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Vol. 16, Issue 9, 3919-3936, September 2005
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Department of Pathology, The University of Chicago, Chicago, IL 60637
Submitted December 20, 2004;
Revised June 1, 2005;
Accepted June 3, 2005
Monitoring Editor: Sandra Schmid
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-actin, occludin, claudin-1, ZO-1, clathrin light chain A1, and caveolin-1 were imaged by time-lapse multidimensional fluorescence microscopy with simultaneous measurement of transepithelial electrical resistance (TER). Actin depolymerization was induced with latrunculin A (LatA). Within minutes of LatA addition TER began to fall. This coincided with occludin redistribution and internalization. In contrast, ZO-1 and claudin-1 redistribution occurred well after maximal TER loss. Occludin internalization and TER loss, but not actin depolymerization, were blocked at 14°C, suggesting that membrane traffic is required for both events. Inhibition of membrane traffic with 0.4 M sucrose also blocked occludin internalization and TER loss. Internalized occludin colocalized with caveolin-1 and dynamin II, but not with clathrin, and internalization was blocked by dominant negative dynamin II (K44A), but not by Eps15
95-295 expression. Inhibition of caveolae-mediated endocytosis by cholesterol extraction prevented both LatA-induced TER loss and occludin internalization. Thus, LatA-induced actin depolymerization causes TJ structural and functional disruption by mechanisms that include caveolae-mediated endocytosis of TJ components. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Colegio et al., 2003), other transmembrane proteins, including occludin and junctional adhesion molecules (Furuse et al., 1993; Martin-Padura et al., 1998), and cytoplasmic plaque proteins such as ZO-1, ZO-2, ZO-3, and cingulin (Stevenson et al., 1986; Citi et al., 1988; Jesaitis and Goodenough, 1994; Haskins et al., 1998). The claudin family of proteins dictates the charge selective permeability of the TJ (Simon et al., 1999; Colegio et al., 2003). In contrast, the specific function of most other TJ protein remains ill-defined. However, studies of cultured cells and genetically modified mice show that additional proteins, including ZO-1 and occludin, are important for proper epithelial function (McCarthy et al., 1996; Chen et al., 1997; Wittchen et al., 1999; Bazzoni et al., 2000; Nusrat et al., 2000a; Ryeom et al., 2000; Saitou et al., 2000; Bruewer et al., 2004; Umeda et al., 2004).
In addition to specific TJ proteins, a ring of filamentous actin encircles each epithelial cell at the level of the apical junctional complex and physically interacts with the TJ (Madara, 1987). The functional dependence of the TJ barrier on this perijunctional actomyosin ring was first recognized 25 years ago when it was shown that pharmacological disruption of this ring disrupts barrier function (Bentzel et al., 1980). This cytoskeletal and TJ disruption is associated with retraction of microfilaments from the plasma membrane, altered cell shape, local accumulations of condensed actin (Meza et al., 1982), disruption of the parallel strands that comprise the TJ as seen by freeze-fracture electron microscopy (Madara et al., 1986), and redistribution of the TJ plaque protein ZO-1 (Madara et al., 1986; Stevenson and Begg, 1994). Because ZO-1 and the transmembrane protein occludin bind directly to actin filaments, most have concluded that the effects of actin depolymerization on TJ structure and function are secondary to loss of these binding interactions with subsequent disruption of molecular TJ structure. However, no data demonstrate this proposed mechanism to be accurate. Moreover, whereas pharmacological actin disruption induces rapid loss of barrier function, the described morphological changes occur more slowly. Thus, we concluded that identification and characterization of specific morphological changes that are temporally correlated with loss of barrier function might provide important new insights into the mechanisms by which this simple model stimulus, actin depolymerization, disrupts TJ function.
We expressed fluorescent fusion proteins of -actin, occludin, claudin-1, ZO-1, clathrin light chain A1, and caveolin-1 in epithelial monolayers. These were imaged using a new apparatus that allows simultaneous measurement of epithelial barrier function and fluorescence microscopy. Although most previously described morphological changes induced by actin depolymerization occurred well after barrier function loss was complete, we noted that vesicular removal of occludin from the TJ correlated well with loss of barrier function. Inhibition of this internalization prevented TJ barrier function loss after actin depolymerization. The data show that actin depolymerization causes TJ disruption by inducing caveolae-mediated endocytosis of TJ components that include occludin.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-cyclodextrin, and water-soluble cholesterol were all from Sigma (St. Louis, MO). LY294002 was from Cell Signaling Technology (Beverly, MA). All other chemicals were of the highest grade available.
Plasmids
The enhanced green fluorescent protein (EGFP)--actin construct was generated by fusing EGFP (BD Biosciences Clontech, Palo Alto, CA) to the amino terminus of human -actin (I.M.A.G.E. clone 769559; American Type Culture Collection, Manassas, VA) and cloning the construct into pcDNA6/TR (Invitrogen, Carlsbad, CA). Monomeric red fluorescent protein 1 (mRFP1), a gift from Roger Y. Tsien and Robert E. Campbell (University of California, San Diego, La Jolla, CA) (Campbell et al., 2002), was cloned into NheI-KpnI sites of pcDNA3.1 zeo (+) (Invitrogen) to form the pmRFP1-C vector. mRFP1-tagged occludin was generated by PCR amplification and cloning of the coding region of human occludin, a gift from Randall J. Mrsny (Cardiff University, Cardiff, United Kingdom) into KpnI-XbaI sites of pmRFP1-C vector in frame with mRFP1. mRFP1-ZO-1 was generated using the coding region of pSK-ZO-1, a kind gift from Alan S. Fanning and James M. Anderson (University of North Carolina, Chapel Hill, NC) by first inserting KpnI sites flanking the coding region and then cloning the coding region into the KpnI site of pmRFP1-C. EGFP-claudin-1 was generated by PCR amplification of coding region of human claudin-1 (I.M.A.G.E. clone 4500534; American Type Culture Collection) and cloning into the KpnI-XbaI sites of pEGFP-C1 (BD Biosciences Clontech). Integrity of the plasmids was verified by restriction digestion and direct sequencing of the expression construct and adjacent regions. Dynamin II-EGFP, dyamin II-K44A-EGFP, EGFP-dyamin I, and EGFP-dyamin I-K44E (Di et al., 2003) were graciously provided by Dr. H. C. Palfrey (The University of Chicago). Caveolin-1-EGFP and enhanced yellow fluorescent protein (EYFP)-clathrin light chain A1 (Massol et al., 2004) were kindly provided by Dr. T. Kirchhausen (Harvard Medical School and the CBR Institute for Biomedical Research, Boston, MA). EGFP-Eps1595-295 (Nichols et al., 2001) was generously provided by Dr. J. Lippincott-Schwartz (National Institutes of Health, Bethesda, MD).
