QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sundberg, U. Articles by Öbrink, B. Search for Related Content PubMed PubMed Citation Articles by Sundberg, U. Articles by Öbrink, B. Social Bookmarking                         What's this?

CEACAM1 isoforms with different cytoplasmic domains show different localization, organization and adhesive properties in polarized epithelial cells

Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden

View larger version (21K): [in a new window]   Fig. 1. Expression of CEACAM1 in transfected MDCK cells. Untransfected MDCK cells and MDCK cells transfected with CEACAM1-L or CEACAM1-S were analyzed by immunoblotting using CC16 antibodies for development. The CEACAM1-L-transfected cells expressed a 120 kDa immunoreactive protein and the CEACAM1-S-transfected cells expressed a 115 kDa immunoreactive protein, whereas untransfected MDCK cells had no immunoreactive protein in the 100-120 kDa range. The molecular masses (in kDa) of marker proteins are shown to the left.

View larger version (83K): [in a new window]   Fig. 7. Confocal microscopy of mixed CEACAM1-transfected and untransfected MDCK cells. Untransfected MDCK cells were mixed 50:50 with either CEACAM1-L-transfected or CEACAM1-S-transfected MDCK cells and grown to confluence on permeable filters. The cells were treated with or without hyperosmotic sucrose, fixed with 3% paraformaldehyde and permeabilized by methanol for 2 minutes at room temperature. Some cultures were stained with anti-CEACAM1 mAb 5.4/Alexa488-conjugated secondary antibodies (green colour), and some cultures were double-stained with anti-CEACAM1 CC16/Alexa 546-conjugated secondary antibodies (red colour) and antioccludin/Alexa488-conjugated secondary antibodies (green colour). The horizontal lines in the xy-composites indicate the plane for the z-reconstructions. Note that lateral expression of CEACAM1-L is only found in the borders between CEACAM1-L expressing cells but not in the borders between CEACAM1-L expressing cells and untransfected cells. CEACAM1-S was not stained by mAb 5.4. Bars: 20 µm.

View larger version (105K): [in a new window]   Fig. 3. Confocal microscopy of polarized CEACAM1-L and CEACAM1-S expressing MDCK cells. CEACAM1-L- (a,c,e,g,i,k) and CEACAM1-S- (b,d,f,h,j,l) expressing cells were immunostained with anti-CEACAM1 CC16 antibodies (a-j) or pre-immune immunoglobulins (k,l) and FITC-labelled secondary antibodies. The cells were either permeabilized (a-f) by methanol for 2 minutes at room temperature or unpermeabilized (g-l) before staining. (a) is a z-reconstruction of the cells shown in xy-projections in (c) and (e), (b) is a z-reconstruction of the cells shown in xy-projections in (d) and (f), (g) is a z-reconstruction of the cells shown in xy-projection in (i), and (h) is a z-reconstruction of the cells shown in xy-projection in (j), respectively. The focal planes of (c,d,i,j,k,l) run through the apical cell surfaces. The focal planes of (e) and (f) run in the middle, between the apical and the basal cell surfaces. Apical staining for CEACAM1-L and CEACAM1-S is seen under all conditions, but lateral staining is only seen for CEACAM1-L and under permeabilizing conditions (a,e). Note that there is no lateral staining for CEACAM1-S (b and f). Bars: 20 µm.

View larger version (95K): [in a new window]   Fig. 5. Differential staining of CEACAM1 by polyclonal and monoclonal antibodies, and effects of hyperosmotic treatment. Confluent, polarized CEACAM1-L- or CEACAM1-S-expressing MDCK cells were treated with or without hyperosmotic sucrose, fixed with 3% paraformaldehyde and immunostained with or without methanol permeabilization. The cells were stained either with the polyclonal antibody CC16 and Alexa546-conjugated secondary antibodies or with the monoclonal antibody 5.4 and Alexa488-conjugated secondary antibodies. For each sample both a z-reconstruction (a'-s') and a composite xy-field (a-s) is shown. The composite xy-fields were constructed by simultaneously showing all xy focal planes of a visual field superimposed in a single, combined xy-field. Lateral staining for CEACAM1-L was seen with CC16 both with and without sucrose treatment (b,b',d,d'), although the staining was weaker after sucrose treatment. MAb 5.4 detected lateral staining of CEACAM1-L only after sucrose treatment (h,h'). Note that mAb 5.4 hardly stained CEACAM1-S at all, whether the cells were treated with hyperosmotic sucrose or not.

View larger version (68K): [in a new window]   Fig. 2. Extractability of CEACAM1-L and CEACAM1-S. (A) Cells were grown on Petri dishes. Fixation was performed with 3% paraformaldehyde for 30 minutes. Upper panel: the cells were extracted with the indicated concentrations of Triton X-100 for 10 minutes at room temperature, and the soluble (S) and insoluble (I) fractions were analyzed by immunoblotting. Lower panel: fixed cells were extracted with absolute methanol for 2 minutes at room temperature or for 6 minutes at -20°C, and the soluble (S) and insoluble (I) fractions were analyzed by immunoblotting. (B) CEACAM1-L-transfected cells were grown on permeable filters, fixed with 3% paraformaldehyde for 30 minutes, stained by CC16 after the indicated permeabilization procedure and viewed by confocal microscopy. (a) Permeabilization with 0.05% Triton X-100 for 10 minutes. (b) Permeabilization with 0.1% Triton X-100 for 10 minutes. (c) Permeabilization with methanol for 6 minutes at -20°C. (d) Permeabilization with methanol for 2 minutes at room temperature. Permeabilization with methanol for 2 minutes at room temperature gave the best preservation of the lateral staining of CEACAM1-L. The xy-fields represent the image in one focal plane (the xy-dimension) parallel to the basal surfaces (the filter plane) of the cells. The images labelled z represent the third, vertical z-dimension, obtained by computer reconstruction of all the focal planes analyzed in the xy-dimension. The lines in the xy-fields indicate the vertical planes that are shown in the z-reconstructions. The lines in the z-reconstructions indicate the level of the focal planes shown in the xy-fields.

