Histochem Cell Biol (2001) 116:371–378 DOI 10.1007/s004180100329
O R I G I N A L PA P E R
Jean-Claude Guillemot · Marianne Naspetti Fabrice Malergue · Philippe Montcourrier Franck Galland · Philippe Naquet
Ep-CAM transfection in thymic epithelial cell lines triggers the formation of dynamic actin-rich protrusions involved in the organization of epithelial cell layers Accepted: 24 August 2001 / Published online: 18 September 2001 © Springer-Verlag 2001
Abstract Thymic epithelium is organized in a highly connected three-dimensional network through which thymocytes differentiate. The molecular mechanisms underlying this organization are still unknown. In thymic medulla, a major site of tolerance induction, the development of the epithelial cell net is tightly regulated by the needs of thymocyte selection. These reticulated epithelial cells express high levels of the Ep-CAM molecule. Using different thymic epithelial cell lines as a model system, we found that transfection of Ep-CAM enhances cell growth and leads to a rapid reorganization of the actin cytoskeleton by inducing the formation of numerous stress fibers and long cell protrusions. Finally, the crosslinking of the extracellular domain of a chimeric CD25ec/Ep-CAMic molecule is sufficient to trigger the formation of protrusions. These results suggest that expression of Ep-CAM might balance the organizing capacity of cadherin molecules and may be participating in the formation of a dynamic stromal cell network in the thymus. Keywords Cell adhesion · Thymus · Ep-CAM · Epithelium
Introduction The development and organization of epithelial tissues involves a complex interplay between a variety of J.C. Guillemot and M. Naspetti are first co-authors M. Naspetti · F. Malergue · F. Galland · P. Naquet (✉) Centre d’Immunologie INSERM-CNRS de Marseille-Luminy, Case 906, Cedex 9, 13288, Marseille, Université de la Méditerranée, France e-mail:
[email protected] Tel.: +33-4-91269462, Fax: +33-4-91269430 J.-C. Guillemot CNRS LNB, Université de Provence, Chemin de J Aiguier, Marseille 13009, France P. Montcourrier CNRS UMR 5539, Université Montpellier II, 34095 Montpellier Cedex 5, France
adhesion molecules allowing the proper segregation of cell types and establishment of tissue polarity. These molecules contribute to the structure of cell–cell or matrix–cell junctions providing stability, tightness, and connectivity to the tissue (Eaton and Simons 1995). They also participate in the plasticity observed during active phases of tissue remodeling by regulating cell activation and motility. Some adhesion molecules such as cadherins play a major role in the establishment of the epithelial phenotype and accumulate at adherens junctions (AJs; Adams and Nelson 1998). Indeed, the formation of AJs is preceded by the accumulation of cortical actin in puncta, where E-cadherin molecules will be recruited in the vicinity of α-catenin molecules. This primary scaffold will progressively allow the formation of stable AJs (Vasioukhin et al. 2000). Then, desmosomes further stabilize cell layers by recruiting keratin filaments at the junction between epithelial cells. During tissue remodeling, the initial events will require the rapid reorganization of cortical actin and the signals involved in the triggering of this process are not fully understood. As for many other epithelial organs, the formation of the thymus is impaired by antibodies interfering with cadherin-dependent cell interactions (Muller et al. 1997). The adhesive function of cadherins can be regulated by a variety of extra- or intracellular signals, including other adhesion molecules. The Ep-CAM molecule is one example of a non-cadherin cell adhesion molecule which does not form junctional complexes by itself, at least in L cell fibroblasts (Balzar et al. 1999a), but regulates cadherin-dependent cell adhesion (Litvinov et al. 1997). It is widely expressed by epithelial tissues and its level of expression is upregulated on actively proliferating benign or malignant tissues (Momburg et al. 1987). EpCAM acts as a homophilic Ca2+-independent molecule via its extracellular domain which contains EGF-like repeats, a thyroglobulin-like and a nidogen-related domain (Balzar et al. 1999b). Upon transfection of EpCAM in fibroblasts, transfected cells segregate from control cells (Litvinov et al. 1994a). This regulatory
372