Cell Culture and Transfection
Madin-Darby canine kidney (MDCK) cells (American Type Culture Collection) were grown in DMEM with 1 g/l glucose (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (Invitrogen) and 15 mM HEPES, pH 7.4. For growth as monolayers cells were plated at confluent density, media were replenished at 24 h, and monolayers were studied 48 h after plating. Stable transfectants were generated by transfection with Lipofectamine 2000 (Invitrogen) in 100-mm dishes, as described previously (Zhao et al., 2004). After growth in medium supplemented with 2.5 mg/ml G418 (Mediatech) or 15 µg/ml blasticidin S (Calbiochem, San Diego, CA) for 2 wk, cells were trypsinized and sorted into 96-well plates using a MoFlow cell sorter (DakoCytomation California, Carpinteria, CA). After expansion, clones with readily-detectable TJ fluorescence were selected for further study. Monolayers of stable transfectants were cultured in media with 7.5 mM sodium butyrate to augment transgene expression, from 24 to 48 h after plating and then transferred to media without sodium butyrate for 4 h before use. Transiently-transfected MDCK cells were plated directly onto Transwells and studied 2 d after transfection.
LatA Treatment
Monolayers were equilibrated in Hank's balanced saline (HBSS) with 15 mM HEPES, pH 7.4, for 1 h at 37°C before study. The basolateral media were then exchanged for HBSS containing LatA. For studies at different temperatures, monolayers were equilibrated at indicated temperatures for 30 min (after equilibration in HBSS at 37°C) before LatA treatment. To synchronize vesicle formation and internalization monolayers were cooled to 14°C before and during 30 min of LatA treatment to allow actin depolymerization. The temperature was then raised to 37°C to allow synchronous endocytosis.
Inhibitor Studies
For inhibitor studies, monolayers were transferred to HBSS as described above. Appropriate inhibitors were added bilaterally and monolayers incubated for 1 h at 37°C before basolateral LatA addition. For cholesterol depletion/repletion experiments monolayers were treated with methyl--cyclodextrin (MBCD) for 45 min followed by transfer to media containing MBCD or 1:10 cholesterol–MBCD complexes (Christian et al., 1997) for an additional 45 min. Monolayers were then treated with LatA as described above.
Transepithelial Electrical Resistance (TER) Measurement
TER was measured using electrodes placed on either side of the monolayer. These were attached to a current clamp (University of Iowa Bioengineering, Iowa City, IA) or EVOM (WPI, Saratosa, FL), as described previously (Turner et al., 1997; Wang et al., 2005).
Determination of F-Actin Content
Confluent MDCK monolayers were washed, equilibrated in HBSS for 1 h at 37°C, and transferred to HBSS at indicated temperatures for 30 min. Monolayers were then transferred to HBSS of the same temperature with LatA and fixed in 3% paraformaldehyde in HBSS at indicated times. After washing with HBSS with 50 mM NH4Cl and permeabilization in HBSS containing 3% bovine serum albumin (BSA) and 0.5% saponin monolayers were incubated with 0.5 µM Alexa Fluor 488-conjugated phalloidin and 0.2 µg/ml Hoescht 33342 in HBSS with 3% BSA and 0.5% saponin for 1 h at room temperature. After five washes in HBSS with 3% BSA and 0.5% saponin and one wash with HBSS, fluorescent intensity was measured (Synergy HT; Bio-Tek Instruments, Winooski, VT). Alexa Fluor 488 fluorescence was normalized to Hoechst 33342 fluorescence to correct for cell number.
Immunofluorescence Staining
Monolayers were fixed with 1% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4, with 1 mM CaCl2 for 30 min at room temperature. After three washes in PBS and a 15-min incubation in PBS with 50 mM NH4Cl cells were permeablized in PBS with 3% BSA and 0.05% saponin. Monolayers were then incubated with primary antibody in PBS with 3% BSA and 0.05% saponin overnight at 4°C; washed five times with PBS, 3% BSA, and 0.05% saponin; and incubated with the appropriate Alexa Fluor 350-, 488-, or 594-conjugated secondary antibodies. After five washes monolayers were rinsed in water and mounted in SlowFade (Molecular Probes).
To retain intracellular vesicles, monolayers were briefly washed with ice-cold HBSS and fixed in methanol overnight at –20°C. After fixation monolayers were air-dried, rehydrated with 100 µM bis(sulfosuccinimidyl)suberate (BS3) in PBS with 0.1% n-octyl-glutaraldehyde (PBS+) for 30 min, washed in PBS+, quenched in 100 mM ethylenediamine, pH 7.5, and washed once more in PBS+. Monolayers were then blocked in 1% nonfat dry milk, 1% fish gelatin, and 1% normal donkey serum in PBS+ for 1 h; incubated with primary antibodies for 2 h; washed; and incubated with the appropriate Alexa Fluor 350-, 488-, or 594-conjugated secondary antibodies for 1 h. After five washes, monolayers were rinsed in water and mounted in SlowFade.
Immunoblotting
MDCK monolayers prepared as described above were washed once with ice-cold HBSS and lysed directly in nonreducing SDS-PAGE sample buffer. After boiling at 95°C for 5 min, aliquots were resolved on SDS-PAGE gels (Bio-Rad, Hercules, CA) and transferred to polyvinylidene difluoride membranes. Membranes were blocked in 5% nonfat dry milk in Tris-buffered saline (TBS), washed in TBS with 0.05% Tween 20 (TBS-T), and incubated with primary antibodies in TBS with 1% nonfat dry milk overnight at room temperature. Membranes were washed with TBS-T, incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology), and washed with TBS-T before detection by enhanced chemiluminescence (SuperSignal; Pierce Chemical, Rockford, IL).
