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First published online 17 February 2004
doi: 10.1242/jcs.00944

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The cytoplasmic domain of CEACAM1-L controls its lateral localization and the organization of desmosomes in polarized epithelial cells

¹ Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, 171 77 Stockholm, Sweden
² McGill Cancer Center, McGill University, Montreal, QC H3G 1Y6, Canada

View larger version (14K): [in a new window]   Fig. 1. Sequence of the cytoplasmic domain of mouse CEACAM1-L. Graphic representation of CEACAM1-L point and deletion mutants. Amino acids are in one letter code.

View larger version (63K): [in a new window]   Fig. 2. Cell surface localization of CEACAM1-L mutants. MDCK cells stably transfected with mouse wild-type CEACAM1-L and CEACAM1-L mutants were grown to confluent, polarized cell layers on permeable filters. Cell layers were fixed, permeabilized, and analyzed by confocal microscopy with antibodies against mouse CEACAM1. A z-reconstruction (top) and an x,y-field between the apical and the basal cell surfaces (bottom) are shown for each CEACAM1-L variant. Lateral cell surface staining is seen in wild-type CEACAM1-L and in the mutants 518, Y488F and V518A, whereas mutants 510, 495, 472, 461, Y515F, Y488,515F, 3K-3A and S503A lack lateral staining for CEACAM1-L. CEACAM1-positive intracellular vesicles are seen in several of the cells.

View larger version (81K): [in a new window]   Fig. 3. Cell surface localization of CEACAM1-L after drug treatment. MDCK cells stably transfected with rat CEACAM1-L were grown to confluent, polarized cell layers on permeable filters and treated with drugs for the time periods indicated. Cell layers were then analyzed for CEACAM1-L expression using confocal microscopy. Both unpermeabilized cell layers and permeabilized cell layers were analyzed. Permeabilization made staining accessible for surface-located CEACAM1-L below tight junctions and intracellular CEACAM1-L. Panels 1-4 in each group show z-reconstructions of unpermeabilized cells. Panels 5-8 in each group show z-reconstructions of permeabilized cells. Panels 9-12 in each group show x,y-planes between the apical and basal surfaces of permeabilized cells. Each x,y-plane shows cells of the same cell layer as the z-reconstruction of the permeabilized cells shown above the respective x,y-plane. (A) Cells treated with pervanadate (100 µM) for 0 minutes (1,5,9), 2.5 minutes (2,6,10), 5 minutes (3,7,11), and 10 minutes (4,8,12). (B) Cells treated with genistein (80 µM) for 0 minutes (1,5,9), 25 minutes (2,6,10), 50 minutes (3,7,11), and 100 minutes (4,8,12). (C) Cells treated with PMA (20 nM) for 0 minutes (1,5,9), 7.5 minutes (2,6,10), 15 minutes (3,7,11), and 30 minutes (4,8,12). (D) Cells treated with staurosporine (20 nM) for 0 minutes (1,5,9), 30 minutes (2,6,10), 60 minutes (3,7,11), and 120 minutes (4,8,12). Notice that the unpermeabilized cell layers were stained only on the apical cell surfaces, indicating that the cell layers were intact, without any leakage of the cells. Both pervanadate and staurosporine provoked complete disappearance of CEACAM1-L from the lateral surfaces, whereas the lateral staining remained intact after incubation with genistein or PMA. Prominent CEACAM1-staining of intracellular vesicles was observed in untreated cells and in cells treated with genistein, PMA or staurosporine, but not in pervanadate-treated cells.