function is in large part assumed by the intracellular domain which possesses alpha-actinin-binding sites connecting Ep-CAM to the actin network (Balzar et al. 1998). Overexpression of Ep-CAM in E-cadherinexpressing cells leads to a disorganization of AJs by accumulation of Ep-CAM in the vicinity of cadherin molecules. Thus, one of the critical parameters of EpCAM function might be related to the level of expression which could act by titrating other regulators of cell adhesion and modifying the dynamics of epithelial tissue organization. Ep-CAM is also expressed by the thymic epithelium (Nelson et al. 1996). This epithelium comprises distinct cell subsets and has the unique feature of forming a three-dimensional network of reticulated cells through which thymocytes migrate and mature (van Ewijk et al. 1999). In the thymic medulla, the presence of a subset of activated epithelial and dendritic cells is required for deletion of autoreactive thymocytes. These cells express high levels of Ep-CAM molecules and in their absence, as in relB-mutant mice, the remaining thymic epithelium is disorganized and tolerance induction is impaired (Laufer et al. 1996; Naspetti et al. 1997). This suggests that a normal medullary structure is a prerequisite to fulfill the needs of thymocyte selection; it also suggests that Ep-CAM expression might be required to organize this microenvironment. We report here that transfection of Ep-CAM or its intracellular domain in thymic epithelial cell lines triggers the appearance of long actin-rich cell filopodia which form rapidly at the proximity of Ep-CAM+-neighboring cells. Ep-CAM expression is associated with enhanced growth properties and disorganization of epithelial cell layers in vitro.
(Gibco-BRL), following the manufacturer’s instructions. Ep-CAM expressing clones were grown under G418 (Gibco-BRL) selection (1 mg/ml) and sorted by flow cytometry (FACStar+; Becton Dickinson). Proliferation assays Cells were grown in a DMEM/10% heat-inactivated FCS with sodium pyruvate medium with 1 mM HEPES. Proliferation assays were performed by plating CFSE (Molecular Probes)stained cell lines in 24-well plates for indicated times and harvesting them by trypsin-EDTA treatment prior to analysis. Cytofluorometric data were analyzed on a FACScan apparatus and different gates were set on cells that had not been cultured (considered as time 0 reflecting 100% of the staining), and on cultured samples in which the decay of CFSE fluorescence was reduced twofold after each cell division. Results corresponding to the percentage of CFSEstained cells in each gate are representative of cell division. Alternatively, the cell lines were seeded at low cell concentrations in 96-well plates and cultures pulsed with 1 µCi tritiated thymidine on day 2. Samples were harvested and thymidine incorporation evaluated using a Packard cell counter. Results represent the mean of triplicate samples.
L-glutamine,
Microscopic analysis of tissue sections and cell cultures Frozen thymic sections were performed as previously described. For co-culture studies, the Ep-CAM–or+ MTE4–14 cells were stained with a CFSE dye prior to culture on plastic wells and mixed with Ep-CAM–or+ MTE1D cells. Pictures of confluent cultures were taken after 48 h either in phase contrast or fluorescent microscopy. For cell culture staining experiments using FITClabeled phalloidin, cell lines were seeded at different densities, grown to subconfluence on uncoated glass coverslips, and permeabilized using 0.1% Triton X-100. The effect of the C3 toxin (300 ng/ml) was assessed after 16 h of treatment followed by actin staining. Texas red–phalloidin staining and SEM analysis were performed as described (Guillemot et al. 1997). In experiments using anti-CD25 or control anti-vanin-1 mAb-coated beads, the CD25/Ep-CAM MTE4–14-expressing cells were incubated with the beads for 20 min and then directly permeabilized and stained with phalloidin.
Materials and methods Antibodies, reagents, and cell lines
Scanning electron microscopy
Texas red-conjugated phalloidin was purchased from Molecular Probes. Cell culture reagents were from Gibco-BRL. The C3 toxin was a gift from P. Boquet. The cell lines MTE1D or MTE4–14 have already been described (Aurrand-Lions et al. 1996). The anti-Ep-CAM G8.8 mAb (Nelson et al. 1996) was provided by B. Kyewski/A. Farr. The anti-vanin-1 407 mAb, and the 29 and 95 mAb to medullary epithelial cell subsets were previously described (Aurrand-Lions et al. 1996; Naspetti et al. 1997). The mAb 29 was recently shown to recognize an Ep-CAM epitope (Naspetti et al. 2000).