Subcellular Fractionation
Monolayers were washed with 37°C HBSS and then scraped into ice-cold HBSS. After recovery by centrifugation at 4°C, cells were resuspended in TBS containing 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.8 mM aprotinin, 0.5 mM bestatin, 15 µM E-64, 20 µM leupeptin, and 10 µM pepstatin A and lysed by 60 passes through a bent 25-gauge needle. Cell disruption was monitored microscopically. Lysates (4 ml) were loaded above 8-ml 10–60% continuous sucrose gradients (in TBS) and centrifuged at 280,000 x g for 18 h at 4°C. Then, 0.5-ml fractions were collected and analyzed for sucrose concentration by refractometry and distribution of specific proteins by SDS-PAGE and immunoblot.
Fluorescence Microscopy with Simultaneous TER Measurement
Monolayers were mounted in a Petri dish in a custom-designed temperature-controlled stage (Brook Industries, Lake Villa, IL). TER was measured using an EVOM and Ag-AgCl electrodes embedded in the Petri dish base and fastened to the side of the microscope objective. Multidimensional imaging was performed by using an epifluorescence microscope (DMLB; Leica Microsystems, Bannockburn, IL), a 63x HCX PL APO L U-V-I aqueous immersion objective, and a 51022 filter set optimized for EGFP/mRFP1 (Chroma Technology, Rockingham, VT). The z-stacks were collected in 0.2–0.5 µm steps using motorized excitation filter wheels, z-motor, and shutter (Ludl, Hawthorne, NY) and a Retiga EXi camera (Q Imaging, Burnaby, British Columbia, Canada) controlled by MetaMorph 6 (Universal Imaging, Downingtown, PA).
For fixed cells, stained monolayers were observed using the same microscope equipped with an 88000 filter set (Chroma Technology) and 63x or 100x PL APO oil immersion objectives. The z-stacks were collected at 0.2-µm intervals. Image stacks were deconvolved using AutoDeblur 9.3 (Autoquant, Watervliet, NY).
Morphometric Analysis
Deconvolved z-stacks were merged, after pseudocolor assignment, using MetaMorph. Vesicles were defined as round or oval structures present in three or more z-planes. The number of vesicles in a single cell was counted over the full height of the cell. Signals were considered to be colocalized if there was 
80% overlap between channels. For each measurement, 20 average-shaped and -sized cells chosen randomly were counted.
Statistical Analysis
All data are presented as average ± SE. All experiments were performed with triplicate or greater samples, and data shown are representative of three or more independent studies. P value was determined by two-tailed Student's t test and was considered to be significant if 
0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) and has been reported to exhibit an unusual biphasic dose dependence: low doses cause mild increases in TER, whereas higher doses cause TER to fall (Stevenson and Begg, 1994; Turner, 2000) (Figure 1A). The mechanism of this biphasic response has not been defined, but it may reflect the multiple mechanisms by which cytochalasins disrupt actin filaments, because cytochalasins cap barbed ends at low doses, whereas at higher concentrations they also sever microfilaments (MacLean-Fletcher and Pollard, 1980; Cooper, 1987; Spector et al., 1989). We therefore compared the effects of cytochalasin D to those of LatA, which causes microfilament depolymerization by a single mechanism, binding to and sequestration of G-actin monomers (Spector et al., 1983; Morton et al., 2000). Like cytochalasin D, LatA caused TER to fall at 0.2 µM (Figure 1A). However, unlike cytochalasin D, low doses of LatA did not cause TER to increase. Because of this simpler monophasic TER response to LatA treatment, LatA was used throughout these studies aimed at defining the mechanisms by which actin depolymerization disrupts TJ structure and function. Kinetic analyses of the effects of LatA on TER and F-actin content showed that LatA reduced F-actin content by 17 ± 0.4% and TER by 17 ± 0.6% within 5 min (Figure 1B). Both F-actin content and TER continued to decrease over time, falling by 42 ± 1.4 and 82 ± 1.1%, respectively, at 30 min. Thus, TER decreases induced by LatA correlated temporally with actin depolymerization.
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Development and Characterization of Fluorescent TJ Fusion Proteins
The data mentioned above demonstrate that traditional immunofluorescence approaches are unsuitable for identification of specific morphological changes that correlate with LatA-induced loss of barrier function. We therefore developed fusion proteins of -actin, occludin, ZO-1, and claudin-1 using two fluorochromes: EGFP and mRFP1 (Campbell et al., 2002). EGFP and mRFP1 fluoresce with high quantum yield at wavelengths that are resolvable from one another, thus allowing their use for double labeling. These fluorescent proteins were linked to the amino termini of -actin and ZO-1 because similar constructs have been shown to be effective (Choidas et al., 1998; Riesen et al., 2002). For claudins, both amino and carboxy-terminus fusions constructs have been reported (Sasaki et al., 2003; Matsuda et al., 2004), but, because the carboxy terminus is required for interaction with PDZ domains, e.g., of ZO-1, the fusion protein was attached to the amino terminus of claudin-1. Similarly, occludin was tagged at the amino terminus, because the carboxy terminus has been reported to be important for interactions with other proteins (Nusrat et al., 2000a; Peng et al., 2003). These fusion proteins were stably expressed in green (EGFP)–red (mRFP1) pairs in MDCK monolayers. Compared with the corresponding endogenous protein, each transfected fusion protein represented no more than one-half of the total of that protein (Figure 2, A–C). Expression of -actin, ZO-1, and claudin-1 fusion proteins did not alter TER of transfected MDCK monolayers (our unpublished data). Expression of occludin increased TER by 21 ± 4%. This effect was similar, but less pronounced, than that reported after expression of chicken occludin in MDCK monolayers (McCarthy et al., 1996), suggesting that the fusion construct is a functional occludin protein. All transfected lines were sensitive to LatA with dose-responses and kinetics similar to nontransfected MDCK monolayers (our unpublished data). Morphological analyses of the distributions of these proteins showed that, in each case, the tagged fusion protein colocalized completely with the endogenous protein (Figure 2, D–F), suggesting that the tagged proteins are correctly targeted. Subcellular fractionation studies provide further evidence for correct targeting of tagged proteins, because both mRFP1-occludin and EGFP-claudin-1 comigrated with endogenous proteins in subcellular fractions in transfected cells and were of the same density as endogenous occludin or claudin-1 in nontransfected cells (Figure 2, G and H). Thus, the fusion proteins do not disrupt TJ assembly, LatA response, or targeting of endogenous TJ proteins and are themselves appropriately targeted, as assessed morphologically and biochemically. Therefore, these fusion proteins are suitable tools for analysis of TJ dynamics in live cells.
