaining epithelial homeostasis in health and disease. A variety of optical techniques, antibodies, and fluorophores are available to image the gate and fence functions of key members

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of the junction. To date, widefield and confocal microscopy have been the most widely utilized imaging techniques complemented by electrophysiological, radioisometric, and biochemical assays. Not only are imaging techniques useful for studies examining the regulation and function of epithelial tight junctions, but they are also necessary for testing the specificity of therapeutic agents that decrease paracellular permeability or alternatively, enhance paracellular permeability for delivery of peptides, proteins, and even gene transfer vectors. Improved knowledge of TJ function and regulation gained from imaging techniques should also advance strategies for targeted disruption of epithelial TJ junctions with minimal toxicity for improved drug delivery. Acknowledgements The author thanks Marguerite C. Applin, Miriam K. Vanhook, and Ashley N. Hardy for their assistance in the preparation of this manuscript. This work was supported by grant HL58342 from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD. References [1] J.M. Anderson, Molecular structure of tight junctions and their role in epithelial transport, News Physiol. Sci. 16 (2001) 126 – 130. [2] M. Furuse, H. Sasaki, S. Tsukita, Manner of interaction of heterogeneous claudin species within and between tight junction strands, J. Cell Biol. 147 (1999) 891 – 903. [3] K. Matter, M.S. Balda, Signalling to and from tight junctions, Nat. Rev., Mol. Cell Biol. 4 (2003) 225 – 236. [4] K. Matter, M.S. Balda, Functional analysis of tight junctions, Methods 30 (2003) 228 – 234. [5] A. Nusrat, J.R. Turner, J.L. Madara, Molecular physiology and pathophysiology of tight junctions: IV. Regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells, Am. J. Physiol.: Gasterointest. Liver Physiol. 279 (2000) G851 – G857. [6] S. Tsukita, M. Furuse, Claudin-based barrier in simple and stratified cellular sheets, Curr. Opin. Cell Biol. 14 (2002) 531 – 536. [7] S. Tsukita, M. Furuse, M. Itoh, Multifunctional strands in tight junctions, Nat. Rev., Mol. Cell Biol. 2 (2001) 285 – 293. [8] S. Tsukita, M. Furuse, Occludin and claudins in tight-junction strands: leading or supporting players? Trends Cell Biol. 9 (1999) 268 – 273.

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