1 Department of Internal Medicine, University of Michigan Medical School, 210 Washtenaw Avenue, Ann Arbor, MI 48109-2216, USA
2 Department of Biological Chemistry, University of Michigan Medical School, 210 Washtenaw Avenue, Ann Arbor, MI 48109-2216, USA
3 Howard Hughes Medical Institute, Howard Hughes Medical Institute, University of Michigan Medical Center, Room 6183, Life Science Institute, 210 Washtenaw Avenue, Ann Arbor, MI 48109-2216, USA
* Author for correspondence (e-mail: bmargoli{at}umich.edu)
Accepted 31 March 2005
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Summary |
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Key words: FERM, Membrane Proteins, Claudin, Mammary, Epithelia, Zona occludens
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Introduction |
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Three isoforms of mammalian Crumbs have been identified, and two have been characterized. Crumbs1 (CRB1) is expressed primarily in the central nervous system (den Hollander et al., 2002; den Hollander et al., 1999
), whereas Crumbs3 (CRB3) is an epithelia-specific isoform (Lemmers et al., 2004
; Makarova et al., 2003
). Both the CRB3 and Par6 complexes have been localized to the tight junctions of mammalian epithelial cells (Izumi et al., 1998
; Joberty et al., 2000
; Roh et al., 2003
). Tight junctions are specialized apical junctional complexes that serve to form a tight seal between adjoining epithelial cells and function as a selective barrier to paracellular diffusion through the intercellular space. Tight junctions also serve to restrict intramembrane diffusion of proteins and lipids between the apical and basolateral domains, and thus are essential to the maintenance of epithelial cell polarity (Schneeberger and Lynch, 2004
; Tsukita et al., 2001
).
Numerous studies now support a role for the proteins of the Par6 complex in the development of epithelial tight junctions (Gao et al., 2002; Hirose et al., 2002
; Joberty et al., 2000
; Mizuno et al., 2003
; Suzuki et al., 2002
; Suzuki et al., 2001
). However, comparatively little is known of the role of the Crumbs complex in epithelial tight junction formation. We have recently shown that the Crumbs/PALS1/PATJ complex is physically linked to the Par6 complex through a direct interaction between PALS1 and Par6 (Hurd et al., 2003
). This suggests that the Crumbs and Par6 complexes may act together to regulate tight junction formation. Indeed, we and others have found that overexpression of CRB3 in MDCKII cells delays the formation of tight junctions in a calcium-switch assay (Lemmers et al., 2004
; Roh et al., 2003
). This effect was found to be dependent upon the C-terminal PDZ binding motif of CRB3; a CRB3 mutant that lacked the C-terminal PDZ binding motif, and thus the ability to bind the PDZ protein PALS1, was unable to affect tight junction formation. This finding implicated PALS1, and potentially the Par6 complex, as an important downstream mediator of CRB3 function in tight junction regulation. Additional evidence that the CRB3 complex is important in tight junction regulation came from a recent study in which RNAi-mediated suppression of PALS1 expression in MDCKII cells also led to defects in tight junction formation (Straight et al., 2004
).
In this paper we present new evidence that the CRB3 protein plays a key role in the development of epithelial tight junctions. Mammary epithelial MCF10A cells express little endogenous CRB3, and are unable to form tight junctions when grown under standard tissue culture conditions. We find that exogenous expression of CRB3 is sufficient for the development of functional tight junctions in these cells. Mutations in both the CRB3 C-terminal PDZ binding motif, and the putative FERM binding motif compromise the ability of CRB3 to induce tight junctions. We propose that CRB3 plays an essential role in tight junction formation, and that interaction with both PALS1 and an unknown FERM protein are required for this process.