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Live Cell Imaging Allows the Identification of Structural Changes That Are Temporally Correlated with LatA-induced Loss of Barrier Function
We analyzed polarized MDCK monolayers expressing fluorescent fusion proteins in an apparatus that allows simultaneous imaging and electrophysiological analysis (Figure 3A). Preliminary experiments showed that both protein localization and TER were generally stable during imaging of these monolayers, although subtle movements of individual TJ proteins within the TJ and minor cell shape changes could be detected (Figure 3B and Supplemental Movie S1). Thus, this apparatus is suitable for real-time analyses of TJ protein distribution and TER.
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-actin and mRFP1-occludin, the latter was uniformly distributed along the TJ before LatA addition. Within 2 min after LatA addition, when TER decreases were first detected, distribution of mRFP1-occludin at the TJ became nonuniform, with alternating zones relatively enriched in or depleted of mRFP1-occludin (Figure 3C and Supplemental Movie S2). This persisted throughout the z-stack and was therefore not due to vertical redistribution or an artifact of imaging. EGFP--actin localization changed similarly and regions with decreased mRFP1-occludin generally also had reduced EGFP--actin fluorescence, suggesting a coordinated redistribution of EGFP--actin and mRFP1-occludin. The small zones enriched in mRFP1-occludin frequently formed small buds and then separated from the TJ in a process that was repeated frequently over the first 15 min after LatA addition (Figure 3D). To further characterize this process of occludin removal from the TJ, imaging was performed at higher rates. These images clearly show that occludin is first concentrated in a small zone within the TJ, followed by local invagination, rounding, and detachment (Figure 3E and Supplemental Movie S3). These data are therefore consistent with endocytic removal of occludin after LatA addition. Analysis of xz reconstructions of fixed monolayers immunostained for occludin, as shown in Figure 3F, demonstrated budding and detached structures near the lower portion of the TJ, i.e., adjacent to the border of the TJ and lateral membrane, in LatA-treated monolayers. Occludin internalization continued as TER fell and was accompanied by progressive increases in numbers of intracellular occludin-containing vesicles. Loss of TER and mRFP1-occludin redistribution were accompanied by EGFP--actin reorganization, including appearance of EGFP--actin aggregates, and changes in cell shape (Figure 3C and Supplemental Movie S2). At later times after LatA addition, residual TJ-associated mRFP1-occludin was only present in dot like structures that also contained EGFP--actin. Thus, endocytic removal from the TJ is the primary morphological change in occludin distribution that correlates with LatA-induced loss of barrier function.
Similar studies were performed using monolayers expressing EGFP--actin and mRFP1-ZO-1 (Figure 4A and Supplemental Movie S4). LatA-induced redistribution of EGFP--actin in these monolayers was similar to that in monolayers expressing EGFP--actin and mRFP1-occludin, thus providing an internal control for comparisons. Although mRFP1-ZO-1 fluorescent intensity did vary along the TJ during first 20 min after LatA addition, this variability was similar to that in control monolayers without LatA addition. As in the fixed immunostained preparations, significant mRFP1-ZO-1 redistribution was only detected >20 min after LatA addition, long after the majority of TER losses had occurred. At these times, mRFP1-ZO-1 was lost from large areas of the TJ. Like mRFP1-occludin, the mRFP1-ZO-1 that remained at the TJ colocalized with the EGFP--actin. However, in contrast to mRFP1-occludin, new intracellular pools of mRFP1-ZO-1 were not identified. Thus, the mRFP1-ZO-1 redistribution detected occurs after the bulk of TER loss and is not associated with occludin internalization.
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-actin and mRFP1-occludin, LatA induced redistribution and internalization of mRFP1-occludin. In contrast, EGFP-claudin-1 was not removed from the TJ during the first 12 minutes after LatA addition. At later time points, the distribution of EGFP-claudin-1 became discontinuous at the TJ. At these times EGFP-claudin-1 at the TJ was present primarily in areas containing mRFP1-occludin. Although claudin internalization has been reported in steady-state epithelial monolayers (Matsuda et al., 2004), in response to calcium depletion (Ivanov et al., 2004), and after cytokine treatment (Wang et al., 2005), increased numbers of EGFP-claudin-1-containing vesicles were not identified after LatA treatment. Moreover, the newly formed mRFP1-occludin intracellular pools did not colocalize with intracellular foci of EGFP-claudin-1 that were present with or without LatA treatment. Thus, it seems that the EGFP-claudin-1 redistribution observed represents a late event that occurs after the bulk of TER loss and is not associated with occludin internalization. Although these data cannot exclude changes in claudin-1 distribution or biochemical interactions that may accompany LatA-induced loss of barrier function, they do show that changes in claudin-1 distribution detectable by fluorescence microscopy do not accompany such TER loss.
One recent study has suggested that immature adherens junctions can be disassembled after latrunculin treatment (Ivanov et al., 2005). To determine whether the loss of TER after LatA treatment could be due to loss of adherens junctions, E-cadherin and occludin were imaged simultaneously in immunostained monolayers. At 20 min after LatA addition, when TER loss was complete, occludin was largely removed from the TJ, whereas the distribution of E-cadherin was unaffected (Figure 4C). Thus, LatA treatment does not cause gross disassembly of adherens junctions in these monolayers under the conditions studied here.
Disruption of Epithelial Barrier Function by LatA Requires Membrane Traffic
The data show that, along with decreased F-actin content, the most prominent early change that accompanies LatA-induced loss of barrier function is the endocytic removal of occludin from the TJ. To test the role of endocytosis in LatA-induced TER loss, we blocked membrane traffic by reducing temperature. Reducing temperature from 32 to 18°C did not affect LatA-induced loss of barrier function. In contrast, LatA-induced TER loss was completely prevented at 14°C (Figure 5A). Similar results were obtained with nontransfected and transfected MDCK monolayers, verifying that this was not an artifact of fusion protein expression. The protective effect was also not due to inhibition of actin depolymerization at 14°C (Figure 5A). This abrupt blockade of LatA-induced TER loss at 14°C suggests that the protective effect is due to inhibition of membrane traffic, rather than effects on protein–protein interactions or enzymatic activity. To determine whether cooling to 14°C also prevented LatA-induced internalization of occludin, we studied monolayers expressing EGFP--actin and mRFP1-occludin. At 14°C, LatA addition did not cause redistribution or endocytosis of mRFP1-occludin (Figure 5B and Supplemental Movie S6). Thus, the LatA-dependent actin depolymerization is dissociated from effects of LatA on TER and occludin internalization at 14°C. These data suggest that endocytosis may be an important step in LatA-induced disruption of TJ barrier function.