Cells were grown on uncoated glass coverslips and fixed in 2.5% glutaraldehyde/0.1 M sodium cacodylate/0.1 M sucrose, pH 7.2, for at least 5 days. Samples were gradually dehydrated using increasing concentrations of ethanol and processed for SEM as described (Brunk et al. 1981), followed by critical-point drying with CO2 and gold sputtering. The samples were observed in a Hitachi S4000 scanning electron microscope at 15 kV.
Ep-CAM cloning and transfection
Ep-CAMhigh epithelial cells in thymic medulla have a reticulated morphology
All constructs were made using standard cloning procedures. The Ep-CAM cDNA was cloned from a thymus cDNA library and fully sequenced. The complete cDNA was inserted in pcDNA3 (Clontech). The Ep-CAM/GFP construct was built by insertion of the Ep-CAM cDNA sequence in the N3 vector, in frame with the GFP epitope (Clontech). The CD25/Ep-CAM construct was made by in frame addition of the extracellular and transmembrane domains of the human CD25 molecule (Castellano et al. 1999) to the intracellular domain of murine Ep-CAM. Stable and transient transfectants were established using lipofectamine transfection
Results
Ep-CAM expression is regulated during thymic development both on lymphocytes and stromal cells (Borkowski et al. 1996; Nelson et al. 1996). More specifically, the molecule is expressed by virtually all thymic epithelial cells but at a much higher level by relB-dependent medullary stromal cells (Naspetti et al. 1997). In Fig. 1, an immunostaining analysis on thymic sections was
373 Ep-CAM+
Fig. 1a, b medullary stromal cells have an irregular morphology. Immunochemical staining on thymic sections using medullary stromal cellspecific mAb 95 (a) or antiEp-CAM 29 (b), revealed by FITC-coupled anti-IgM antiserum (×20). Arrows indicate the presence of long Ep-CAM+ cell extensions
performed using the anti-medullary epithelial subset mAb 95 or the anti-Ep-CAM mAb 29 which distinguish two subpopulations of medullary epithelial cells. Whereas most 95+ cells have a few thick extensions giving them a triangular shape (Fig. 1a), 29+ (Ep-CAMhigh) cells are larger and have a highly reticulated morphology displaying numerous thin cell protrusions forming a net around the cells as well as longer and thicker cell extensions (Fig. 1b). The development of 29+ cells and of thymic medulla aborts in the absence of the relB transcription factor. In relB-deficient mice, Ep-CAM expression by medullary epithelial cells remains low, the 95+ epithelial cells scatter in the cortex, and the corticomedullary junction is disorganized. So Ep-CAM overexpression is a feature of an organized thymic medulla and we wondered whether Ep-CAM might play an active role in epithelial cell organization. This possibility was tested using different thymic epithelial cell lines. Transfection of Ep-CAM in thymic epithelial cell lines is associated with increased cell proliferation Although Ep-CAM is expressed at a low level by most thymic epithelial cells in situ, this expression is often lost when these cells are grown as cell lines. Two naturally transformed thymic epithelial cell lines were studied which differ by their morphological features. The MTE1D cell line grows as an E-cadherin+ pavimentous cell line forming extremely tight intercellular junctions and exhibiting a high proliferation rate with contact inhibition (Lepesant et al. 1990). In contrast, the MTE4–14 cell line grows slower and displays less compact intercellular junctions with barely detectable E-cadherin expression. Stable Ep-CAM-transfected clones of both cell lines were established (MTE1D/Ep-CAM, MTE4–14/Ep-CAM) with comparable levels of Ep-CAM expression (relative fluorescence intensities were 40 and 20 for transfected versus less than 5 for untransfected cells using the anti-Ep-CAM 29 or G8.8 mAb). Transfection of Ep-CAM in the MTE4–14 cell line dramatically enhanced cell proliferation (Fig. 2). This was measured by the cytofluorometric decay of the CFSE dye staining (Fig. 2a) representative of cell division. A 75% reduction was observed within 2 versus 4 days in transfected