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Materials and Methods |
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Cell culture
MCF-7, T47D, SKBr3, MDA-MB-231 and MDCKII cells were cultured in DMEM plus 10% fetal bovine serum supplemented with penicillin, streptomycin and L-glutamine. MCF10A cells were cultured essentially as described (Debnath et al., 2003) and grown in DMEM/F12 plus 5% horse serum supplemented with penicillin, streptomycin, L-glutamine, 20 ng/ml epidermal growth factor, 0.5 µg/ml hydrocortisone, 10 µg/ml insulin and 100 ng/ml cholera toxin. All cells were maintained on standard tissue culture plastic in an 8% CO2 humidified incubator at 37°C. For immunostaining experiments, cells were seeded onto permeable 24-mm Transwell clear polyester filters, 0.4 µm pore size (Corning, NY), and allowed to grow until confluency. Thereafter, media was changed every day until desired time points were reached.
Immunostaining and analysis
Cell monolayers grown on permeable Transwell filters were fixed in 4% paraformaldehyde and permeabilized with 1% SDS in PBS for 10 minutes at room temperature, except for samples immunostained for mycCRB3 with the myc 4A6 antibody. These cells were instead permeabilized with 0.1% Triton X-100 in PBS to visualize CRB3 expression. Samples were then washed with PBS and immunostained as previously described (Straight et al., 2004). Regular fluorescence microscopy was performed with an inverted Leica DM IRB fluorescent microscope and data analyzed with SPOT image software (Diagnostic Instruments). Confocal images were taken with a Zeiss lSM 510 Axiovert 100 M inverted confocal microscope or with an Olympus FV500 inverted confocal microscope. Confocal images were analyzed with the Zeiss LSM 5 Image Browser software. All fluorescent images were prepared for publication with Adobe Photoshop and Adobe Illustrator software (San Jose, CA).
Retrovirus construction and infection
The png retroviral vector was generously provided by Steven Ethier (Barbara Ann Karmanos Cancer Institute, Detroit, MI). The mycCRB3-png, mycCRB3ERLI-png, mycCRB3mutFERM-png and mycCRB3mutFERM
ERLI-png retroviral constructs were produced by respective PCR amplification from the previously described mycCRB3-pSecTag, mycCRB3
ERLI-pSecTag, mycCRB3FERMmut-pSecTag and mycCRB3FERMmut
ERLI-pSecTag constructs (Roh et al., 2003
). The amplified mycCRB3 PCR products were cloned into the NotI/XhoI sites of the png retroviral vector. The mycCRB3mutRP-png construct was produced by PCR-directed site-specific mutagenesis using the mycCRB3-pSecTag vector as template. The mycCRB3mutRP sequence was then cloned into the NotI/XhoI sites of the png vector. The sequences of all constructs were verified by automated sequencing at the University of Michigan DNA Sequencing Core.
Amphotropic retroviruses were produced by transfection of DNA constructs into the retroviral producer Phoenix-Ampho cell line. Viral supernatants were collected 48 hours post-transfection, filtered through a 0.45 µm membrane, and either used immediately or stored at 80°C. MCF10A cells were infected with viral supernatants according to standard procedures as described (Debnath et al., 2003). 48 hours post-infection cells were passaged into selection media containing 1 µg/ml puromycin to select for cells stably expressing the retroviral vector. Cells were selected for 5-6 days before use, or until all mock-infected cells had died.
Immunoprecipitation and immunoblotting
Cell lysates were prepared from confluent plates of cells as previously described (Straight et al., 2004). Protein concentrations of cell lysates were determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories). For immunoprecipitations, 1 mg protein from each cell lysate sample was incubated overnight with 5 µl purified antibody at 4°C. The next day samples were incubated with 60 µl of 50% slurry protein A-sepharose (Zymed) for an additional 1-2 hours, then washed three times with wash buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% Triton X-100 and 10% glycerol) and finally resuspended in LDS loading buffer (Invitrogen). Resolution of samples by gel electrophoresis was performed using the NuPage Novex gel system (Invitrogen) as previously reported (Straight et al., 2004
). Gels were transferred to nitrocellulose membranes and immunoblotted as previously described (Straight et al., 2004
). Equal loading was confirmed by Ponceau S staining.