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). Incubation of monolayers in media made hypertonic with 0.4 M sucrose caused TER to fall in the absence of LatA. However, no further loss of TER was induced by addition of LatA (Figure 5C), consistent with a requirement for vesicular transport in LatA-induced barrier dysfunction.
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) successfully preserved intracellular occludin vesicles during fixation and subsequent immunostaining. This fixation protocol was used to assess intracellular occludin pools induced by LatA treatment in HBSS at 37 or 14°C or at 37°C in hypertonic buffer (Figure 5D). At 37°C, LatA treatment induced the accumulation of intracellular occludin-containing vesicles in fixed cells in a manner identical to that seen during live cell imaging, with an increase from 28 ± 1.2 to 43 ± 1.6 vesicles per cell (Figure 5, D and E; p < 0.01). In monolayers cooled to 14°C, LatA did not cause an increase in occludin-containing intracellular vesicles (Figure 5, D and E). Similarly, LatA did not cause occludin internalization in monolayers incubated in hypertonic buffer (Figure 5, D and E). Thus, LatA-induced TER loss and occludin internalization were both blocked when membrane traffic was inhibited at 14°C or with hypertonic media. These data show that LatA-induced TJ disruption requires membrane traffic that correlates with occludin internalization.
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; Altschuler et al., 1998; Oh et al., 1998). In control monolayers, 6.8 ± 0.6 vesicles per cell labeled for both occludin and dynamin II. This represented 22 ± 1.4% of the 31 ± 1.7 occludin-containing vesicles present in each cell. LatA treatment increased the number of occludin-containing vesicles that also contained dynamin II to 16 ± 0.9 per cell (p < 0.01), or 36 ± 1.3% of all occludin-containing vesicles (Figure 6, A and B). Dynamin II localization at the TJ also was occasionally seen in LatA-treated cells, but not in the absence of LatA treatment. These data suggest that dynamin II may be involved in the occludin endocytosis that occurs after LatA treatment.
To directly test the role of dynamin II in occludin endocytosis, MDCK cells were transiently transfected with dynamin II-EGFP or dynamin II K44A-EGFP. The latter is a dominant negative dynamin II that lacks GTPase activity and blocks both caveolae- and clathrin-mediated endocytosis (van der Bliek et al., 1993; Oh et al., 1998). In cells expressing dynamin II-EGFP, occludin was delivered to the TJ normally (Figure 6C), and LatA treatment caused the number of intracellular occludin vesicles to increase from 27 ± 2.4 to 45 ± 2.2 per cell (Figure 6D; p < 0.01). Monolayers expressing dynamin II K44A-EGFP had a reduced baseline TER that was 71 ± 3% of those expressing dynamin II-EGFP. Dynamin II K44A-EGFP-expressing cells displayed discontinuous occludin staining at the TJ (Figure 6E), with decreased intensity of TJ occludin staining and increased occludin-containing intracellular vesicles to 48 ± 2.5 per cell in the absence of LatA treatment (Figure 6, D and E). These data suggest that dynamin II may be important for basal delivery and recycling of occludin. When cells expressing dynamin II K44A-EGFP were treated with LatA there was no significant increase in the number of occludin-containing intracellular vesicles (Figure 6, D and E). In contrast, expression of dominant negative dynamin I was unable to prevent occludin internalization (our unpublished observations). Because dominant negative dynamin I inhibits apical but not basolateral endocytic events in polarized epithelia (Altschuler et al., 1998), this observation is consistent with the apparent origin of occludin-containing vesicles from the lower portion of the TJ adjacent to the border with the lateral membrane. Thus, these data demonstrate that LatA-induced occludin endocytosis occurs by a process that requires dynamin II function.
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; Oh et al., 1998). Intracellular occludin has been identified in association with both caveolin-1 (Nusrat et al., 2000b) and clathrin (Ivanov et al., 2004). Thus, although the data show that dynamin II-mediated endocytosis is involved in LatA-induced occludin internalization and TER loss, they do not differentiate clathrin- and caveolae-mediated endocytic pathways. To evaluate the association of clathrin or caveolin with occludin during LatA-induced internalization, monolayers expressing mRFP1-occludin and either EYFP-clathrin light chain A1 or caveolin-1-EGFP were analyzed. mRFP1-occludin internalization was frequently observed in EYFP-clathrin light chain A1-expressing cells, but clathrin light chain A1 never colocalized with mRFP1-occludin during internalization (Figure 7A). In contrast, caveolin-1-EGFP colocalized with mRFP1-occludin in surface membrane invaginations and detaching vesicles in nearly all observed instances of LatA-induced occludin internalization (Figure 7B and Supplemental Movie S7). At later time points, some mRFP1-occludin could be seen to segregate away from caveolin-1-EGFP (Figure 7C and Supplemental Movie S8). Initially, a rim of occludin devoid of caveolin-1 was noticeable. Later, a separate vesicle containing occludin, but not caveolin-1, seemed to detach from the initial vesicle, resulting in two vesicles; one containing mRFP1-occludin and caveolin-1-EGFP and a second containing only mRFP1-occludin (Figure 7C).
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A similar approach was used to evaluate the potential role of clathrin-mediated occludin endocytosis. In contrast to caveolin, LatA induced only a small increase in the number of occludin-containing vesicles positive for clathrin heavy chain, and this number was similar at both 10 and 20 min after warming (Figures 8B and 9). Trafficking of occludin to early and late endosomes also was assessed using EEA1 (Figure 8C) and LAMP-1 (Figure 8D), respectively, as markers of these compartments. In each case, increased labeling of occludin-positive vesicles was only apparent 20 min after warming (Figure 9), at which time colocalization of caveolin-1 and occludin was decreasing. These data suggest that clathrin is not involved in the initial internalization of occludin and that this initial internalization does not involve EEA1-positive early endosomes. The observed separation of occludin from caveolin-1 and small increases in colocalization of occludin with EEA1 and LAMP-1 at later times raises the possibility that, like cholera toxin (Pelkmans et al., 2004), a fraction of occludin internalized through caveolae can be subsequently directed to other endocytic compartments.