Fig. 2a, b Ep-CAM transfection enhances MTE4–14 proliferation. Cell proliferation was analyzed either by incubation of CFDA-SE dye (a) or by thymidine incubation (b). In a, the exponential decay of cell staining followed by flow cytometry (FACScan) reflects the comparative rate of cell division between control and Ep-CAMtransfected cells. The rapid loss of fluorescence is associated with a high cell division rate. In b, the analysis of cell division was performed by thymidine incorporation on day 2, of two independent Ep-CAM cell clones and parental cell lines showing again the characteristic enhancement of cell division of Ep-CAM-transfected MTE4–14 cell line. The high rate of cell division of both parental or transfected MTE1D cell lines explains the rapid decay of CFDA-SE fluorescence and the low thymidine incorporation when cultures have reached confluence on day 2
versus non-transfected cells, respectively. Similar results were obtained using thymidine incorporation where MTE4–14 transfected cells incorporate four- to tenfold more thymidine in the 12-h in vitro pulse (Fig. 2b). This property was observed for several independent Ep-CAM-
374 Fig. 3a–d Ep-CAM expression regulated epithelial cell segregation. Co-cultures of control (a, b) or Ep-CAM-transfected (c, d) MTE1D and MTE4–14 cell lines were established and analyzed at confluence by phase contrast (a, c) or fluorescence (b, d) microscopy. MTE4–14 cells were stained with the CFDA-SE dye prior to culture. Arrows indicate areas where MTE4–14 cells grow in aggregates with little contact with the plastic dish whereas EpCAM-transfected cells mix in a single cell layer (×10)
transfected clones but not for control vanin-1 or JAMtransfected clones (data not shown). In contrast, under different experimental conditions, we were unable to document any increase in the rate of cell growth of the MTE1D cell line after Ep-CAM transfection. In Fig. 2a, one sees that the rate of MTE1D CFSE staining decreases equally rapidly in Ep-CAM+or– cells within 2 days till cells reach confluence and thereafter incorporate little thymidine due to contact inhibition (Fig. 2b).
progressively lose contact with the plastic and grow in compact aggregates on top of MTE1D cells (Fig. 3a, b). Transfection of Ep-CAM in either MTE1D or MTE4–14 alone does not significantly affect this segregating behavior (data not shown). In contrast, when both cell types are transfected, MTE1D/Ep-CAM and MTE4–14/Ep-CAM cells intermingle into a heterogeneous cell layer (Fig. 3c, d). Thus, in this system, the presence of Ep-CAM on both cell types prevents cell segregation.
Ep-CAM expression regulates cell segregation Previous reports have demonstrated that the transfection of Ep-CAM in E-cadherin+or– fibroblasts provoked the segregation of Ep-CAM transfectants from parental cells. This effect has been correlated with the ability of Ep-CAM molecules to disturb E-cadherin association with cytoskeletal components and formation of AJs (Balzar et al. 1999b). Since Ep-CAM expression is highly variable among thymic epithelial cells in situ, we evaluated its ability to interfere with the segregation of distinct epithelial cells in vitro. For this purpose, co-cultures of the MTE1D and MTE4–14 thymic epithelial cell lines and the corresponding Ep-CAM transfectants were established on uncoated plastic wells. The fate of the two cell lines was followed by staining only MTE4–14 cells with the CFSE dye prior to culture. When co-cultured with the MTE4–14 cell line, the MTE1D cells remain organized in a tight cell layer that prevents the formation of the MTE4–14 cell layer; consequently, the later cells
