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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON
Institute for Molecular Bioscience, University of Queensland, St. Lucia, Queensland, Australia
Submitted 17 February 2008 ; accepted in final form 21 June 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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cyst; epithelia; cadherin; MDCK
-catenin, to form the cadherin-catenin complex (43), which associates with the actin cytoskeleton and with signaling proteins (35). Continual trafficking of newly synthesized E-cadherin to and from the cell surface is essential to ensure the dynamic formation of adherens junctions and to modulate cadherin-based adhesion during morphogenesis and wound healing (5). The correct delivery of E-cadherin specifically to the lateral cell membrane is a critical step in its trafficking in polarized cells (28). Previous studies from our laboratory revealed that E-cadherin in HeLa and Madin-Darby canine kidney (MDCK) cells is trafficked from the trans-Golgi network (TGN) to the recycling endosome (RE) before its delivery to the basolateral membrane (24). The RE compartment was initially designated for recycling proteins, such as transferrin-loaded transferrin receptor (TfR), back to the cell surface following endocytosis (31, 42). More recently, the RE has been increasingly recognized as a way station for post-Golgi exocytosis and sorting of membrane and soluble cargo proteins destined for the cell surface (2, 6, 12, 17, 25, 29). In nonpolarized or early-polarized cells, basolateral proteins travel from the Golgi complex to the cell surface via the RE (2, 24), where, according to recent studies, these proteins are also sorted (12). Some apical proteins also appear to traverse the RE, although this route remains more controversial (10). Studies tracking basolateral proteins including newly synthesized TfR and vesicular stomatitis virus G (VSV-G) protein suggest that the RE may only be transiently used as a basolateral exocytic route before epithelial polarization, after which a more direct route to the cell surface is favored (17). Whether E-cadherin trafficking via the RE occurs throughout the whole process of epithelial cell polarization and morphogenesis remains to be tested, and this issue formed the focus for the current studies.
The small GTPase, Rab11a (referred to here as simply Rab11), is a well-known marker of the apical RE (15), and it operates to regulate epithelial polarity and membrane traffic into and out of the RE (11, 37). Rab11 acts through a variety of effectors including members of the Rab11-interacting proteins family, Rab11-FIPs (36), and myosin Vb (20). Experiments in nonpolarized mammalian cells showed perturbed E-cadherin trafficking in the presence of Rab11 mutants (24). Rab11 also interacts with components of the exocyst complex as part of the trafficking machinery at the RE (32), and loss of function of exocyst in Drosophila epithelial tissues implicated Rab11 in Drosophila epithelial-cadherin (DE-cadherin) trafficking throughout morphogenesis (19).
In the current study, we have used Rab11 mutants to investigate the further requirement for Rab11 and the RE in E-cadherin trafficking throughout the development of polarity and cyst formation in epithelia. The results revealed a continued dependence on an intact and functioning RE and on normal Rab11 GTP cycling for correct basolateral delivery of E-cadherin. Additionally, we showed that the RE is more globally required for the critical early stage of lumen formation during epithelia cyst morphogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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S1-hE-cad-GFP or S1-hE-cad-Cherry) have been described previously (24, 27). The hE-cad and S1-hE-cad were tagged in the hE-cad-GFP or S1-hE-cad-GFP plasmids by replacing the GFP-encoding sequence with a PCR-generated mCherry fragment from the pRSETB-Cherry vector (a generous gift from Dr. R. Tsien, University of California, San Diego). Monolayers of wild-type MDCK strain II cells and MDCK cells stably expressing Rab11-GFP or DN-Rab11-GFP were grown and passaged in Dulbecco's modified Eagle's medium (GIBCO Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum and 4 mM L-glutamine at 37°C, in an atmosphere of 5% CO2 and 95% air as previously described (28). Cells were plated on glass coverslips or on Transwell polycarbonate filters (Corning Costar, Cambridge, MA) at various densities and were allowed to polarize for up to 7 days.