LatA-induced Barrier Dysfunction Is Not Prevented by Inhibition of Clathrin-mediated Endocytosis or Macropinocytosis
To functionally define the role of clathrin-mediated endocytosis and macropinocytosis in LatA-induced TER loss, we assessed the effect of inhibitors of these pathways. Macropinocytosis was prevented with amiloride, which inhibits Na+/H+ exchange and blocks macropinocytosis without affecting clathrin-mediated endocytosis (West et al., 1989). LY294002 inhibits phosphoinositide 3-kinase and blocks maintenance, maturation, and translocation of macropinocytic vesicles (Araki et al., 1996; Murray et al., 2000). Neither of these drugs prevented LatA-induced barrier dysfunction (Figure 10A). Thus, although each of these drugs has additional effects unrelated to macropinocytosis, the failure of both to block LatA-induced barrier loss suggests that macropinocytosis does not play an important role in this process.
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). Although chlorpromazine increased TER to 175 ± 6.6% of control monolayers in the absence of LatA, it did not prevent LatA-induced TER loss (Figure 10B), suggesting that clathrin-mediated endocytosis is not involved in LatA-induced TJ disruption. Moreover, specific inhibition of clathrin-mediated endocytosis by expression of EGFP-Eps1595-295 (Benmerah et al., 1999; Nichols et al., 2001) failed to block LatA-induced occludin internalization (Figure 10C). Thus, both pharmacological and dominant negative inhibition of clathrin-mediated endocytosis failed to prevent LatA-induced TER loss and occludin internalization, suggesting that this pathway is not essential for LatA-induced TJ disruption.
LatA-induced Barrier Dysfunction Is Prevented by Cholesterol Extraction
The data suggest that dynamin II-dependent occludin internalization is responsible for LatA-induced TER loss. The morphological and morphometric data suggest that this occludin internalization may occur via caveolae-mediated endocytosis. Caveolae-mediated endocytosis depends on the specialized lipid composition of caveolae. To functionally define the role of caveolae-mediated endocytosis in LatA-induced TER loss, we extracted membrane cholesterol using either MBCD or filipin III, both of which have been shown to block caveolae-mediated endocytosis (Pike and Casey, 2002; Shigematsu et al., 2003). MBCD and filipin III caused TER to decrease by 12 ± 0.6 and 25 ± 0.4%, respectively, consistent with a previous report (Francis et al., 1999). However, further TER decreases were not induced by LatA in MBCD- or filipin III-treated monolayers (Figure 11A). Repletion of cholesterol restored the sensitivity of barrier function to LatA treatment, confirming that the effect of MBCD was due to cholesterol extraction (Figure 11B). Morphological analysis confirmed that MBCD prevented LatA-induced occludin internalization and that cholesterol repletion restored LatA-induced occludin internalization (Figure 11C). Moreover, morphometric analysis showed that although MBCD treatment alone caused the number of occludin-containing vesicles to increase slightly, from 28 ± 1.2 to 33 ± 1.7 vesicles per cell, there was no further increase upon LatA addition (Figure 11D). Thus, disruption of caveolae-mediated endocytosis prevents both LatA-induced barrier dysfunction and occludin internalization. Therefore, the functional and morphological data both indicate a critical role for caveolae-mediated endocytosis of TJ components, including occludin, in LatA-induced barrier disruption.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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We first sought to develop appropriate fluorescent TJ protein fusion constructs. We selected ZO-1, the first identified TJ plaque protein, occludin, the first identified TJ transmembrane protein, and claudin-1, one of the first two identified members of the claudin family of proteins, as representatives of three classes of TJ proteins, plaque proteins, nonclaudin transmembrane proteins, and claudins, respectively. Each protein was tagged at a site remote from regions known to participate in protein–protein interactions or to otherwise be involved in function. Stably transfected cell lines expressing the fusion proteins at levels similar to or less than the endogenous proteins were chosen for further analyses to avoid artifacts due to simple protein overexpression. These lines all developed normal epithelial barrier function and responded to LatA in a dose-dependent manner that was nearly identical to that of the parental line. Immunofluorescent analyses and subcellular fractionation studies showed that the fusion proteins were targeted identically to the corresponding endogenous TJ proteins. These fusion proteins also were redistributed by actin depolymerization in a manner similar to the endogenous proteins. Thus, the fusion proteins represent a suitable trace-label for tracking the movement of TJ proteins that does not significantly alter TJ morphology or barrier function.
Our initial studies of paraformaldehyde-fixed monolayers failed to identify morphological TJ disruption at early times after LatA addition, when barrier function was falling rapidly. Analysis of claudin-1 and ZO-1 fusion constructs also failed to show redistribution of these proteins that correlated with barrier loss. Subsequent studies using methanol fixation also failed to show redistribution of claudin-1, claudin-4, or ZO-1 until well after barrier loss had occurred. This should not be interpreted as indicating that these proteins are not involved in loss of barrier function, because changes not detectable by fluorescence microscopy, including altered protein–protein interactions, may still play a role. However, the most striking morphological change that correlated precisely with loss of barrier function was the concentration of occludin at distinct sites with the TJ that were then internalized into vesicular cytoplasmic structures. This initially puzzled us, because these vesicles were not detected in our preliminary immunofluorescent studies using paraformaldehyde-fixed monolayers. The loss of these vesicles was resolved as a fixation artifact, because we found that vesicles containing endogenous occludin were detectable in LatA-treated monolayers fixed using a methanol-based protocol. Using this approach, we were able to confirm the occludin internalization seen in our live cell analyses.
Although occludin and claudins have been previously identified in intracellular vesicles, the mechanisms and functional significance of this localization remain uncertain. For example, although a peculiar transcellular endocytosis of EGFP-claudin-3 has been reported, little is known of the mechanisms or functional consequences of this observation (Matsuda et al., 2004). Occludin and claudin internalization has been reported in immunofluorescent studies of fixed cell preparations (Hopkins et al., 2003; Ivanov et al., 2004; Wang et al., 2005), and data suggest that, after calcium chelation, occludin is internalized via a clathrin-mediated process (Ivanov et al., 2004). In contrast, constitutive activation of rho, rac, and cdc42 using Escherichia coli cytotoxic necrotizing factor-1 has been associated with occludin internalization into caveolin-1-containing vesicles along with removal of ZO-1 and junction adhesion molecule-1 from the TJ (Hopkins et al., 2003; Bruewer et al., 2004). However, neither clathrinnor caveolae-mediated occludin endocytosis was linked to changes in epithelial barrier function. Thus, although multiple mechanisms for internalization of occludin and other transmembrane TJ proteins have been reported, the relationship of these membrane trafficking events to TJ barrier function has not been explored previously. Given the striking temporal correlation we observed between occludin internalization and loss of barrier function, we sought to determine whether the membrane traffic this represented was essential to LatA-induced TJ barrier dysfunction.