Ep-CAM induces the formation of actin-rich protrusions and abundant stress fibers Modifications in cell morphology involve actin cytoskeletal reorganization. As shown in Fig. 4a, b, transfection of Ep-CAM in the MTE4–14 cell line leads to the appearance of numerous long actin-rich Ep-CAM+ protrusions, resulting in a profound modification of the aspect of the cell layer, where it becomes difficult to define individual cells. Similarly, the untransfected MTE1D cell line shows a regular pavimentous organization with a homogeneous cortical actin staining (Fig. 4c, e). Upon transfection of Ep-CAM in MTE1D cells (Fig. 4d) there is little dissociation of cell contacts but actin staining is weaker, more irregular, and cell size is increased. Furthermore, transfected cells display numerous actin bundles and protrusions (Fig. 4f). The protrusions remain in the absence of serum (data not shown) and when the cells are cultured in the presence of the C3
375
Fig. 4a–g Ep-CAM-expressing cells exhibit drastic modification of actin organization. MTE4–14 (a), MTE4–14 Ep-CAM (b), MTE1D (c, e), and MTE1D Ep-CAM (d, f, g) cells were permeabilized, methanol-fixed, and stained with FITC-coupled phalloidin to reveal polymerized actin (×20). In g, transfected MTE1D cells were treated with the C3 toxin prior to staining (×63). Arrow and arrowheads show stress fibers and cell protrusions, respectively
toxin which inhibits the GTPase activity of the Rho protein (Fig. 4g). A morphological analysis of the transfected MTE4–14 cell line was performed by scanning electron microscopy. A striking difference exists between the wild-type MTE4–14 (Fig. 5a) and the Ep-CAM-transfected cells. The wild-type cells exhibit a clear separation between the cells whereas it is almost impossible to distinguish the border between transfected cells. As shown in Fig. 5b–d, these protrusions are thick (up to 2 µm), strongly bound to the underlying cell, and can reach long distances (>30 µm), exceeding the diameter of the cell (around 20 µM). Their extremities are ramified in numerous thinner dendrites attached to distinct cell targets.
Crosslinking of chimeric huCD25 molecules bearing the Ep-CAM intracellular domain triggers the formation of protrusions The intracellular domain of Ep-CAM controls its localization at cell–cell boundaries and interacts with α-actinin, a major regulator of actin cytoskeletal organization. To test whether the induction of the previously described phenotype could be triggered by the engagement of the intracellular domain, chimeric cDNA constructs containing the extracellular and the transmembrane segments of human CD25 and intracytoplasmic moieties of Ep-CAM were transfected in the MTE4–14 cell line. The CD25/Ep-CAM-transfected cells exhibit a wild-type phenotype in culture in the absence of crosslinking. Thus, the cells were incubated in the presence of antihuCD25 mAb-coated beads to crosslink membrane chimeric molecules while being able to localize the engaged molecules. As shown in Fig. 6b, c, beads are found at the extremity or on the side of long cell extensions where actin polymerization is detectable. The extensions
376 Fig. 5a–d Ep-CAM transfectants show abundant stress fibers and long cell protrusions. Scanning electron microscopy analysis of control (a) or EpCAM-transfected (b–d) MTE4–14 cells. The rectangular areas on b are enlarged in c and d, respectively, for proper visualization of cell extensions indicated by arrows
Fig. 6a–c The intracellular domain of Ep-CAM is sufficient for the formation of cell protrusion. MTE4–14 cells transfected with CD25/Ep-CAM chimeric construct were incubated with biotinylated control anti-vanin (a) or CD25 (b, c) mAb and SAV-coated beads for 20 min. Actin staining was revealed as in Fig. 4 (×63, zoom ×2)
were not found in control conditions with an irrelevant anti-vanin-1 mAb where beads remain bound to the cell surface (Fig. 6a). This observation shows that the modification of the concentration of the intracytoplasmic domain of Ep-CAM at the plasma membrane induces the
polymerization of actin resulting in the formation of long protrusions.