View larger version (62K): [in a new window]   Fig. 5. Effects of wortmannin on drug-induced disappearance of CEACAM1-L. Confluent, polarized MDCK cells expressing rat CEACAM1-L were incubated with (right column) or without (left column) 50 nM wortmannin for 30 minutes at 37°C. Cells were then not treated (A) or treated with 100 µM pervanadate for 5 minutes (B) or with 20 nM staurosporine for 2 hours (C). Cells were then analyzed for CEACAM1-L expression by confocal microscopy using antibody against rat CEACAM1. Wortmannin inhibited the CEACAM1-L lateral disappearance induced by pervanadate but not that induced by staurosporine. Prominent intracellular vesicular staining for CEACAM1-L is seen in the staurosporine-treated cells. A-C each show a z-section, and x,y-sections between the apical and basal surfaces of permeabilized cells.

View larger version (42K): [in a new window]   Fig. 4. Effects of pervanadate on CEACAM1-L Tyr phosphorylation. Untransfected MDCK cells and MDCK cells stably transfected with rat CEACAM1-L or CEACAM1-S were grown to confluence in Petri dishes and treated with pervanadate (100 µM) for the indicated times. The cells were then analyzed by immunoblot for CEACAM1 expression and Tyr-phosphorylation of CEACAM1 using antibody against CEACAM1 (-CEACAM1) and antibody against phosphorylated-Tyr (-PY), respectively. Maximal Tyr-phosphorylation of CEACAM1-L occurred after 4 min. CEACAM1-S was not Tyr-phosphorylated.

View larger version (83K): [in a new window]   Fig. 6. Fate of CEACAM1-L after pervanadate- and staurosporine-treatment. Confluent, polarized MDCK cells expressing rat CEACAM1-L or rat CEACAM1-S were treated with pervanadate for up to 15 minutes or staurosporine for up to 2 hours and analyzed by quantitative immunoblotting (A) or by confocal microscopy (B, C). (A) Total cellular CEACAM1 expression levels were determined in triplicate; the data shown represent numerical averages±s.d. Comparison of the expression levels of cells left untreated (U), treated with staurosporine for 2 hours (S) and treated with pervanadate for 15 minutes (P) was analyzed statistically by Student's t-test. Only the staurosporine-treated CEACAM1-L-expressing cells exhibited a barely significant rise in expression levels when compared to untreated cells (*, P<0.05). Expression levels in the other groups were not significantly different from levels in the corresponding untreated cells (0). Identical results were obtained in a separate series of triplicate determinations of CEACAM1 expression levels. AU, arbitrary units. (B, C) Single focal x,y-planes from within CEACAM1-L-expressing cells, recorded above the level of the nucleus. Smaller panels represent higher magnifications of the marked fields in the larger panels. Bars, 20 µm. (B) Double-staining for CEACAM1-L (red) and EEA-1 (green) of untreated cells, cells treated with pervanadate for 60 seconds and cells treated with staurosporine for 60 minutes. In untreated and pervanadate-treated cells vesicular structures with closely associated CEACAM1-L and EEA-1 were observed in low abundance. Cells treated with staurosporine showed a significant increase in the abundance of vesicular structures with colocalized CEACAM1-L and EEA-1. (C) Double-staining for CEACAM1-L (red) and LAMP-1 (green) of untreated cells, cells treated with pervanadate for 60 seconds and cells treated with staurosporine for 60 minutes. In untreated cells a low abundance of structures with closely associated CEACAM1-L and LAMP-1 was observed. Pervanadate-treatment led to a significant increase of larger vesicular structures with colocalized CEACAM1-L and LAMP-1. In staurosporine-treated cells abundant intracellular vesicles occurred that showed prominent colocalization of CEACAM1-L and LAMP-1.

View larger version (160K): [in a new window]   Fig. 7. Colocalization of CEACAM1-L with adaptor proteins AP-1 and AP-2. Confluent CEACAM1-L-expressing cells were double-stained for CEACAM1-L (red) and AP-1 (green) or AP-2 (green). Single focal x,y-planes from within CEACAM1-L-expressing cells, recorded above the level of the nucleus are shown. Bars, 10 µm. (A) Cells cultured on coverslips. (Left) AP-1 was closely associated with CEACAM1-L in structures that were observed predominantly within the cells (encircled area). (Right) AP-2 colocalized with CEACAM1-L in the plasma membrane (encircled area) and in vesicular structures close to the plasma membrane (encircled area). (B) Cells cultured on filters. In cells treated with pervanadate for 2 minutes, prominent colocalization of CEACAM1-L and AP-1 was seen in large vesicular structures (left). In cells treated with staurosporine for 60 minutes, clusters of small vesicular structures occurred that showed close association of CEACAM1-L and AP-2 (right).