Transfection and microinjection. MDCK cells were plated at subconfluent densities 24 h before being transfected with the appropriate plasmid(s) using the LipofectAMINE 2000 system (Invitrogen) following the manufacturer's instructions. Cells were typically left to recover for 4–36 h posttransfection before use. For stable expression, transfected cells were passaged and maintained in medium containing Geneticin (G418 Sulphate; Invitrogen) for 10–14 days. Surviving cells were ring-cloned, and clonal lines with varying expression levels were grown. Clones with relatively low expression levels were selected by immunofluorescence microscopy and immunoblotting for experiments here; two or more clonal lines were used for most experiments.
Microinjection was carried out on confluent monolayers grown on glass coverslips of either MDCK cells transiently expressing hTfR and left to recover in fresh medium for 18 h, or MDCK cells stably expressing Rab11-GFP. Individual coverslips were transferred to a 35-mm dish with CO2-independent medium and placed under an Axiovert 200 inverted microscope (Carl Zeiss) to select target cells for microinjection. Purified hE-cad-GFP or hE-cad-Cherry (at 0.1 µg/µl) were microinjected into the cytoplasm using an AIS-2 Micromanipulation Injection System (Cell Biology Trading, Hamburg, Germany; Dr. Rudolph Kern). Microinjection needles were made from borosilicate glass 1.2-mm capillary tubes (Harvard Apparatus, Kent, UK) drawn into fine tips using a Flaming/Brown Micropipette Puller-P97 (Sutter Instrument). Cells stably expressing Rab11-GFP were microinjected with hE-cad-Cherry and left to recover for 6 h. Microinjected cells transiently expressing hTfR were left to recover for 4 h to allow expression of hE-cad-GFP, before labeling of the RE using human transferrin (hTfn) as described in Recycling endosome labeling with transferrin and endosomal inactivation assay. Cells were then fixed and assayed.
Cystogenesis in three-dimensional cultures.
Untransfected MDCK cells and lines stably transfected with Rab11-GFP, DN-Rab11-GFP, or S1-hE-cad-GFP were grown in three-dimensional (3D) cultures by plating them as an overlay on a thin base of extracellular matrix using a glass chamber slide support (26). Matrigel HGF (BD Biosciences, San Jose, CA) extracellular matrix was spread as a thin layer on precooled eight-well glass chamber slides (Nunc LabTek ll, In Vitro Technologies) and allowed to gel at 37°C. Cell monolayers were trypsinized and resuspended at 2 x 104 cells/ml in standard MDCK growth media. The single-cell suspension was mixed 1:1 with normal media containing 4% Matrigel (2% final concentration), and 200 µl of this was overlaid onto the set Matrigel base. The cells were fed every 3 days with 2% Matrigel in fresh media and grown for up to 10 days before being fixed and assayed.
Recycling endosome labeling with transferrin and endosomal inactivation assay. To specifically label the RE with hTfn, MDCK cells were transfected with hTfR and left to recover in fresh media for 18 h, and 10 µg/ml Texas Red-labeled hTfn (TR-hTfn) was then added for 1.5 h and chased for 45 min with fresh media before fixation or live imaging recording.
A horseradish peroxidase (HRP) inactivation assay was modified from the protocol of Ang et al. (2) and used here as previously described (29). In brief, hTfR-transfected MDCK cells were incubated with 10 µg/ml HRP-hTfn in media plus 1 µg/ml TR-Tfn (for visualization of the uptake) for 30 min in the dark at 37°C. Cells were washed twice in ice-cold PBS, and surface-bound HRP-hTfn and TR-hTfn were removed by two 5-min washes with 0.15 M NaCl and 20 mM citric acid, pH 5. Cells were then washed twice with ice-cold PBS and incubated in the dark for 1 h with PBS containing 0.1 mg/ml diaminobenzidine (DAB), and 0.025% H2O2 was added to the inactivation sample (the control contained DAB but no H2O2). Cells were finally washed twice in PBS containing 1% BSA to stop the reaction and were incubated in prewarmed media for up to 4 days.
Immunofluorescence and fluorescence imaging. Cells grown on coverslips were fixed in 4% paraformaldehyde (PFA) in PBS for 60 min, permeabilized using 0.1% Triton X-100 for 10 min, and then stained as previously described (28). Fixation and immunostaining of cells grown in 3D cultures used a modification of published methods (26). Briefly, cells on glass chamber slides were fixed in 4% PFA/PBS for 30 min, permeabilized in 0.5% Triton X-100 for 10 min, blocked in PBS/fish skin gelatin/Saponin, then stained with primary antibodies and fluorescently tagged secondary antibodies for microscopic investigations. For antibodies requiring methanol fixation, incubation with –20°C cold methanol for 10 min was used.