Discussion Ep-CAM has initially been described as a homophilic Ca2+-independent cell adhesion molecule (Litvinov et al. 1994b). Its expression is preferential on epithelial tissues and its level of expression significantly enhanced on a variety of carcinomas (Momburg et al. 1987). Trans-
377
fection of Ep-CAM in E-cadherin+ cells was shown to lead to a reduction of intercellular adhesion strength due to a decrease in the number of AJs (Balzar et al. 1999b). Although the adhesive function depends upon the extracellular domain, its localization at cell boundaries and its regulatory activity are due to the interaction of the intracellular domain with α-actinin (Balzar et al. 1998). So, Ep-CAM seems to play a dual role in cell adhesion, first by promoting homotypic interactions leading to cell segregation but also by destabilizing cadherin-dependent cell adhesion. Our results document a novel function of the Ep-CAM molecule. Transfection of Ep-CAM in thymic epithelial cell lines triggers a dynamic reorganization of the actin cytoskeleton associated with a disorganization of epithelial cell layers and enhanced cell growth. Ep-CAM expression has two effects on actin remodeling in thymic epithelial cells. First stress fibers are induced in a Rho-dependent manner; this process might link Ep-CAM to specific transduction pathways leading to cell growth (Bourdoulous et al. 1998; Frost et al. 1997). Secondly and more specifically, long Ep-CAM+ protrusions develop in a dynamic process probably triggered by the engagement of homophilic Ep-CAM interactions. Indeed, using a chimeric CD25/Ep-CAM molecule which only contains the intracellular moiety of Ep-CAM, the crosslinking of CD25 is required to trigger the formation of protrusions. This result is in agreement with the fact that two α-actinin binding sites are present in the intracellular region of the Ep-CAM molecule and are required for the connection with the actin cytoskeleton (Balzar et al. 1998). The formation of protrusions might be regulated by downstream effector proteins such as PKN, a Rho-activated serine-threonine kinase which binds to α-actinin (Mukai et al. 1997). Thus, expression of Ep-CAM triggers cell membrane activity preventing it from forming stable and more definitive cell contacts with neighboring cells. The fact that Ep-CAM expression is precisely regulated on thymic epithelium might be related to this property. Indeed, thymic epithelium has the unique feature among epithelial tissues to organize into a weakly polarized three-dimensional network of interconnected cells (van Ewijk et al. 1999). The stability of the mesh, through which thymocytes migrate, is maintained by adherens and tight junctions. In mutant mice lacking mature thymocytes, thymic medullary epithelial cells scatter and lose their high level of Ep-CAM expression; this phenotype is reversible following bone marrow engraftment and is accompanied by epithelial cell expansion (van Ewijk 1991; Naquet et al. 1999). So this microenvironment is dynamically shaped by the presence of thymocytes and provides a functional network of interconnected epithelial cells which is optimal for tolerance induction. Our results using thymic epithelial cell lines suggest that Ep-CAM might be a key organizer of this dynamic process. First, growth of Ep-CAM-transfected thymic epithelial cells is significantly enhanced. This could play an important role in the organization of medulla which contains high amounts of laminin known to regulate cell