To elucidate the role of membrane traffic in LatA-induced TJ barrier dysfunction, we used diverse inhibitors of membrane traffic. The data show that reduced temperature and hypertonicity prevent both the barrier dysfunction and occludin redistribution caused by LatA. Thus, these data are the first to show the absolute requirement for membrane traffic in TJ disruption induced by actin depolymerization. Given the range of membrane transport processes recently implicated in TJ protein internalization, we sought to identify the specific endocytic mechanisms that contribute to this TJ disruption. Based on lack of colocalization of internalized occludin with clathrin, the failure of the clathrin-mediated endocytosis inhibitor chlorpromazine to prevent LatA-induced barrier dysfunction, and the failure of Eps1595-295 to prevent occludin internalization, we conclude that clathrin-mediated endocytosis is not critical to LatA-induced TJ disruption and occludin redistribution. Similarly, two separate inhibitors of fluid-phase macropinocytosis, amiloride and LY294002, were unable to prevent LatA-induced barrier dysfunction. We therefore also excluded macropinocytosis as a mechanism of TJ protein internalization and barrier dysfunction after actin depolymerization.
In contrast to clathrin-mediated endocytosis and macropinocytosis, several pieces of data point to caveolae-mediated endocytosis as the mechanism of occludin internalization in this process. First, extraction of plasma membrane cholesterol, which disrupts caveolae, prevented both barrier dysfunction and occludin redistribution after actin depolymerization. Second, both high-frequency imaging and morphometric analyses show that internalized occludin colocalizes with caveolin-1. Third, internalized occludin also colocalized with dynamin II and occludin internalization depended on dynamin II function, because dominant negative dynamin II prevented LatA-induced occludin internalization. Thus, although caveolae have not been reported within TJs, we conclude that similar caveolin- and dynamin II-dependent events are required for internalization of TJ proteins. This is also consistent with the observations that TJ proteins are present in detergent-resistant glycolipid- and cholesterol-rich membrane fractions (Nusrat et al., 2000b) and that aggressive cholesterol extraction, using higher MBCD doses than those used here, disrupts barrier function (Francis et al., 1999).
Thus, the data provide an alternative, although not mutually exclusive, explanation to the assumption that disruption of the direct binding interactions between TJ proteins and actin filaments explains the effects of actin depolymerization on barrier function. The data suggest that actin depolymerization triggers internalization of cholesterol-rich membranes, leading to TJ disruption. This is consistent with reports that cytochalasin D treatment increases lateral mobility of caveolin-1 and causes caveolae clustering (Thomsen et al., 2002) and that SV40 virus binding induces transient actin depolymerization and dynamin-II recruitment that are necessary for formation of caveolae-derived endocytic vesicles (Pelkmans et al., 2001, 2002). The phenomenon of clustering at the plasma membrane also may be related to the clustered occludin distribution and reduced barrier function we observed in dynamin II K44A-expressing monolayers. Based on the data presented here, one could hypothesize that depolymerization of actin allows increased lateral mobility of occludin, resulting in occludin clustering within TJ subdomains adjacent to lateral membrane. These regions then become the sites at which caveolae-mediated occludin internalization occurs using the endocytic machinery specialized for basolateral membrane domains. Although the data using a broad spectrum of inhibitors suggest that it is this process of endocytosis that causes loss of TJ barrier function, we cannot entirely exclude the possibility that barrier function is also compromised as occludin diffuses toward the lateral membrane.
In summary, these data show the critical role of membrane traffic in the functional and structural TJ disruption that is induced by actin depolymerization. Moreover, the data suggest that this membrane traffic is required for caveolae- and dynamin-mediated endocytosis of TJ components, including occludin. Thus, these data provide new understanding of the 25-year-old observation that actin depolymerization causes structural and functional TJ disruption. Moreover, although LatA-induced actin depolymerization is an artificial stimulus, it is possible that the mechanisms defined here are involved in the actomyosin-dependent TJ regulation that occurs throughout physiological and pathophysiological processes (Turner, 2000; Clayburgh et al., 2004).
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Abbreviations used: BS3, bis(sulfosuccinimidyl)suberate; EGFP, enhanced green fluorescent protein; HBSS, Hank's balanced saline solution; LatA, latrunculin A; MBCD, methyl--cyclodextrin; MDCK, Madin-Darby canine kidney; mRFP1, monomeric red fluorescent protein 1; TER, transepithelial resistance; TJ, tight junction.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Jerrold R. Turner (jturner{at}bsd.uchicago.edu).