View larger version (75K): [in a new window]   Fig. 8. Localization of CEACAM1-L in relation to occludin, ZO-1, E-cadherin and ß-catenin. Confluent, filter-grown polarized cells expressing rat CEACAM1-L were fixed, permeabilized and analyzed using confocal microscopy. Staining is shown in individual and merged vertical z-reconstructions and in merged horizontal x,y-planes. Arrowheads indicate the position of the z-reconstruction in the corresponding x,y-plane, and the position of the x,y-plane in the corresponding z-reconstruction. (Top group of panels) Staining for occludin (green), ZO-1 (green) and CEACAM1-L (red). No colocalization of CEACAM1-L with either occludin or ZO-1 was observed. (Bottom group of panels) Staining for E-cadherin (green), ß-catenin (green) and CEACAM1-L (red). Notice the prominent colocalization (yellow) of CEACAM1-L with E-cadherin and ß-catenin in the apical parts of the lateral cell surfaces at sites corresponding to adherens junctions. Bars, 10 µm.

View larger version (63K): [in a new window]   Fig. 9. Perturbation of desmosomes and cytokeratin by CEACAM1-L. Confluent, filter-grown polarized cells expressing CEACAM1 were fixed, permeabilized and analyzed by confocal microscopy. (A) Untransfected cells, cells expressing mouse or rat CEACAM1-L, and cells expressing rat CEACAM1-S were stained for desmoplakin (green). To visualize the distribution of the desmosomal plaques, 3-dimensional reconstructions were made, in which the apical cell surfaces were excluded. The data are shown as slightly tilted, three-dimensional reconstructions of the cell layers. The apical and basal borders of the tilted lateral surfaces are marked by pairs of arrow and arrowhead, where arrows mark apical borders and arrowheads mark basal borders. In untransfected cells the desmosomes occurred as one population of closely arranged plaques with a belt-like distribution around the cells in the apical part of the lateral surfaces, and another population of scattered plaques in the remainder of the lateral surfaces. Cells expressing rat CEACAM1-S exhibited identical abundance and organization of the desmosomal plaques to untransfected cells. In cells expressing mouse or rat CEACAM1-L the apical band of desmosomes remained (although the plaques seemed smaller) but scattered desmosomes had almost disappeared. A double-stained x,y-plane of cells transfected with mouse CEACAM1-L shows no colocalization of CEACAM1-L and desmoplakin in the remaining desmosomal plaques. (B) Scattered desmosomal plaques were quantified in MDCK cells expressing various forms of rat and mouse CEACAM1-L, and are given as the number of plaques per 25 µm2. The data represent numerical averages±s.d. Comparison of the expression levels of transfected and untransfected cells was analyzed statistically by Student's t-test (***, P<0.001; *, P<0.05; , P>0.1). (C) Untransfected cells and cells expressing rat CEACAM1-L or rat CEACAM1-S were stained for cytokeratin (green) and CEACAM1 (red). x,y-planes between the apical and basal surfaces are shown. In untransfected cells and CEACAM1-S-expressing cells, the cytokeratin was highly organized in concentrated bundles underneath the plasma membrane. By contrast, CEACAM1-L-expressing cells had lost the membrane-associated bundles and the cytokeratin was now diffusely distributed in the cytoplasm. The upper panels show staining for cytokeratin alone, lower panels show double-staining for cytokeratin and CEACAM1. Notice the lateral staining for CEACAM1-L in the lateral cell borders. Bar, 20 µm.