growth (Panayotou et al. 1989). Secondly, expression of Ep-CAM forces the mixing of two distinct epithelial cell lines that would naturally tend to segregate from each other. This might bridge heterogeneous cell populations such as epithelial and dendritic cells which both express Ep-CAM in situ. Thirdly, the protrusions induced by EpCAM overexpression disorganize the establishment of a two-dimensional cell layer. The intensity of these effects is dependent upon the cellular environment. Indeed, in the E-cadherinhigh MTE1D cell line, Ep-CAM expression is not sufficient to totally disorganize the cell layers despite the appearance of numerous stress fibers and a partial loss of cortical filamentous actin; in contrast, in the less-organized MTE4–14 cell line, the Ep-CAM-mediated disorganizing effects are dramatic. Thus, we favor a model in which medullary stroma would lie on a scaffold of laminin matrix, allowing cells to engage discrete intercellular contacts where the balance between cadherin and Ep-CAM-dependent adhesion would be finely tuned. Such a mechanism might involve the α-catenin protein which has been shown to regulate the morphological transition involving cadherin-dependent cell adhesion processes (Gimond et al. 1999). This would create a permeable three-dimensional structure for developing thymocytes. The function of these protrusions remains hypothetical. They might be involved in the transmission of information between cell types as cytonemes described in Drosophila imaginal discs (Ramirez-Weber and Kornberg 1999). Ep-CAM-induced structures might serve similar purposes in thymic medulla such as the exposure of antigenic signals to developing thymocytes in the medulla. They might also participate in the maintenance of a dynamic plasticity in the constitution of a reticulated epithelial cell network. Indeed, a recent report has shown that the formation of cadherin-dependent cell junctions is preceded by the constitution of an adhesion zipper made of several filopodia between adjacent cells; on this scaffold, AJ-linked cytoskeletal proteins such as α-catenin, vinculin, or VASP accumulate (Vasioukhin et al. 2000). In our biological system, a local accumulation of similar proteins at the contact site could not be documented, as also described for Ep-CAM-dependent adhesion in a human mammary epithelial cell line (Balzar et al. 1998). Thus, given the antagonist effect of Ep-CAM-dependent adhesion on cadherin-mediated contacts, the primary function of these cell processes might rather prevent epithelial cells from forming tight and confluent contacts that would interfere with the constitution of a threedimensional mesh. In cancer cells, a similar mechanism would tend to give a growth advantage to cells that would lose lateral contacts with other epithelial cells and might better disseminate. Acknowledgements J.C. Guillemot was supported by the Institut Electricité Santé and M. Naspetti had a fellowship from Association pour la Recherche contre le Cancer (ARC). F. Malergue is a recipient of a Fellowship from the Ministry of Research. We thank Marc Barad and Mathieu Fallet for their help in microscopy analysis and the Centre Régional d’Imagerie Cellulaire de Montpellier for scanning electron microscopy analysis.
378
References Adams CL, Nelson WJ (1998) Cytomechanics of cadherin-mediated cell-cell adhesion. Curr Opin Cell Biol 10:572–577 Aurrand-Lions M, Galland F, Bazin H, Zakharyev V, Imhof BA, Naquet P (1996) Vanin-1, a novel GPI-linked perivascular molecule involved in thymus homing. Immunity 5:391–405 Balzar M, Bakker HA, Briaire-de-Bruijn IH, Fleuren GJ, Warnaar SO, Litvinov SV (1998) Cytoplasmic tail regulates the intercellular adhesion function of the epithelial cell adhesion molecule. Mol Cell Biol 18:4833–4843 Balzar M, Prins FA, Bakker HA, Fleuren GJ, Warnaar SO, Litvinov SV (1999a) The structural analysis of adhesions mediated by Ep-CAM. Exp Cell Res 246:108–121 Balzar M, Winter MJ, Boer CJ de, Litvinov SV (1999b) The biology of the 17–1A antigen (Ep-CAM). J Mol Med 77:699–712 Borkowski TA, Nelson AJ, Farr AG, Udey MC (1996) Expression of gp40, the murine homologue of human epithelial cell adhesion molecule (Ep-CAM), by murine dendritic cells. Eur J Immunol 26:110–114 Bourdoulous S, Orend G, MacKenna DA, Pasqualini R, Ruoslahti E (1998) Fibronectin matrix regulates activation of RHO and CDC42 GTPases and cell cycle progression. J Cell Biol 143: 267–276 Brunk U, Collins VP, Arro E (1981) The fixation, dehydration, drying and coating of cultured cells of SEM. J Microsc 123: 121–131 Castellano F, Montcourrier P, Guillemot JC, Gouin E, Machesky L, Cossart P, Chavrier P (1999) Inducible recruitment of Cdc42 or WASP to a cell-surface receptor triggers actin polymerization and filopodium formation. Curr Biol 9:351–360 Eaton S, Simons K (1995) Apical, basal, and lateral cues for epithelial polarization. Cell 82:5–8 Ewijk W van (1991) T-cell differentiation is influenced by thymic microenvironments. Semin Immunol 9:591–615 Ewijk W van, Wang B, Hollander G, Kawamoto H, Spanopoulou E, Itoi M, Amagai T, Jiang YF, Germeraad WT, Chen WF, Katsura Y (1999) Thymic microenvironments, 3-D versus 2-D? Semin Immunol 11:57–64 Frost JA, Steen H, Shapiro P, Lewis T, Ahn N, Shaw PE, Cobb MH (1997) Cross-cascade activation of ERKs and ternary complex factors by Rho family proteins. Embo J 16:6426–6438 Gimond C, Flier A van der, Delft S van, Brakebusch C, Kuikman I, Collard J, Fässler R, Sonnenberg A (1999) Induction of cell scattering by expression of β1 integrins in β1-deficient epithelial cells requires activation of members of the Rho family of GTPases and downregulation of cadherin and catenin function. J Cell Biol 147:1325–1340 Guillemot JC, Montcourrier P, Vivier E, Davoust J, Chavrier P (1997) Selective control of membrane ruffling and actin plaque assembly by the Rho GTPases Rac1 and CDC42 in FcepsilonRI-activated rat basophilic leukemia (RBL-2H3) cells. J Cell Sci 110:2215–2225
Laufer TM, DeKoning J, Markowitz JS, Lo D, Glimcher LH (1996) Unopposed positive selection and autoreactivity in mice expressing class II MHC only on thymic cortex. Nature 383:81–85 Lepesant H, Reggio H, Pierres M, Naquet P (1990) Mouse thymic epithelial cell lines interact with and select a CD3lowCD4+CD8+ thymocyte subset through an LFA-1-dependent adhesion–de-adhesion mechanism. Int Immunol 2:1021–1032 Litvinov SV, Bakker HA, Gourevitch MM, Velders MP, Warnaar SO (1994a) Evidence for a role of the epithelial glycoprotein 40 (Ep-CAM) in epithelial cell-cell adhesion. Cell Adhes Commun 2:417–428 Litvinov SV, Velders MP, Bakker HA, Fleuren GJ, Warnaar SO (1994b) Ep-CAM: a human epithelial antigen is a homophilic cell-cell adhesion molecule. J Cell Biol 125:437–446 Litvinov SV, Balzar M, Winter MJ, Bakker HA, Briaire-de Bruijn IH, Prins F, Fleuren GJ, Warnaar SO (1997) Epithelial cell adhesion molecule (Ep-CAM) modulates cell-cell interactions mediated by classic cadherins. J Cell Biol 139:1337–1348 Momburg F, Moldenhauer G, Hammerling GJ, Moller P (1987) Immunohistochemical study of the expression of an Mr 34,000 human epithelium-specific surface glycoprotein in normal and malignant tissues. Cancer Res 47:2883–2891 Mukai H, Toshimori M, Shibata H, Takanaga H, Kitagawa M, Miyahara M, Shimakawa M, Ono Y (1997) Interaction of PKN with alpha-actinin. J Biol Chem 272:4740–4746 Muller KM, Luedecker CJ, Udey MC, Farr AG (1997) Involvement of E-cadherin in thymus organogenesis and thymocyte maturation. Immunity 6:257–264 Naquet P, Naspetti M, Boyd R (1999) Development, organization and function of the thymic medulla in normal, immunodeficient or autoimmune mice. Semin Immunol 11:47–55 Naspetti M, Aurrand-Lions M, DeKoning J, Malissen M, Galland F, Lo D, Naquet P (1997) Thymocytes and RelB-dependent medullary epithelial cells provide growth-promoting and organization signals, respectively, to thymic medullary stromal cells. Eur J Immunol 27:1392–1397 Naspetti M, Martin F, Biancotto A, Malergue F, Mansuelle P, Galland F, Naquet P (2000) A novel anti-Ep-CAM antibody to analyze the organization of thymic medulla in autoimmunity (in process citation). Curr Top Microbiol Immunol 251: 109–117 Nelson AJ, Dunn RJ, Peach R, Aruffo A, Farr AG (1996) The murine homolog of human Ep-CAM, a homotypic adhesion molecule, is expressed by thymocytes and thymic epithelial cells. Eur J Immunol 26:401–408 Panayotou G, End P, Aumailley M, Timpl R, Engel J (1989) Domains of laminin with growth-factor activity. Cell 56:93–101 Ramirez-Weber FA, Kornberg TB (1999) Cytonemes: cellular processes that project to the principal signaling center in Drosophila imaginal discs. Cell 97:599–607 Vasioukhin V, Bauer C, Yin M, Fuchs E (2000) Directed actin polymerization is the driving force for epithelial cell-cell adhesion. Cell 100:209–219