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 Y. Tang, D. R. Clayburgh, N. Mittal, T. Goretsky, R. Dirisina, Z. Zhang, M. Kron, D. Ivancic, R. B. Katzman, G. Grimm, et al. Epithelial NF-{kappa}B Enhances Transmucosal Fluid Movement by Altering Tight Junction Protein Composition after T Cell Activation Am. J. Pathol., January 1, 2010; 176(1): 158 - 167. [Abstract] [Full Text] [PDF]
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 C. M. Van Itallie, A. S. Fanning, A. Bridges, and J. M. Anderson ZO-1 Stabilizes the Tight Junction Solute Barrier through Coupling to the Perijunctional Cytoskeleton Mol. Biol. Cell, September 1, 2009; 20(17): 3930 - 3940. [Abstract] [Full Text] [PDF]
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 T. Murakami, E. A. Felinski, and D. A. Antonetti Occludin Phosphorylation and Ubiquitination Regulate Tight Junction Trafficking and Vascular Endothelial Growth Factor-induced Permeability J. Biol. Chem., July 31, 2009; 284(31): 21036 - 21046. [Abstract] [Full Text] [PDF]
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S. M. Stamatovic, R. F. Keep, M. M. Wang, I. Jankovic, and A. V. Andjelkovic Caveolae-mediated Internalization of Occludin and Claudin-5 during CCL2-induced Tight Junction Remodeling in Brain Endothelial Cells J. Biol. Chem., July 10, 2009; 284(28): 19053 - 19066. [Abstract] [Full Text] [PDF]
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 V. A. Swystun, B. Renaux, F. Moreau, S. Wen, M. A. Peplowski, M. D. Hollenberg, and W. K. MacNaughton Serine proteases decrease intestinal epithelial ion permeability by activation of protein kinase C{zeta} Am J Physiol Gastrointest Liver Physiol, July 1, 2009; 297(1): G60 - G70. [Abstract] [Full Text] [PDF]
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 D. C. Brady, J. K. Alan, J. P. Madigan, A. S. Fanning, and A. D. Cox The Transforming Rho Family GTPase Wrch-1 Disrupts Epithelial Cell Tight Junctions and Epithelial Morphogenesis Mol. Cell. Biol., February 15, 2009; 29(4): 1035 - 1049. [Abstract] [Full Text] [PDF]
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 J. S. Young, J. A. Guttman, K. S. Vaid, and A. W. Vogl Tubulobulbar Complexes Are Intercellular Podosome-Like Structures That Internalize Intact Intercellular Junctions During Epithelial Remodeling Events in the Rat Testis Biol Reprod, January 1, 2009; 80(1): 162 - 174. [Abstract] [Full Text] [PDF]
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 L. Shen, C. R. Weber, and J. R. Turner The tight junction protein complex undergoes rapid and continuous molecular remodeling at steady state J. Cell Biol., October 17, 2008; 181(4): 683 - 695. [Abstract] [Full Text] [PDF]
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R. K. Gill, L. Shen, J. R. Turner, S. Saksena, W. A. Alrefai, N. Pant, A. Esmaili, A. Dwivedi, K. Ramaswamy, and P. K. Dudeja Serotonin modifies cytoskeleton and brush-border membrane architecture in human intestinal epithelial cells Am J Physiol Gastrointest Liver Physiol, October 1, 2008; 295(4): G700 - G708. [Abstract] [Full Text] [PDF]
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 J. Peralta-Ramirez, J. M. Hernandez, R. Manning-Cela, J. Luna-Munoz, C. Garcia-Tovar, J.-P. Nougayrede, E. Oswald, and F. Navarro-Garcia EspF Interacts with Nucleation-Promoting Factors To Recruit Junctional Proteins into Pedestals for Pedestal Maturation and Disruption of Paracellular Permeability Infect. Immun., September 1, 2008; 76(9): 3854 - 3868. [Abstract] [Full Text] [PDF]
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 Y. Zhong, E. J. Smart, B. Weksler, P.-O. Couraud, B. Hennig, and M. Toborek Caveolin-1 Regulates Human Immunodeficiency Virus-1 Tat-Induced Alterations of Tight Junction Protein Expression via Modulation of the Ras Signaling J. Neurosci., July 30, 2008; 28(31): 7788 - 7796. [Abstract] [Full Text] [PDF]
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H. Nakatsuji, N. Nishimura, R. Yamamura, H.-o. Kanayama, and T. Sasaki Involvement of Actinin-4 in the Recruitment of JRAB/MICAL-L2 to Cell-Cell Junctions and the Formation of Functional Tight Junctions Mol. Cell. Biol., May 15, 2008; 28(10): 3324 - 3335. [Abstract] [Full Text] [PDF]
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W. Fries, C. Muja, C. Crisafulli, S. Cuzzocrea, and E. Mazzon Dynamics of enterocyte tight junctions: effect of experimental colitis and two different anti-TNF strategies Am J Physiol Gastrointest Liver Physiol, April 1, 2008; 294(4): G938 - G947. [Abstract] [Full Text] [PDF]
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 G. Schreibelt, G. Kooij, A. Reijerkerk, R. van Doorn, S. I. Gringhuis, S. van der Pol, B. B. Weksler, I. A. Romero, P.-O. Couraud, J. Piontek, et al. Reactive oxygen species alter brain endothelial tight junction dynamics via RhoA, PI3 kinase, and PKB signaling FASEB J, November 1, 2007; 21(13): 3666 - 3676. [Abstract] [Full Text] [PDF]
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V. S. Subramanian, J. S. Marchant, D. Ye, T. Y. Ma, and H. M. Said Tight junction targeting and intracellular trafficking of occludin in polarized epithelial cells Am J Physiol Cell Physiol, November 1, 2007; 293(5): C1717 - C1726. [Abstract] [Full Text] [PDF]
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 D. Roosterman, O. J. Kreuzer, N. Brune, G. S. Cottrell, N. W. Bunnett, W. Meyerhof, and M. Steinhoff Agonist-Induced Endocytosis of Rat Somatostatin Receptor 1 Endocrinology, March 1, 2007; 148(3): 1050 - 1058. [Abstract] [Full Text] [PDF]
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C. T. Capaldo and I. G. Macara Depletion of E-Cadherin Disrupts Establishment but Not Maintenance of Cell Junctions in Madin-Darby Canine Kidney Epithelial Cells Mol. Biol. Cell, January 1, 2007; 18(1): 189 - 200. [Abstract] [Full Text] [PDF]
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 P. P Y Lie, W. Xia, C. Q F Wang, D. D Mruk, H. H N Yan, C.-h. Wong, W. M Lee, and C Y. Cheng Dynamin II interacts with the cadherin- and occludin-based protein complexes at the blood-testis barrier in adult rat testes J. Endocrinol., December 1, 2006; 191(3): 571 - 586. [Abstract] [Full Text] [PDF]
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J. R. Turner Molecular Basis of Epithelial Barrier Regulation: From Basic Mechanisms to Clinical Application Am. J. Pathol., December 1, 2006; 169(6): 1901 - 1909. [Abstract] [Full Text] [PDF]
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 L. Shen, E. D. Black, E. D. Witkowski, W. I. Lencer, V. Guerriero, E. E. Schneeberger, and J. R. Turner Myosin light chain phosphorylation regulates barrier function by remodeling tight junction structure J. Cell Sci., May 15, 2006; 119(10): 2095 - 2106. [Abstract] [Full Text] [PDF]
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T. Terai, N. Nishimura, I. Kanda, N. Yasui, and T. Sasaki JRAB/MICAL-L2 Is a Junctional Rab13-binding Protein Mediating the Endocytic Recycling of Occludin Mol. Biol. Cell, May 1, 2006; 17(5): 2465 - 2475. [Abstract] [Full Text] [PDF]
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L. Shen and J. R. Turner Role of Epithelial Cells in Initiation and Propagation of Intestinal Inflammation. Eliminating the static: tight junction dynamics exposed Am J Physiol Gastrointest Liver Physiol, April 1, 2006; 290(4): G577 - G582. [Abstract] [Full Text] [PDF]
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