View larger version (26K): [in a new window]   Fig. 4. Surface labelling of CEACAM1-L and CEACAM1-S expressing MDCK cells. Untransfected MDCK cells and MDCK cells transfected with CEACAM1-L or CEACAM1-S were grown to confluence on permeable filters and incubated with anti-CEACAM1 Fab fragments from either the apical (A) or the basolateral (BL) compartments. The cells were solubilized, bound Fab fragments were retrieved, and complexed CEACAM1 was detected by immunoblotting. No immunoreactive proteins were detected in the untransfected cells. In the transfected cells, CEACAM1-L was retrieved both from the apical and the basolateral surfaces, whereas CEACAM1-S was retrieved only from the apical surfaces.

View larger version (118K): [in a new window]   Fig. 6. Double-staining for CEACAM1 and occludin. Confluent, polarized CEACAM1-L- or CEACAM1-S-expressing MDCK cells were treated with or without hyperosmotic sucrose, fixed with 3% paraformaldehyde and permeabilized by methanol for 2 minutes at room temperature. They were then double-stained with an anti-occludin antibody and Alexa488-conjugated secondary antibodies (green colour) and anti-CEACAM1 (CC16) and Alexa546-conjugated secondary antibodies (red colour). The z-reconstructions show the stainings for both CEACAM1 (red) and occludin (green). The xy-fields show focal planes at the level of the tight junctions; only the staining for occludin (green) is shown in these fields. The tight junctions were intact after treatment with hyperosmotic sucrose, as judged by the occludin staining. Note also that the lateral staining for CEACAM1-L is weaker after sucrose treatment. Bars: 20 µm.

View larger version (81K): [in a new window]   Fig. 8. Double-staining for CEACAM1-L with monoclonal and polyclonal anti-CEACAM1 antibodies. Confluent, polarized CEACAM1-L-expressing cells were first stained with mAb 5.4/Alexa488-conjugated secondary antibodies (green colour), then with CC16/Alexa546-conjugated secondary antibodies (red colour). The yellow colour indicates apparent colocalization. (A) Non-permeabilized cells, which were treated with or without hyperosmotic sucrose, were analyzed at standard resolution (voxel: 0.07x0.07x0.35 µm3). A composite of all the focal planes for the entire apical region above the tight junction is shown for each sample. (B) Non-permeabilized cells, treated with hyperosmotic sucrose. Several focal planes (0.2 µm) were scanned through the apically located microvillar region, at two different xy-resolutions. In the upper part of the figure the higher resolution corresponds to a voxel size of 0.04x0.04x0.2 µm3; in the lower part of the figure the same cells were scanned at lower resolution corresponding to a voxel size of 0.07x0.07x0.2 µm3. The z-reconstructions through the microvillar region are from the same z-plane, as indicated by the horisontal lines in the xy-panels. Note that the apparent colocalization of the different epitopes at the lower resolution disappeared at the higher resolution. (C) Permeabilized cells treated with or without hyperosmotic sucrose. Voxel size, 0.04x0.04x0.2 µm3. The upper panels (marked `Above tight junction') represent an xy-plane through the apical portion of two adjacent cells. The middle panels (marked `Below tight junction') represent an xy-plane through the middle portion of the same cells, half-way between the apical and the basal surfaces. The lower panels (marked z) represent a z-reconstruction through the border between the adjacent cells at the location indicated by the arrows in the upper and middle panels. Note that in the non-treated cells only the polyclonal epitopes (O-epitopes) were exposed, whereas in cells treated with hyperosmotic sucrose the monoclonal epitope (N-epitope) became exposed in the area below the tight junctions. (D) A model of apical microvilli analyzed at two pixel sizes. The microvilli of the apical surfaces in kidney tubular cells are uniform in size and organized in a hexagonal pattern. Here we show an xy-plane through the apical region, where we have drawn the microvilli, the intermicrovillar distances and CEACAM1 to scale. The average diameter of brush border microvilli is 150 nm, the smallest intermicrovillar distance is around 30 nm and the length of the 4 Ig domain extracellular domain of CEACAM1 is approximately 15 nm. The hexagonal arrangement of the microvilli allow for antiparallel CEACAM1-CEACAM1 binding at the narrowest inter-microvillar distances, while at other locations CEACAM1 would not be engaged in antiparallel binding. In the upper part of the figure we put a raster on this model with squares corresponding to 0.04x0.04 µm2, in the lower part of the figure we put a raster on the model with squares corresponding to 0.07x0.07 µm2, that is, the dimensions of the pixels in Fig. 8B. Squares (pixels) that contained only antiparallel, bound CEACAM1 (corresponding to the state with masked N-epitope and exposed O-epitopes) were given a red color, squares (pixels) that contained only CEACAM1 not engaged in antiparallel binding (corresponding to the state with exposed N-epitope and masked O-epitopes) were given a green color and squares (pixels) that contained both states of CEACAM1 were given a yellow color. This showed that with a pixel size of 0.07x0.07 µm2, the majority of the pixels became yellow, whereas the pixels of 0.04x0.04 µm2 became almost exclusively red or green. See the Discussion for further details.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter