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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.
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