Transmission electron microscopy
Cells on Transwell filters were fixed with 2.5% glutaraldehyde in 0.1 M Sorensen's buffer pH 7.4 containing 0.1% Ruthenium Red for 30 minutes at 4°C. After washing twice with Sorensen's buffer, the filters were post-fixed in 1% osmium tetroxide in Sorensen's buffer for 15 minutes at 4°C. After two further washes with Sorensen's buffer, cells were stained with 3% uranyl acetate in water for 15 minutes at room temperature and dehydrated in ethanol/water mixtures. The filters were embedded in LX112 resin (Ladd Research). Ultrathin sections (50-70 nm) were placed on carbon/Formvar-coated copper grids and stained post sectioning with uranyl acetate and Reynold's lead citrate. The grids were imaged under a Philip CM100 electron microscope.
Transepithelial electrical resistance measurements
Cells were grown on permeable Transwell filters, and transepithelial electrical resistance (TER) measurements were determined with a Millicell-ERS volt-ohm meter (Millipore, Billerica, MA) according to manufacturer's instructions. Background resistance was determined using cell-free filters. Samples for each time point were measured in triplicate.
Quantification of tight junction formation
For quantification purposes, a tight junction structure was defined as a completely enclosed ring of smooth, contiguous, apical ZO-1 staining. Cell samples were immunostained for ZO-1 and examined with a Leica DM IRB fluorescent microscope. For each cell sample, the number of tight junction structures were counted in three different fields under 20 x magnification (>100 cells per field) and the mean number of tight junction structures per field was determined. Experiments were performed three times and the mean number of tight junction structures per field from three independent experiments was calculated. Data were plotted using the GraphPad Prism data analysis software (GraphPad Software). Error bars represent s.e.m.
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Results |
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We co-stained mycCRB3-expressing MCF10A cells for ZO-1 and a variety of known tight junction marker proteins. PALS1 and PATJ are cytoplasmic CRB3-associated proteins, which are specifically localized to tight junctions in epithelial cells (Lemmers et al., 2002; Roh et al., 2002
). Claudin-1 and occludin are integral membrane proteins that constitute the intermembrane strands of the tight junction complex; the claudin proteins are believed to form pores that directly mediate the selective permeability properties of tight junctions (Schneeberger and Lynch, 2004
). PALS1 and PATJ display a diffuse, cytoplasmic staining pattern in control MCF10A cells, whereas claudin-1 and occludin display a fragmented apical junctional staining (Fig. 4A). Upon expression of exogenous CRB3, all four proteins were recruited to tight junction structures containing ZO-1, and all displayed a smooth, continuous staining pattern (Fig. 4A). Western blotting experiments showed that the introduction of exogenous CRB3 does not alter the total expression levels of these tight junction proteins, or of ZO-1 (Fig. 4B).
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We undertook a mutagenesis study to determine the sequences of CRB3 that are required for the induction of tight junctions. The CRB3 intracellular sequence contains two motifs that are conserved with Drosophila Crumbs and all identified Crumbs isoforms: a juxtamembrane FERM binding motif with similarity to that found in glycophorin C, and a C-terminal PDZ binding motif, `ERLI' (Izaddoost et al., 2002; Klebes and Knust, 2000
; Makarova et al., 2003
). A comparison of the CRB3 sequence across several mammalian species (mouse, human, dog, pig and cow) also revealed an intracellular conserved `RxPPxP' sequence, which is specific to the CRB3 isoform and has similarity to an SH3 domain binding site (see Fig. S1 in supplementary material). We generated specific mutations in these three motifs (Fig. 7A) and expressed these CRB3 mutants as myc-epitope tagged proteins from retroviral vectors. These retroviral constructs were used to infect MCF10A cells and stably expressing pools were selected. Western blotting of these pools showed that all mycCRB3 mutants were expressed at the same level as wild-type mycCRB3 (Fig. 7B). Immunofluorescence analysis showed comparative levels of protein expression in individual cells, and also similar cellular localization (data not shown). As expected, deletion of the ERLI PDZ binding motif abolished interaction between CRB3 and PALS1 in MCF10A cells. Mutations in the FERM binding motif and RxPPxP motif, however, had no effect on CRB3 binding to PALS1 in these cells (Fig. 7C).
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MCF10A cells expressing the different mycCRB3 constructs were seeded onto permeable membranes and grown for 4 days past confluence as before. Staining for the ZO-1 protein revealed a marked difference in tight junction formation between the different cell lines. Point mutations in the RxPPxP motif did not affect the ability of CRB3 to induce tight junction structures in MCF10A cells; however, loss of the ERLI sequence, or point mutations in the FERM binding motif, severely compromised the ability of CRB3 to induce tight junctions (Fig. 8A-C). Mutations in the FERM binding motif appeared to result in a more severe defect than loss of the PDZ binding motif, as tight junctions were still sometimes seen in the mycCRB3delERLI cells (Fig. 8A, arrows). In order to verify this, we quantified the number of tight junction structures induced by the different CRB3 constructs. A tight junction structure was defined as an enclosed ring of contiguous apical ZO-1 staining; Fig. 8B shows the average number of such tight junction structures formed per field in 4 days post-confluent cells. These quantitative results confirm that the mycCRB3ERLI protein retains a partial, if weak, ability to induce tight junctions above levels seen in the vector control MCF10A cells. In contrast, the mycCRB3mutFERM protein is almost completely unable to induce tight junctions at this time point.
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We have noted that the number of tight junction formed in MCF10A cells increases with time in culture. Therefore, we cultured our cells for 9 days post confluence and compared the mycCRB3ERLI and myCRB3mutFERM cell lines at this time point (Fig. 8C). After 9 days, patches of ZO-1-positive tight junction-like structures have expanded to cover the entire sample of wild-type mycCRB3-expressing cells. However, these structures continue to be very rare in the control vector MCF10A cells, even after 21 days in culture (Fig. 8C and data not shown). By 9 days post confluence a small number of tight junction structures could be seen forming in the mycCRB3mutFERM cells. However, more tight junction structures as defined by ZO-1 staining had formed in the mycCRB3
ERLI cells. These results are consistent with those seen at the earlier 4-day time point, and show that both the CRB3
ERLI and CRB3mutFERM proteins have partial, albeit weak ability to induce ZO-1-containing junctions. In contrast, a mycCRB3 construct which lacked both the ERLI sequence and contained mutations in the FERM binding motif was completely unable to induce these junctions in MCF10A cells, even after 9 days post-confluent growth (Fig. 8C).
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Discussion |
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How may CRB3 act to promote the formation of tight junctions? Numerous studies now support a critical role for the proteins of the Par6 complex in the development of epithelial tight junctions. One possibility is that CRB3 acts to recruit and/or stabilize the Par6 complex at the developing tight junction. Genetic evidence in Drosophila suggests that the proteins of the Crumbs and Par6 complexes are mutually dependent upon one another for proper targeting and stabilization at the subapical domain (SAC), or marginal zone, a domain in Drosophila epithelia that is spatially analogous to the mammalian tight junction (Bilder et al., 2003; Tanentzapf and Tepass, 2003
). We have recently shown that the mammalian Crumbs complex is physically linked to the Par6 complex through a direct interaction between PALS1 and Par6 (Hurd et al., 2003
). In MDCK cells, mislocalization of PALS1 or inhibition of its expression both result in the mislocalization of aPKC
, a member of the Par6 complex, away from tight junctions (Hurd et al., 2003
; Straight et al., 2004
). Defects in tight junction development also result. It may be that CRB3 is required to recruit/stabilize PALS1 at tight junctions, which in turn leads to the recruitment and stabilization of the Par6 complex there. This hypothesis is consistent with genetic evidence in Drosophila that Crumbs, Stardust (the Drosophila PALS1 homologue), and PATJ are all mutually dependent upon one another for proper localization to the subapical complex (Bachmann et al., 2001
; Hong et al., 2001
; Tepass et al., 2001
).