Confocal imaging of fixed cells and live cell imaging were performed using an LSM 510 META confocal microscope (Carl Zeiss) using optical spectral separation. Single images were captured with an optical thickness of 0.7–1.7 µm. For Z series, a 0.34- to 0.5-µm step interval was used. The thresholds for the fluorescence intensity of each channel were carefully adjusted to most closely represent the signal strength of the original 2D images collected. Analysis was performed using LSM510 META software (Carl Zeiss MicroImaging) and Photoshop CS2 (Adobe).
Quantification of colocalization between hTfn and biosynthetic hE-cad, based on the Pearson's correlation coefficient (R), was performed on 3D cell reconstructions generated in Volocity v3.7 (Improvision). The thresholds of intensities for each fluorophore were predetermined for each cell using ImageJ v1.37p (National Institutes of Health, Bethesda, MD). For specifically measuring the degree of colocalization of hE-cad with hTfn in the RE, staining of E-cadherin at the membrane was removed from the equation by cropping the image before calculation.
Live cell imaging was performed on individual live cells grown on 35-mm dishes (MatTek). During imaging, the cells were immersed in CO2-independent medium at 37°C using a heated microscope stage mount. The selective photobleaching of E-cadherin at the plasma membrane was performed using the Zeiss LSM 510 software. On a still image of the cell to be recorded, an area of interest was manually drawn around the plasma membrane and was specifically bleached by illumination in the green channel with maximum laser power reiterated 50 times. Recording was performed on the confocal microscope, with excitation in the green and red channels and with frames recorded every 8 s. Videos were analyzed, cropped, and constructed using ImageJ v1.37p, Volocity v3.7, and Photoshop CS2 and were exported as Quick-Time videos (Apple) with a playback speed of 10 frames/s. In the supplemental data (available online at American Journal of Physiology-Cell Physiology website), Video 1 shows trafficking of vesicles containing newly synthesized hE-cad-GFP (green) in transfected MDCK cells, from the Golgi to the RE labeled with TR-hTfn (red), and toward the cell surface to make contact with the cell membrane.
Antibodies and reagents. Canine E-cadherin was recognized using the monoclonal antibody 3B8, obtained from Dr. W. Gallin (University of Alberta, Edmonton, Canada). Anti-human E-cadherin is a kind gift from Dr. A. Yap (University of Queensland, Brisbane, Australia). Antibody against Rab11 was obtained from BD Biosciences Pharmingen, and known markers of apical polarity and tight junction formation included rabbit anti-phosphorylated ezrin/radixin/moesin (pERM) polyclonal antibody (Chemicon) and rabbit anti-zonula occludens-1 (ZO-1) (N-term)-polyclonal antibody (Zymed). Secondary antibodies included Cy3-conjugated sheep anti-mouse and goat anti-rabbit IgGs (Jackson ImmunoResearch Laboratories, West Grove, PA), Alexa 647-conjugated goat anti-rabbit and goat anti-mouse (Molecular Probes), and Alexa 488-conjugated goat anti-rabbit and goat anti-mouse IgGs (Molecular Probes). Alexa 488-conjugated phalloidin and Texas Red-conjugated phalloidin were used to label F-actin, and 4,6-diamidino-2-phenylindole (DAPI) was used to label nuclei (Molecular Probes, Eugene, OR). HRP-hTfn was purchased from Jomar Diagnostics (Stepney, Australia), and TR-hTfn was purchased from Molecular Probes.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Overlap between staining of TR-Tfn and hE-cad-GFP was also studied in fixed cells (Fig. 1D). Preconfluent, nonpolarized transfected cells were examined after TR-Tfn uptake, and, to compare this to E-cadherin trafficking in fully polarized cells, individual cells in polarized monolayers were microinjected with hE-cad-GFP and allowed to take up TR-Tfn. In both cases we see significant colocalization of hE-cad-GFP with TR-Tfn in REs. Furthermore, the degree of overlap between hE-cad-GFP and the TR-hTfn-labeled RE was assessed on 3D reconstructions of preconfluent or fully polarized MDCK cells to account for the total pools of each marker in cells (Fig. 1D). Overlap of the two fluorescent signals was quantified as Pearson's correlation coefficient (R). Preconfluent cells showed significant colocalization of hE-cad-GFP and TR-hTfn with an average Pearson's coefficient of R = 0.354 ± 0.089. Other structures in the cells were labeled individually for hE-cad-GFP or TR-hTfn representing nonoverlapping transport steps. In polarized MDCK cells, a correlation coefficient of R = 0.268 ± 0.025 also indicated a significant overlap of these markers as evidence that trafficking of E-cadherin via the RE persists in fully polarized cells.
