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Am J Physiol Cell Physiol 293: C1717-C1726, 2007. First published September 13, 2007; doi:10.1152/ajpcell.00309.2007
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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Tight junction targeting and intracellular trafficking of occludin in polarized epithelial cells

Veedamali S. Subramanian,1,2 Jonathan S. Marchant,3 Dongmei Ye,4 Thomas Y. Ma,4 and Hamid M. Said5

Departments of 1Medicine and 2Physiology and Biophysics, University of California, Irvine, California; 3Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota; 4Department of Internal Medicine, University of New Mexico, Albuquerque, New Mexico; and 5Department of Veterans Affairs Medical Center, Long Beach, California

Submitted 19 July 2007 ; accepted in final form 4 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Occludin, a transmembrane (TM)-spanning protein, is an integral component of the tight junctional (TJ) complexes that regulate epithelial integrity and paracellular barrier function. However, the molecular determinants that dictate occludin targeting and delivery to the TJs remain unclear. Here, using live cell imaging of yellow fluorescent protein-labeled occludin fragments, we resolved the intracellular trafficking of occludin-fusion proteins in polarized Madin-Darby canine kidney and Caco-2 cells to delineate the regions within the occludin polypeptide that are important for occludin targeting to the TJs. Live cell confocal imaging showed that complete or partial truncation of the COOH-terminal tail of the occludin polypeptide did not prevent occludin targeting to the TJs in epithelial cell lines. Progressive truncations into the COOH-terminal tail decreased the efficiency of occludin expression; after the removal of the regions proximal to the fourth transmembrane domain (TM4), the efficiency of expression increased. However, further deletions into the TM4 abolished TJ targeting, which resulted in constructs that were retained intracellularly within the endoplasmic reticulum. The full-length occludin polypeptide trafficked to the cell surface within a heterogenous population of intracellular vesicles that delivered occludin to the plasma membrane in a microtubule- and temperature-dependent manner. In contrast, the steady-state localization of occludin at the cell surface was dependent on intact microfilaments but not microtubules.

epithelium

TIGHT JUNCTIONS (TJs) play a critical role in regulating paracellular permeability across epithelial surfaces (1, 20). The TJ complex consists of multiple proteins, of which occludin is a transmembrane (TM) component that interacts with other components of the TJ assembly. Although the exact roles of occludin in the TJ complex are not completely understood, previous studies have shown that occludin plays an important role in TJ barrier function and signaling processes (1, 11,19, 20, 24, 33, 39).

Studies in polarized epithelial cells have shown that the targeting and intracellular trafficking of plasma membrane proteins to particular domains of the cell surface are highly regulated events mediated by diverse targeting signals/domains that can be distributed throughout the sequence of the polypeptide (14, 28, 34, 36). However, despite several previous studies (2, 5, 8–10, 15, 21, 23, 25), the mechanisms responsible for occludin targeting to the TJ complex remain unclear. For example, some studies implicate the occludin COOH-terminal domain in TJ targeting (15, 25). Furuse et al. (8) reported that the COOH-terminal cytoplasmic domain of occludin, and particularly that of the zonula occludens ZO-1 binding region (amino acids 358–504), was necessary for TJ localization. However, other reports suggest that occludin mutants lacking the COOH-terminal domain can localize to the TJs (2). Mankertz et al. (21) reported that naturally occurring occludin splice variants lacking the fourth TM domain (TM4) and only the proximal 32 amino acids (266–297) of the COOH-terminal domain failed to localize at the TJs, despite containing the distal part of the COOH-terminal domain. Therefore, it remains unclear which molecular signals/domains mediate occludin targeting to the TJs. Thus, in the present study, we used live cell confocal microscopy of fluorescent protein–tagged occludin constructs to visualize the intracellular trafficking and cell surface targeting of occludin mutants in both kidney [Madin-Darby canine kidney (MDCK)] and intestinal (Caco-2) epithelial cell lines. Both cell lines have retained the ability to target proteins with appropriate polarity in vitro (36, 37). Our aims in the present study were to investigate the region(s) of occludin polypeptide that is important for its targeting to the TJ and to determine the internal and extrinsic factors that regulate occludin intracellular trafficking and its steady-state maintenance at the TJ of intestinal and renal epithelial cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals and reagents. The enhanced green fluorescent protein- and yellow fluorescent protein-tagging vectors (GFP-C3 and YFP-N1) and the DsRed-ER plasmid [the red fluorescent protein is targeted to the lumen of the endoplasmic reticulum (ER) both by in-frame fusion to the ER-targeting sequence of calreticulin and by tagging with the ER retention sequence KDEL] were from BD Biosciences (Palo Alto, CA). G418 was from Invitrogen (Carlsbad, CA). Cytoskeletal agents were from Calbiochem (La Jolla, CA). Rhodamine phalloidin was from Molecular Probes (Eugene, OR). MDCK (NBL-2) and Caco-2 cells were from ATCC (Manassas, VA). Occludin polyclonal antibodies were from Zymed (San Francisco, CA). Anti-rabbit [tetramethylrhodamine isothiocyanate (TRITC)]-conjugated secondary antibodies, myosin light chain kinase (MLCK) inhibitor ML-7, and all other reagents were from Sigma (St. Louis, MO).