Our mutagenesis experiments support the idea that CRB3 binding to PALS1 is crucial to its ability to promote tight junction formation. Loss of the conserved PALS1 binding ERLI sequence severely compromises the ability of CRB3 to induce tight junctions. In addition to binding Par6, PALS1 also mediates association between CRB3 and PATJ. PATJ contains multiple PDZ domains and binding partners for many of these domains remain unidentified. It is possible that PATJ, too, may act downstream of PALS1 to regulate tight junction formation through as yet unknown effectors. A recent study reported that the Drosophila homologue of PATJ can interact directly with Drosophila Par6, potentially providing another mechanism by which PALS1 may indirectly promote association of CRB3 with the Par6 complex (Nam and Choi, 2003). Moreover, another recent study has reported that the ERLI sequence of CRB3 may itself bind directly to Par6 (Lemmers et al., 2004
). Thus, the conserved ERLI motif may link CRB3 to the Par6 complex through multiple mechanisms.
Although loss of the ERLI sequence resulted in defects in the ability of CRB3 to induce tight junctions, we observed that the CRB3delERLI protein retained a partial ability to induce tight junction formation. This suggests that other domains in CRB3 are also involved in tight junction formation. We found that mutations in a conserved `RxPPxP' motif did not affect the ability of CRB3 to induce tight junctions. However, mutations in the FERM binding motif severely disturbed the ability of CRB3 to promote tight junction formation and led to a more severe loss of function than did loss of the ERLI sequence. The CRB3mutFERM protein retained the ability to interact with PALS1, suggesting that a PALS1-independent mechanism is required for tight junction formation. This is supported by experiments in which we have found that overexpression of PALS1 alone in MCF10A cells was not sufficient to induce tight junction formation (data not shown).
The FERM binding motif of CRB3 is expected to interact with a protein containing a FERM domain. The FERM domain is a protein-protein interaction domain found in a diverse set of proteins, many of which function as adaptors that link transmembrane proteins to the cortical actin cytoskeleton (Chishti et al., 1998). Such a FERM protein may be involved in linking CRB3 to the actin cytoskeleton, and such linkage may stabilize the CRB3 complex at the plasma membrane. Additionally, recruitment and reorganization of cytoskeletal elements through the FERM protein may be involved in tight junction formation. At this moment, the FERM protein that binds to the Crumbs FERM binding motif is still unknown. In Drosophila embryos and S2 cells, a physical interaction has been reported between Crumbs and the FERM-containing protein dMoesin, a protein that has homology to the mammalian ezrin/radixin/moesin family of ERM proteins (Medina et al., 2002
). However, definitive evidence that dMoesin functions in the Crumbs polarity pathway is still lacking. An interesting new study identified Mosaic Eyes, or Moe (not to be confused with moesin) as a novel FERM protein required for tight junction formation in the zebrafish retinal pigmented epithelium (Jensen and Westerfield, 2004
). Genetic experiments in zebrafish suggest that Moe may act in the same pathway as Crumbs. However, a physical interaction between these proteins has not been confirmed. In our own studies, we have found that the FERM binding domain of CRB3 is able to interact with several different FERM proteins in vitro (our unpublished data). Thus the identification of the functional FERM protein partner of Crumbs remains an important challenge.