Thus, in both live and fixed MDCK cells, the transient overlap of hE-cad-GFP with TR-hTfn is new evidence that the RE is a way station for E-cadherin en route to the cell surface. Tracking microinjected hE-cad-GFP in fully polarized monolayers also shows that this route is maintained after the cells have become polarized.
E-cadherin surface delivery is dependent on the functional integrity of the recycling endosome. We next asked whether the RE is a requisite compartment for E-cadherin surface delivery and E-cadherin-based adhesion during the formation of cell monolayers. Preconfluent MDCK cells were cotransfected with hE-cad-GFP and hTfR and allowed to recover for 12 h posttransfection. The RE compartment was chemically inactivated by allowing the cells to take up HRP-hTfn, along with lower amounts of TR-hTfn (to visualize the uptake), and then treating with DAB and peroxide to inactivate the endosomes, as previously described (2, 29). Control cells were treated in the absence of peroxide, and in these cells, the RE remained functional and the cells grew to form a patent monolayer of adhesive cells, showing the typical pattern of endogenous E-cadherin staining with cobblestone boundaries and some intracellular staining (Fig. 2). Exogenous hE-cad-GFP was colocalized with endogenous E-cadherin at the cell membranes and inside the cells, and both proteins showed some overlap with TR-hTfn in the cytoplasm. In contrast, the inactivated cells showed mislocalization of both hE-cad-GFP and endogenous E-cadherin, with neither delivered to the cell membrane. Instead, the majority of hE-cad-GFP and depleted endogenous E-cadherin was concentrated intracellularly in a tightly defined TR-hTfn-positive compartment. The cells themselves were dysmorphic, with disrupted cell-cell contacts concomitant with reduced or absent E-cadherin staining at the surface. Cells expressing only endogenous E-cadherin retained their surface staining but showed increased intracellular accumulation and overgrowth of neighboring cells. Noticeably, individual cells that had not ingested HRP-hTfn, and therefore had functional REs, still had prominent surface staining of recombinant and endogenous E-cadherin.
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Recycling endosome regulates E-cadherin trafficking during the maturation of MDCK monolayers. Recent studies suggested that the RE is bypassed during the trafficking of some basolateral cargo, with a more direct route to the surface used in the later stages of cell polarization (17). We therefore investigated the role of the RE in E-cadherin trafficking during the establishment and maturation of polarized monolayers using Rab11 mutants to disrupt RE trafficking. Stable MDCK cells expressing low levels of either Rab11-GFP or dominant-negative DN-Rab11-GFP (GDP-locked form) were evaluated daily as they developed from sparse cells to a polarized monolayer. Endogenous E-cadherin and Rab11-GFP were localized at each time point (Fig. 3A). Preconfluent cells showed Rab11-GFP-labeled vesicles dispersed throughout the cytoplasm. E-cadherin staining in these cells was apparent in perinuclear vesicles, some of which were also positive for Rab11-GFP. By day 4, the cells were polarized and Rab11-GFP was increasingly expressed at the apical pole, emulating the very tight subapical location of the (apical) RE previously described in polarized cells (15). E-cadherin first appeared on lateral membranes in regions of cell-cell contact and then progressed over time to mark the entire basolateral cell borders in more confluent monolayers (Fig. 3A). Both E-cadherin and Rab11-GFP staining resembled those for endogenous E-cadherin and Rab11, respectively, in untransfected MDCK cells (data not shown).
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We next examined whether Rab11 plays a role in E-cadherin transport and sorting once the cells are already polarized in a monolayer. Fully polarized Rab11-GFP stable cells were microinjected with hE-cad-Cherry and allowed to recover for 6 h before analysis (Fig. 3C). Confocal microscopy of the monolayer in the XZ plane revealed colocalization of the apical Rab11-GFP compartment with vesicles positive for hE-cad-Cherry, which was also present at the basolateral plasma membrane. This suggested that in polarized MDCK cells, biosynthetic hE-cad-Cherry traffics through the apical RE visualized by Rab11 on its way to the basolateral membrane. Wild-type MDCK cells transiently expressing hE-cad-Cherry with and without Rab11-GFP, dominant-negative DN-Rab11-GFP, or S1-E-cad-Cherry were then examined (Fig. 3D). The recombinant hE-cad appeared correctly on the basolateral cell membrane together with endogenous E-cadherin. Some hE-cad-Cherry was also observed in the apical cytoplasm, consistent with our previous observation of it being in the Golgi or REs, en route to the plasma membrane. Cotransfection of Rab11-GFP did not perturb the basolateral expression of hE-cad-Cherry, as previously observed in Fig. 3C. However, coexpression of DN-Rab11-GFP in the subapical RE caused the missorting of newly synthesized hE-cad-Cherry to the apical membrane of polarized cells (Fig. 3D). The effects of DN-Rab11 on the trafficking of E-cadherin in fully polarized cells implicates the RE as an exocytic destination for this cargo, even in polarized cells. Finally, as further evidence that DN-Rab11 is causing missorting of E-cadherin during its exocytosis, we compared the fate of hE-cad in cells expressing DN-Rab11 with that of a targeting mutant of E-cadherin. The S1-E-cad mutant has a critical dileucine motif ablated, and, as a result, it is missorted in MDCK cells, appearing on the apical membrane (28). Expression of S1-E-cad-Cherry in MDCK cells here gives apical staining in pattern reminiscent of that caused by expression of DN-Rab11.
Rab11 is essential for E-cadherin trafficking and lumen formation during cyst morphogenesis. Many features of cells undergoing morphogenesis into polarized epithelia are revealed optimally during the growth of epithelial cysts (18). To examine the ability of Rab11-GFP (at low expression levels) and DN-Rab11-GFP MDCK cell lines to form cysts, cells were plated in Matrigel according to established protocols (26). Cells were grown for up to 10 days and were then fixed, stained, and examined by confocal imaging at various intervening times.
At 3 days, the Rab11-GFP cells began to form spherical structures with appearance of a lumen, delineated by strong actin staining (Fig. 4A). Rab11-GFP was expressed in the cytoplasm, in an increasing gradient from the basal to apical pole. By day 10, mature, patent Rab11-GFP cysts were formed with a single layer of cells organized spherically around a central hollow lumen. Rab11-GFP was localized in a tight band at the apical pole immediately underneath the actin staining (Fig. 4A), similar to the location observed for endogenous Rab11 in wild-type MDCK cysts (Fig. 4B). Endogenous E-cadherin localized at the basolateral membrane from the earliest time point and throughout cyst development (Fig. 4C). As further markers of polarity, we stained for ezrin-radixin-moesin in its phosphorylated form (pERM) and the tight junction-associated protein zonula occludens-1 ZO-1 (16). The pERM proteins are restricted to the subapical compartment of polarized epithelia where they link the plasma membrane and the cytoskeleton (for review, see Ref. 13). Accordingly, pERM staining appeared in a subapical pattern colocalized with the actin band (Fig. 4C). ZO-1 staining appeared in typical, single puncta on the apicolateral membranes between adjacent cells (Fig. 4D). Thus, cell polarity and cyst morphogenesis were unchanged in cells overexpressing modest levels of Rab11-GFP compared with cysts formed from untransfected cells examined over the same time course (Fig. 4B). In addition, the expression of Rab11-GFP did not perturb E-cadherin targeting to the basolateral membrane, one of the earliest events seen in cell polarization.
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Mistargeting of E-cadherin results in dysmorphic cysts.
Since lateral cell surface placement of E-cadherin is an early event during cyst formation as shown above, E-cadherin missorting is likely to be a factor responsible for the abortion of cyst formation observed with the DN-Rab11-GFP MDCK cells. To address this, the S1-E-cad sorting mutant (see Fig. 3D) was used to investigate the link between E-cadherin targeting and cyst formation. We generated a stable cell line expressing low amounts of S1-E-cadherin-GFP and cultured them as cysts for up to 10 days (Fig. 6). Compared with untransfected cells, cyst formation in S1-E-cadherin-GFP-expressing cells was impaired and resulted in dysmorphic cysts with, again, disrupted lumen formation. Indeed, most cysts remained as a ball of unordered cells with actin and pERM randomly localized around cell surfaces. Figure 6 demonstrates that within a cyst harboring a pseudolumen, S1-E-cadherin-GFP was found on the apical side of cells facing the lumen.
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S1-E-cad-GFP mutant also leads to loss of lumen formation and loss of cyst cell polarity. This further suggests that the correct sorting and trafficking of E-cadherin, via the RE, is critical for epithelial polarity and cyst formation. In addition, we have newly identified E-cadherin targeting via the RE as a critical event in epithelial lumen formation. |
DISCUSSION |
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Our findings support the growing concept of the RE as an important compartment for exocytosis of multiple proteins; the RE is often associated with the polarized delivery of proteins to cell surface domains in epithelial cells and in macrophages (2, 25, 29). In MDCK cells, the RE mediates the exocytic delivery of other basolateral proteins, VSV-G and TfR (2, 6, 17), although Gravotta et al. (17) showed TfR bypassing the RE on its direct journey to the cell surface in fully polarized epithelia. In the current study, newly synthesized E-cadherin still appears to traverse the RE throughout polarity. E-cadherin expressed in already polarized cells in monolayers colocalized transiently with the RE on its way to the cell surface. E-cadherin also maintained this dynamic overlap with the RE in cells forming cysts. Taken together our results suggest that E-cadherin needs a functional RE compartment and GDP-GTP cycling Rab11 for its correct targeting to the basolateral membrane even in polarized cells. Our results reveal distinct differences in the dependence of different cargo on the RE for trafficking in polarized cells. Such a difference may depend on cargo sorting into different exit carriers leaving the TGN, where E-cadherin is loaded into golgin-97 carriers for transport to the RE (23); the carriers for other basolateral cargo are unknown. Further studies on the post-TGN carriers and the TGN or RE-based sorting requirements for different basolateral cargo proteins may reveal different mechanisms for their routes of cell surface delivery.
Increasing evidence suggests that some sorting does indeed occur within the RE. Sorting adaptors have been localized to the RE, including subunits of the AP1B complex which have been linked to the RE sorting of VSV-G protein and TfR (2, 6). Basolateral sorting of E-cadherin by a membrane proximal dileucine motif (28), or by a type 1
phosphatidylinositol phosphate kinase (22), also appears to occur at the RE. Other trafficking components such as the VAMP3 SNARE protein, also implicated in adaptor-mediated sorting and trafficking from the RE, might additionally regulate E-cadherin trafficking (12).
Although there is significant overlap between E-cadherin and RE markers, this represents only a small proportion of total cellular E-cadherin and is suggestive of a transient residence in the RE for this newly synthesized protein. Indeed, a requisite role for the RE in E-cadherin trafficking has been described in Drosophila, despite a rather limited colocalization of E-cadherin with Rab11 in pupal epithelial cells of the dorsal thorax (19). Similarly, E-cadherin was rarely found colocalized with Rab11 in germline stem cells of the Drosophila ovary, although Rab11-null animals exhibited less E-cadherin at the membrane than controls (4). These studies all point to a crucial but fleeting functional relationship between E-cadherin and RE proteins, during the relatively rapid transit of new proteins, as described previously for transport of cargos between the TGN and the RE (6). There may be different routes through the RE itself, with different exocytic cargo accessing different RE compartments and machinery. Thus, VSV-G is regulated by and colocalizes with Rab8 in the RE (1) but E-cadherin traffic is unaffected by Rab8 mutants (data not shown) and is instead linked here with Rab11 in the RE. In macrophages, we have previously demonstrated that exocytic cargo, in the form of different cytokines, is seemingly segregated within the RE (25). In MDCK cells, the RE has subcompartments or even adjacent organelles typically defined as the apical RE classically attributed to Rab11 localization and a more basal common RE marked by Tfn (40). E-cadherin appears to localize transiently with both Rab11 and Tfn and further high-resolution imaging will be needed to determine whether this is sequential movement through distinct RE domains or whether it involves dynamic or joint subdomains of the RE. Mapping other RE markers will eventually help to define the precise role of this compartment and its physical relationship with sorting and exocytic trafficking. Thus, specific marker(s) of the RE can be attributed to specific trafficking events, as recently illustrated by the Arf6-dependent, and Rab11- or Rab27-independent pathways identified for recycling lipid rafts out of the RE to the plasma membrane during re-adhesion (3).
Disrupting RE function mistargeted E-cadherin to the apical membrane both in MDCK cells monolayers and in cysts. In assessing other markers of cell polarity, we found that ZO-1 at tight junctions was not disrupted in DN-Rab11 cells, suggesting that ZO-1 location is independent of the RE and is preserved in the presence of mistrafficked E-cadherin. Since ZO-1 itself is a cytoplasmic protein, it is unlikely to need the RE for trafficking to the plasma membrane. Despite the disrupted trafficking of E-cadherin in DN-Rab11 cysts, it appears that sufficient E-cadherin was still delivered to lateral membranes to support adherens junction formation and the subsequent assembly of tight junctions. Parenthetically, DN-Rab11 cells did have aberrant localization of normally apical pERM and F-actin, consistent with the novel finding here that apical membrane development is hindered by disruption of the RE and by E-cadherin mistrafficking. Of note, Rab11 is required for vesicular insertion along the lateral surface and for membrane growth during Drosophila cellularization (33). Reinforcing this notion, our mutant DN-Rab11 cells never formed a normal cyst structure, instead displaying cells of diverse sizes and heights consistent with disrupted membrane insertion. The aberrant apical delivery of E-cadherin in DN-Rab11 cells, emulated the missorting induced by the S1-E-cad sorting mutant. Because this mutant still binds β-catenin, which also gets missorted in MDCK monolayers (27), we speculate that the Rab11 mutation is disrupting cystogenesis by having potential adhesive E-cadherin all around the cell perimeter instead of confined to lateral membranes.
Nonetheless, the aberrant delivery of E-cadherin to the apical membrane with DN-Rab11-GFP or with the sorting mutant S1-E-cadherin has serious consequences for cyst formation, in particular the early, critical process of lumen formation. Thus, one important consequence of sorting in the RE is the exclusive targeting of E-cadherin to the basolateral membrane to engage epithelial polarity from early stages and to ensure morphogenesis is on track thereafter. Recent studies suggest an essential role for E-cadherin early on in establishing cell polarity (7, 30), and one that can be independent of cell-cell adhesion (9). Cells in these situations often retained cell-cell contacts. Our findings are consistent with earlier suggestions (9) that lateral membrane E-cadherin mediates a targeting patch for establishing cell polarity and lumen formation. During cyst morphogenesis, lumen formation is an early and critical step as it denotes the acquisition of the polarized phenotype of the cells. In epithelial cells, PTEN induces the apical segregation of phosphoinositides, apical membrane proteins, and Cdc42, for lumen formation (26). Our results suggest that the RE's critical role in lumen formation probably involves the trafficking of some of these proteins in addition to E-cadherin. The current study of MDCK cyst morphogenesis served to link lumen formation to Rab11 and RE function, and both of these aspects to E-cadherin targeting. Thus, this study provided new evidence in a mammalian system for the roles of the RE in E-cadherin trafficking and epithelial morphogenesis that were previously shown in Drosophila (8, 19, 39).
In conclusion, our study expands the function of the RE as a necessary compartment for the correct trafficking and sorting of E-cadherin to the plasma membrane during epithelial polarization and in mature epithelia. During morphogenesis, Rab11 is required for cyst formation including normal trafficking of E-cadherin, for development of polarity and for lumen formation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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