Generation of GFP-occludin, occludin-YFP, and truncated constructs. The open reading frame for occludin (1,566 bp) was amplified by RT-PCR from total RNA isolated from Caco-2 cells by using gene-specific primers (Table 1). The full-length GFP-occludin (GFP-OCC) or occludin-YFP (OCC-YFP) and the truncated constructs were generated by PCR by using the primer combinations shown in Table 1 under previously described conditions (36, 37). The PCR products and the GFP-C3 and YFP-N1 vectors were subsequently digested with HindIII and SacII, and the products were gel separated and ligated together to generate in-frame fusion proteins, with the fluorescent proteins fused to either the NH2 terminus (GFP-C3-OCC) or COOH terminus (OCC-YFP-N1) of each construct. The nucleotide sequence of each construct was verified by sequencing (Laragen, Los Angeles, CA).


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  		Table 1. Gene-specific primers used to generate the truncations in occludin constructs by PCR

 
Cell culture, transient transfection, and stable transfection. MDCK cells were maintained in MEM, and human adenocarcinoma (Caco-2) cells were maintained in DMEM. All media were supplemented with 10%-20% fetal bovine serum, glutamine (0.29 g/l), sodium bicarbonate (2.2 g/l), penicillin (100,000 U/l), and streptomycin (10 mg/l). Both MDCK and Caco-2 cells were transiently transfected. Cells were grown on sterile glass-bottomed Petri dishes (MatTek, Ashland, MA) and were transiently transfected at 95% confluency with 2 µg plasmid DNA by using 2 µl Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Stable MDCK cells expressing GFP-OCC and OCC-YFP plasmids and GFP and YFP vectors alone were generated by G418 selection (0.8 mg/ml) for 4–6 wk.

Immunofluorescence. Stable GFP-OCC-expressing MDCK cells either untreated or treated with Ca2+- and Mg2+-free solution followed by the reintroduction of Ca2+ and Mg2+ were fixed for 10 min in 4% paraformaldehyde solution (Electron Microscopy Sciences, Washington, PA). Cells were permeabilized with 2% Triton X-100 and were blocked in 1% bovine serum albumin for 30 min at room temperature. Cells were then incubated with occludin antibodies (Zymed) in PBS (2 h at room temperature) and were finally incubated with anti-rabbit TRITC-conjugated secondary antibodies in PBS (1 h). For the visualization of immunofluorescence, cells were mounted by using Fluoromount reagent (Southern Biotechnology, Birmingham, AL) and were imaged by using confocal microscopy.

Live cell confocal microscopy. GFP-OCC- or OCC-YFP–expressing cells were imaged by using a Nikon C1 confocal scanner head attached to a Nikon inverted phase-contrast microscope or a Bio-Rad MRC 1024 confocal scanner Olympus Ax70 microscope. Fluorophores were excited by using the 488-nm line from an argon-ion laser, and emitted fluorescence was monitored with the use of a 530 ± 20 nm band-pass filter (YFP or GFP) or a 620-nm long-pass filter (DsRed or actin dye or TRITC). We previously showed no difference in polarized targeting of apical and basolateral constructs in our MDCK clone grown on coverglass or filter support (3). For the imaging of vesicular trafficking, GFP-OCC fluorescence was monitored by using a custom-built confocal microscope equipped with a x60 oil-immersion objective (4). Images were captured at ≥30 Hz (≥1 frame/33 ms) and were digitized by using VideoSavant processing software (IO Industries, London, Ontario, Canada). For processing the motion of individual vesicles, the frame-to-frame tracking function in MetaMorph was used (Universal Imaging, Downingtown, PA). The fluorescence distribution was quantified with the use of IDL analysis software (Research Systems, Boulder, CO).