Previous experiments in MDCKII cells have suggested that CRB3 acts to regulate epithelial tight junction formation. Overexpression of CRB3 in MDCKII cells leads to a delay in tight junction formation in the calcium switch assay. This phenotype was dependent on the presence of the ERLI sequence: a CRB3 construct that lacked the ERLI sequence was unable to delay tight junction formation when overexpressed in these cells (Lemmers et al., 2004; Roh et al., 2003
). However, mutations in the FERM binding motif did not affect the ability of overexpressed CRB3 to disturb tight junction formation in MDCKII cells (Roh et al., 2003
). Our current study thus provides the first evidence that the CRB3 FERM binding motif is involved in tight junction formation. Experiments in Drosophila have shown the importance of the FERM binding motif in epithelial cell polarization, and have also shown that the Crumbs FERM and PDZ binding motifs can act separately to regulate distinct processes. Both the FERM binding and PDZ binding motifs are required for the rescue of epithelial cell polarity in Drosophila embryos mutant for the crumbs gene. Crumbs overexpression as well as underexpression also leads to defects in epithelial cell polarity in Drosophila; however, the FERM binding motif, but not the ERLI sequence, is dispensable for the overexpression phenotype (Klebes and Knust, 2000
). Recently, the function of Crumbs has also been studied in the development of Drosophila photoreceptors. Overexpression in Drosophila photoreceptors of a Crumbs construct lacking the ERLI sequence but retaining the FERM binding domain led to ectopic localization of adherens junctions, but did not alter the localization of the Drosophila PATJ homologue. On the other hand, overexpression of a Crumbs construct containing the ERLI sequence but lacking the FERM binding motif led to mislocalization of PATJ, but not the mislocalization of adherens junctions (Izaddoost et al., 2002
).
It is not known whether the FERM and PDZ binding motifs act through completely parallel signaling pathways that are able to partially compensate for one another, or if some level of cross-talk exists between pathways that may partially compensate for mutations in either motif. In this respect, it is interesting to note that PALS1 also contains a FERM binding motif (Kamberov et al., 2000), although PALS1 interaction with a FERM protein has not been demonstrated. FERM domains may simultaneously interact with multiple partners (Han et al., 2000
); it is therefore possible that a FERM protein may simultaneously interact with both CRB3 and PALS1 and act to help stabilize the complex in vivo. Such a FERM protein may act as a bridge to recruit a low level of PALS1 into the complex even in the absence of the CRB3 ERLI sequence. Similarly, PALS1 may act to recruit a small amount of FERM protein into the complex even if CRB3 itself contains mutations in its FERM binding motif. Such interactions may explain the partial activities we see in our CRB3 mutants. We found that loss of both the PDZ binding and FERM binding motifs led to a complete loss of the ability of CRB3 to induce tight junction formation in our MCF10A cell system.
The formation of tight junctions is intimately involved with the development and maintenance of epithelial cell polarity. When cultured in a three-dimensional matrix of collagen or reconstituted basement membrane, MDCKII cells are able to form multicellular cysts with defined lumens and apico-basal polarity (O'Brien et al., 2001). We and others have shown that overexpression of CRB3 or PALS1 in MDCKII cells disrupts the polarization of such cysts, and leads to defects in lumen formation (Lemmers et al., 2004
; Straight et al., 2004
). Mammary MCF10A cells also form hollow, polarized cysts when cultured in a three-dimensional matrix of reconstituted basement membrane (Debnath et al., 2003
; Muthuswamy et al., 2001
). However, we have found that overexpression of CRB3 appears to have little effect on the development and polarization of MCF10A cysts (our unpublished data). Immunostaining for ZO-1 and other tight junction markers in MCF10A cysts has not been reported, and our own attempts have been complicated by technical difficulties; therefore, the effect of exogenous CRB3 expression on tight junction formation in three-dimensional MCF10A cyst structures cannot be assessed.
In summary, we have found that expression of exogenous CRB3 is sufficient to induce tight junction formation in a cell line that normally lacks tight junctions. When grown in monolayer culture, wild-type MCF10A cells express some tight junction marker proteins such as ZO-1, claudin-1 and occludin at the apical/basolateral boundary; however, these proteins appear only in a fragmented staining pattern. This fragmented staining may represent nascent tight junction structures in MCF10A cells. Expression of exogenous CRB3 leads to a coalescence of such nascent structures into the smooth, contiguous, apical structures typical of tight junctions. This leads us to conclude that Crumbs3 expression appears crucial for proper tight junction formation.
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Acknowledgments |
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Footnotes |
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References |
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