Flow cytometry. Flow cytometry was performed by using a FACScalibur Benchtop cytometer (BD Biosciences, San Jose, CA). The OCC-YFP wild-type and truncated constructs transiently expressing MDCK cells were grown in T25 tissue culture flasks. MDCK cells were trypsinized, pelleted, and resuspended in 1 ml of Dulbecco's PBS with Ca2+ and Mg2+ at a density of 1 x 106 cells/ml, as described previously (22). In all flow cytometry experiments, the samples of untransfected and YFP alone-transfected MDCK cells were run in parallel with the samples transiently expressing occludin wild-type and truncated constructs to calibrate optical parameters for identifying the intact, transfected cell population.

Statistical analysis. The results are expressed as means ± SE. All experiments were repeated a minimum of three times to ensure reproducibility, and representative experiments are shown.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Occludin targets to the TJs in renal and intestinal epithelia. Figure 1A shows a schematic representation of the OCC-YFP full-length construct to show the domain organization of the occludin polypeptide. The full-length occludin comprises a short intracellular NH2-terminal domain (1–66, amino acid residues); a TM domain containing four predicted membrane-spanning domains, with one intracellular loop and two extracellular loops (67–265, amino acid residues); and a long cytoplasmic COOH-terminal domain (266–522, amino acid residues). YFP was fused to the COOH terminus of the occludin polypeptide. To investigate the TJ targeting of the full-length OCC-YFP, this construct was transiently transfected into MDCK and Caco-2 epithelial cells, where it targeted to the TJ region, as evidenced by live cell confocal imaging (Fig. 1B). In contrast, YFP-transfected cells showed fluorescence emission from the entire cytoplasmic volume (Fig. 1C).


Figure 1
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  		Fig. 1. Tight junction (TJ) targeting of occludin-yellow fluorescent protein (OCC-YFP) in renal and intestinal cells. A: schematic representation of the amino acid sequence of the full-length occludin protein (1–522 amino acids) with YFP fused to the COOH terminus. TM, transmembrane. B: targeting of OCC-YFP in Madin-Darby canine kidney (MDCK; left) and Caco-2 (right) cells in lateral (xy) and axial (xz) sections. All axial (xz) images were captured from the cells shown in the associated lateral (xy) images. C: expression of YFP in MDCK (left) and Caco-2 (right) cells.

 
The cytoplasmic COOH-terminal tail of occludin is not required for TJ targeting. We used a truncation approach (Fig. 2A) to investigate the region(s) within the occludin polypeptide that is important for TJ targeting in MDCK cells: the full-length polypeptide was progressively shortened from the COOH terminus, and the TJ localization of each truncated construct was resolved by transient transfection in MDCK cells. Neither the partial [OCC (1–346)-YFP, OCC (1–322)-YFP, OCC (1–298)-YFP, OCC (1–281)-YFP, OCC (1–273)-YFP] nor complete [OCC (1–265)-YFP] truncation of the COOH-terminal region prevented the targeting of occludin to the TJs in live MDCK cells (Fig. 2B), which were imaged after transient transfection. Furthermore, it was not surprising that the COOH terminus alone [OCC (266–522)-YFP or GFP-OCC (266–522)] showed cytoplasmic localization (Fig. 2B). From these results, we conclude that the cytoplasmic COOH tail of occludin is neither essential nor sufficient for TJ targeting and therefore does not contain the targeting signals/domains responsible for targeting the full-length occludin protein. Although the COOH-terminal region did not affect the TJ targeting profile of the occludin truncations, progressive truncations into the COOH-terminal tail decreased the efficiency of occludin expression at the TJ (Fig. 2C). The removal of most of the distal region (~75%, ~200 amino acids) of the COOH-terminal tail reduced the expression efficiency by about half. Removal of the next ~25 amino acids (298–322 amino acids) was associated with a further halving of expression efficiency. Finally, the deletions immediately proximal to TM4 salvaged the expression efficiency, with the fluorescence emission increasing to ~80% of wild type.


Figure 2
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  		Fig. 2. The cytoplasmic COOH-terminal region of occludin is not necessary for TJ targeting. A: schematic representation of truncation mutants (i–vii) within the cytoplasmic tail of OCC-YFP. B: lateral (xy) and axial (xz) scans showing expression of COOH-terminal tail truncated constructs (i–iii) imaged in MDCK cells 24–48 h after transfection. C: results from flow cytometry analysis showing mean population fluorescence intensity of COOH-terminal truncations (bottom) relative to full-length OCC-YFP, plotted alongside the extent of the truncation (top).

 
TJ targeting of occludin is dependent on the integrity of the polypeptide backbone. Although the partial or complete deletion of the COOH-terminal tail of occludin did not affect TJ targeting, the deletion of a further 6 amino acids (260–265) into the TM4 domain of the occludin polypeptide resulted in a pleiotropic phenotype, with most of the cells showing cytoplasmic localization and only few cells displaying TJ targeting (Fig. 3A). The deletion of a further five amino acids [OCC (1–254)-YFP] resulted in a complete loss of TJ targeting and retention within the ER (Fig. 3A). These results show that integrity of TM4 is necessary for occludin targeting to the TJ. Finally, a construct encompassing solely TM4 and the COOH-terminal tail of occludin [OCC (244–522)-YFP] was retained within the ER, which was evidenced by the colocalization with a red fluorescence protein construct targeted to the ER lumen (DsRed-ER; Fig. 3, B and C). Therefore, these results show that integrity of TM4 is necessary but is not in itself sufficient for the targeting of occludin to the TJs.


Figure 3
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  		Fig. 3. Systematic truncation of the occludin protein backbone abolishes TJ targeting in MDCK cells. A: cellular expression of two occludin polypeptide backbone truncations. B: intracellular expression of OCC (244–522)-YFP construct. C: live confocal images of MDCK cells expressing OCC-YFP (wild-type; top), OCC(1–254)-YFP (middle), and OCC(245–522)-YFP (bottom) together with DsRed-endoplasmic reticulum (ER). YFP (left) and DsRed (center) channels were superimposed to generate an overlay image (right).

 
Intracellular trafficking of occludin in MDCK cells. Next, we analyzed the distribution of the full-length GFP-OCC construct at various time points after transient transfection into MDCK cells. At early time points (~6–18 h), many discrete intracellular GFP-positive structures were apparent in the cytosol of transfected cells (Fig. 4Ai), with comparatively little fluorescence staining of the plasma membrane. By 24 h, "patchy" staining of the lateral cell membrane was evident in confocal sections throughout the cellular volume (Fig. 4Aii). At later times, this staining became brighter and more homogenous, such that the lateral cell surface, especially at multicellular contacts, exhibited bright GFP-OCC expression (Fig. 4A, iii and iv). Staining of the lateral membranes in GFP-OCC-expressing cells was clearly evident in the axial sections compared with the distribution of GFP alone (Fig. 4Bi). Furthermore, the selective targeting of GFP-OCC to the lateral membranes was established early after GFP-OCC expression (Fig. 4Bii), which supports a model in which the TJ proteins are targeted directly to the lateral cell surface domain, rather than indirectly through the apical or basal cell surface. Finally, the population of intracellular structures containing GFP-OCC was heterogeneous. In addition to the numerous vesicle-like structures, many cells contained a small number of "plaque"- or "raft"-like assemblies of GFP-OCC often observed intracelluarly, but in close apposition to the lateral cell surface (Fig. 4C).


Figure 4
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  		Fig. 4. Trafficking and targeting of green fluorescent protein (GFP)-OCC in MDCK cells. A: lateral (xy) images of MDCK expressing GFP-OCC and imaged at specific times (i, 12 h; ii, 24 h; iii, 36 h; iv, 48 h) after transient transfection. Bi, left: axial (xz) images of GFP and GFP-OCC expression in MDCK cells 48 h after transient transfection. A, apical; B, basal, L, left, R, right. Bi, right: representative profile of fluorescence measured laterally (L–R; *) across axial image of GFP-OCC distribution. Bii: analysis of polarized distribution of GFP-OCC through comparison of peak fluorescence distributions at apical, basal, and lateral (left and right) surfaces, measured from cells that were transfected for 24 h (solid bars) and 48 h (open bars). C: higher-magnification images, highlighting the "plaque"-like intracellular structures of GFP-OCC (*), in the peripheral cytoplasm (i and ii), and seemingly integrating with the lateral cell surface membrane (iii). D: analysis of motility of intracellular GFP-OCC-containing particles. The motion of discreet vesicular puncta was resolved by using an automated tracking algorithm, which was modified from that reported previously (22, 36), owing to the lower overall level of apparent vesicular motion in MDCK cells compared with other cell lines. Tracks were normalized spatially to an identical origin, and motion was scored as the fraction of vesicles with a path length breeching either a 2-µm radius (blue*) or a 4-µm radius (red*) circle during 30 s of real-time recording at 37°C (i), 22°C (ii), 37°C but preincubated with 10 µM nocodazole for 10 min (**scale bar = 0.8 µm) (iii), or 37°C but preincubated with 15 µM cytochalasin D for 10 min (iv). Bar charts represent the records of >30 vesicles in a single transfection.

 
To study the role of the cytoskeleton in trafficking GFP-OCC to the lateral membrane domains, we used confocal imaging methods (<100-ms frame rates) to monitor the dynamics of individual GFP-OCC-positive vesicles (see supplemental movie in the online version of this article). Because resolution of vesicular motility was more difficult in polarized MDCK cells (owing to a lower proportion of mobile vesicles) than in other cell types previously used by us in trafficking studies (36), we used a more qualitative approach to assess overall vesicular motility. Individual tracks of vesicular motion were aligned from a common origin, and the proportion of vesicles that moved specific distances from the origin were monitored over a set time. For example, in MDCK cells maintained at 37°C, 75 ± 10% and 42 ± 8% of vesicles showed net displacements of >2 and >4 µm from the origin in a 20-s recording period (Fig. 4Di and supplemental movie). These fractions were inhibited by incubation at room temperature (Fig. 4Dii) with the microtubule-disrupting drug nocodazole (Fig. 4Diii) and, albeit to a lesser extent, with the microfilament-disrupting drug cytochalasin D (CytoD) (which tended to immobilize the vesicles in the vicinity of the plasma membrane; Fig. 4Div and supplemental movie).

Role of cytoskeletal network in steady-state localization of occludin protein in polarized MDCK cells. To examine the role of the cytoskeleton in the steady-state localization of occludin in confluent MDCK cells, we treated a stable GFP-OCC-expressing MDCK cell line with nocodazole (10 µM), colchicine (10 µM), or CytoD (10 µM) for 10–90 min. Nocodazole- or colchicine-treated MDCK monolayers showed little or no alteration in preestablished TJ localization of occludin (Fig. 5A). In contrast, CytoD-treated cells showed marked disruption of preestablished occludin TJ localization and noticeable discontinuity in the peripheral occludin band (Fig. 5Bii) compared with control (Fig. 5Bi). CytoD, an actin-depolymerizing agent, caused a marked disruption in junctional actin microfilament localized with disassembly of perijunctional actin filaments and formation of discrete small actin clumps throughout the cytoplasm, with only sparse localization at the cellular borders (Fig. 5Bii). Because previous studies have suggested that MLCK activation plays an important role in CytoD modulation of TJ barrier function (18, 19), we examined the involvement of MLCK activation in CytoD disruption of occludin localization. The GFP-OCC-expressing MDCK monolayer was pretreated with an MLCK inhibitor (ML-7, 10 µM) for 30 min, followed by CytoD (10 µM) treatment for 30–90 min (Fig. 5Biii). ML-7 prevented the CytoD-induced disruption of both occludin and actin (Fig. 5Biii), which suggests that MLCK activation and actin-myosin interaction were also required for the disturbance of occludin localization to the TJs.


Figure 5
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  		Fig. 5. Effect of cytoskeletal drugs on steady-state TJ expression of occludin in MDCK cells. A: control cells (untreated) or nocodazole- or colchicine (10 µM, 10–90 min)-treated GFP-OCC-expressing MDCK cells. B: GFP-OCC-expressing MDCK cell either untreated (i), incubated with cytochalasin D (CytoD; 10 µM, 10–90 min) (ii), or treated with MLCK inhibitor (ML-7, 10 µM, 30 min) and CytoD (10 µM, 10–90 min) (iii). Images show GFP fluorescence (left), rhodamine phalloidin staining (center), and fluorescence overlay (right).

 
Effect of Ca2+ and Mg2+ on steady-state expression of occludin in MDCK cells. Previous studies showed that extracellular Ca2+ (and Mg2+) plays an important role in TJ formation and maintenance of TJ barrier function (17, 27, 29; unpublished observation from our laboratories). Next, we examined the roles of Ca2+ and Mg2+ on steady-state expression of occludin in stable GFP-OCC-expressing MDCK cells. In control cells, GFP-OCC was present at the TJ of MDCK cells and showed significant overlap with fluorescence staining resolved by using an occludin antibody (Fig. 6A), which confirms appropriate processing of the fusion construct. However, incubation of GFP-OCC-expressing cells in Ca2+- and Mg2+-free HBSS for 20 min resulted in a rapid, progressive internalization of GFP-OCC (Fig. 6B). Internalized GFP fluorescence again showed significant overlap with occludin antibody staining (Fig. 6B). During this process, the disassembly and separation of occludin from the apical-lateral borders and formation of large paracellular gaps between the adjacent cells (absence of occludin staining) was evident (Fig. 6B). The reincubation of the GFP-OCC-expressing cells in medium containing 1.8 mM Ca2+ (in the absence of Mg2+) for 2 h resulted in the reassembly of GFP-OCC at the apical cellular borders and the reclosure of the large paracellular gaps between the adjacent cells (Fig. 6C). The reincubation of cells with 1.8 mM Mg2+-containing HBSS in the absence of Ca2+ resulted in only the partial restoration of GFP-OCC at the cellular border, and large aggregations of GFP-OCC were evident in the cytoplasm (Fig. 6D). The morphological changes were also reversed by the reincubation of GFP-OCC-expressing cells with 1.8 mM Ca2+ and Mg2+ together for 2 h (Fig. 6E). Similar results were obtained by live cell confocal imaging on Ca2+- and Mg2+-free or reintroduced stable GFP-OCC-expressing MDCK cells (data not shown).


Figure 6
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  		Fig. 6. Effects of Ca2+ and Mg2+ on steady-state TJ expression of occludin in MDCK cells. A: lateral (xy) scans of GFP-OCC-expressing control cells (left) were labeled by using occludin-specific antibody (center) and overlays of green and red channels (right). Lateral (xy) scans of GFP-OCC-expressing MDCK cells treated with Ca2+- and Mg 2+-free HBSS for 20 min (B), reintroduced Ca2+ (1.8 mM, 2 h) alone (C), reintroduced Mg2+ (1.8 mM, 2 h) alone (D), or Ca2+ and Mg2+ together (left, E) were labeled by using occludin-specific antibody (center) and overlays (right).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Occludin is an integral TJ protein that plays an important role in TJ barrier function and cell signaling. However, the precise molecular determinants/domains that are responsible for occludin targeting/trafficking to TJs remain unclear. Previous studies did not reach a consensus: several reports suggested that the lengthy COOH-terminal domain of occludin mediates targeting to the TJs (5, 8, 15, 23, 25), whereas others suggested that TJ targeting can occur in the absence of the cytoplasmic COOH-terminal domain (2, 21). An understanding of the role of the long (~50% of the polypeptide length) COOH-terminal tail is important, because it has been implicated in several models of occludin targeting. For example, the COOH tail is the target for phosphorylation by multiple kinases, several of which modulate TJ reorganization (6, 13, 26, 32, 35). The increased phosphorylation of occludin has been shown to correlate with localization within TJs in MDCK cells (31). Furthermore, the COOH-terminal domain binds to a variety of accessory proteins [e.g., cingulin, casein kinase 2 and F-actin, and several zonula occludens (ZO) proteins (7, 8, 12, 26, 38)]. However, Mankertz et al. (21) found that a naturally occurring occludin splice variant lacking 54 amino acids of TM4 and the proximal component of the COOH-terminal tail failed to localize at the TJs. Balda et al. (2) showed that, in MDCK cells, a COOH-terminal domain truncated chicken occludin was able to incorporate into TJs. Therefore, although the cytoplasmic domains of occludin are likely important mediators of protein scaffolding at TJs, the role of this region in targeting occludin to TJs remains controversial.

Here, we used live cell imaging of fluorescent protein-tagged truncations to resolve occludin targeting and trafficking in two commonly used polarized epithelial cell systems, MDCK (kidney) and Caco-2 (intestinal) cells. Our results show that sequence determinants within TM4 are necessary, but not in themselves sufficient, for TJ localization. Neither partial nor complete deletions of the cytoplasmic COOH-terminal domain of OCC (amino acids 266–522) prevented TJ targeting (Fig. 2). The expression efficiency of these constructs did vary (Fig. 2C), which provides a possible explanation—decreased detection efficiency—for previous reports that supported a role for the cytoplasmic COOH-terminal region. Deletions into the fourth TM domain, however, strongly affected TJ targeting. The deletion of six amino acids (260–265) in the midportion of TM4 caused a partial impairment of TJ targeting, whereas extending the deletion to include a further 5 amino acids (255–259) resulted in a complete loss of TJ targeting. These data indicate that the integrity of TM4 was required for TJ targeting. However, TM4 is not by itself sufficient for TJ targeting, because a mutant construct comprising an intact TM4 and COOH-terminal domain failed to target to the TJs (Fig. 3B).

By using live cell imaging methods with high spatial and temporal resolution to resolve vesicular motility, we resolved the distribution of occludin within a population of intracellular structures, which included a heterogeneous population of vesicles (see supplemental movie) and plaquelike/raftlike assemblies close to the lateral membrane surface. These larger structures may be indicative of intracellular preassemblies of TJ complexes or, alternatively, endocytic trafficking of occludin-positive structures. Our data indicate that intracellular motility of occludin-containing vesicles was closely dependent on intact microtubular network, because pharmacological disruption of the microtubule network with nocodazole almost completely inhibited the intracellular motility of occludin-containing vesicles (Fig. 4D and supplemental movie). This is consistent with previous data on the trafficking of cell surface proteins toward (35–37) and in close proximity to the cell surface (30). The disruption of actin microfilaments with CytoD also interfered with occludin vesicular motility, but to a lesser extent than following microtubule disruption (supplemental movie).

Under steady-state conditions, the disruption of the microtubular network with nocodazole or colchicine did not have any effect on occludin junction localization (Fig. 5A) or on Caco-2 TJ barrier function (16, 18). In contrast, the disruption of actin microfilaments caused a disassembly of occludin from the junctional regions, with the formation of large gaps in the continuity of occludin junctional localization (Fig. 5Bii). The disturbance in actin microfilaments and the junctional dislocation of occludin protein correlated with a functional disturbance in TJ barrier function (16, 18, 35). The inhibition of MLCK activity with ML-7 prevented the CytoD-induced disturbance in junctional localization of occludin, which suggests that MLCK activity was required for the disassembly of occludin from the junctional regions (Fig. 5Bii). These findings are consistent with previous results from our laboratory, which showed the requirement of MLCK activation in mediating the CytoD-induced opening of the TJ barrier. Our results also indicated that the exposure of MDCK cells to low extracellular Ca2+ and Mg2+ environment leads to a separation or centripedal retraction of occludin protein away from the cellular junctions. The low extracellular Ca2+- and Mg2+-induced retraction of occludin proteins was accompanied by visible gaps in occludin localization between adjacent cells and a loss of TJ barrier function (Fig. 6). The reintroduction of Ca2+ rapidly restored occludin junctional localization and TJ barrier function, whereas Mg2+ only partially reversed the occludin junctional localization and did not have any effect on restoring the barrier function as assessed by transepithelial electrical resistance measurements. These studies would suggest that the partial restoration of occludin junctional localization is inadequate to restore the barrier function.

In conclusion, our data indicate that complete or partial truncation of the COOH-terminal tail of occludin does not prevent TJ targeting. The integrity of TM4 was required but was not sufficient for TJ targeting. The membrane trafficking of occludin vesicles was dependent on intact microtubule and actin microfilament network. The steady-state junctional localization of occludin required intact actin microfilaments and extracellular Ca2+ but not microtubular network. Thus, our data provide new insight into the molecular determinants of occludin and the internal and extrinsic factors that regulate occludin membrane trafficking and steady-state junctional localization.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Department of Veterans Affairs and by National Institutes of Health Grants DK-64165 (to T. Y. Ma and H. M. Said), DK-71538 (to V. S. Subramanian), and NS-046783 (to J. S. Marchant).


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. M. Said, UCI/VA Medical Program, VA Medical Center-151, Long Beach, CA 90822 (e-mail: hmsaid{at}uci.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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