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First published online March 2, 2004
doi: 10.1242/10.1242/jcs.00972

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A peculiar internalization of claudins, tight junction-specific adhesion molecules, during the intercellular movement of epithelial cells

Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan

View larger version (75K): [in a new window]   Fig. 1. Eph4 transfectants expressing GFP-claudin-3. (A) A membrane folding model for GFP-claudin-3 (NGFP-Cld3), in which GFP was fused to the N-terminus of mouse claudin-3. (B) Expression levels of endogenous claudin-3 (endogenous Cld3) and exogenous NGFP-Cld3 (GFP-Cld3). The whole cell lysate of parental Eph4 cells, as well as two independent Eph4 clones expressing NGFP-Cld3 (Eph4-GFP-Cld3#1, Eph4-GFP-Cld3#2), was immunoblotted with anti-claudin-3 pAb or anti-GFP pAb. In each lane, the same amount of total protein was applied. The amounts of endogenous claudin-3 (Q1) and NGFP-Cld3 (Q2) in two stable clones were quantified as described in Materials and Methods, and their Q2/Q1 ratios were calculated. (C) Colocalization of NGFP-Cld3 with endogenous claudin-4 and ZO-1 in Eph4:NGFP-Cld3 cells. Cells were stained with anti-claudin-4 mAb or anti-ZO-1 mAb. (D) Confirmation of the correct membrane folding of NGFP-Cld3 in Eph4:NGFP-Cld3 cells. In the presence (+) or absence (–) of Triton-X 100, cells were stained with anti-GFP pAb (left panels) or anti-claudin-3 pAb, which recognizes the C-terminal tail of claudin-3 (right panels). Bars: 10 µm (C); 10 µm (D).

View larger version (92K): [in a new window]   Fig. 2. Intercellular motility within the confluent sheet of Eph4:NGFP-Cld3 cells. (A) Time-lapse images of the dynamic behavior of NGFP-Cld3 in Eph4:NGFP-Cld3 cells under confluent conditions. Elapsed time is indicated at the bottom (min). The right panel is a merged image of 0-minute and 90-minute frames in green and red, respectively. During this time-lapse series, one cell (a) was divided into two daughter cells (a'). See Movie 1 (http://jcs.biologists.org/supplemental/). (B) Close time-lapse observation of the dividing cell. Bars, 10 µm (A); 10 µm (B).

View larger version (154K): [in a new window]   Fig. 3. Time-lapse observation of the remodeling of GFP-positive TJs in Eph4:NGFP-Cld3 cells under confluent conditions. Elapsed time is indicated at the bottom (in minutes:seconds). (A) Shortening of TJs between adjacent cells. Owing to intercellular motility, TJs (between arrows) decreased in length during a 120 minute period. (B) Close time-lapse observation of shortening TJs. GFP-positive granules budded from the TJ and moved into the cytoplasm (arrowheads). See Movie 2 (http://jcs.biologists.org/supplemental/). Bars, 10 µm (A); 10 µm (B).

View larger version (38K): [in a new window]   Fig. 4. Two possible models for the endocytosis of TJs. In Model #1, the two apposed membranes are detached, i.e. claudin-based cell adhesion is released, and then individual membranes are endocytosed into their own cells. In Model #2, the two apposed membranes are not detached, but co-endocytosed into one of the adjacent cells.

View larger version (85K): [in a new window]   Fig. 5. `Eat-each-other' endocytosis of claudins. (A,B) Time-lapse observation. Elapsed time is indicated at the bottom (in minutes:seconds). Eph4:NGFP-Cld3 cells were co-cultured with parental Eph4 cells, and a border between these two distinct types of cells was observed. In the first frame (A), four Eph4:NGFP-Cld3 cells (G) and four Eph4 cells (asterisks) were identified by fluorescence microscopy (left panel) and phase contrast image microscopy (right panel). From frame 35:00 to 70:30, one GFP-positive granule (arrowhead) budded off from the GFP-positive TJ, and moved into parental Eph4 cells. See Movie 3 (http://jcs.biologists.org/supplemental/). (C,D) Co-culture of MDCK transfectants exogenously expressing nontagged claudin-3 (MDCK-Cld3) or N-terminally FLAG-tagged claudin-1 (MDCK-FCld1). Parental MDCK cells expressed no endogenous claudin-3. Confluent cell sheets were double stained with anti-claudin-3 pAb and anti-FLAG mAb in green and red, respectively. As schematically depicted in C, the border between MDCK-Cld3 (Cld3) and MDCK-FCld1 (FLAG-Cld1) cells was focused on. Claudin-3-posive granules (arrowheads; green) were detected in MDCK-FCld1 cells, and FLAG-positive granules (arrows; red) were scattered in the cytoplasm of MDCK-Cld3 cells. Bars, 10 µm (A); 10 µm (B); 10 µm (D).

View larger version (110K): [in a new window]   Fig. 6. Time-lapse observation of the process of cell detachment of adjoining CFP-claudin-1- and YFP-claudin-1-expressing MDCK cells. As these cells were mix-cultured at a low cell density, they were dissociated as a result of their motility. Before detachment, the TJ between two cells looked yellow, because both CFP- and YFP-claudin-1 contributed to this TJ. Even after detachment, many of the torn-off fragments of TJs on individual cells also looked yellow (arrows). Elapsed time is indicated at the bottom (in minutes:seconds). See Movie 4 (http://jcs.biologists.org/supplemental/). Bar, 10 µm.

View larger version (177K): [in a new window]   Fig. 7. Possible ultrastructural counterparts of the endocytosed vesicles of TJs, as observed by electron microscopy. Confluent sheets of Eph4 cells were cut tangentially to the apical surface of cells, and the area around the belt of TJs (arrows) was closely observed. Boxed areas in the left panels are enlarged in the corresponding right panels. These vesicles were characterized by double membranes with no gaps. Bars, 200 nm (left panels); 40 nm (right panels).

View larger version (134K): [in a new window]   Fig. 8. Internalized claudins and endosome markers. MDCK cells expressing GFP-claludin-4 were stained with anti-EEA1 (early endosome antigen 1) mAb (A) or antibodies specific for late endosome markers such as LBPA (lysobisphosphatidic acid) and Rab7 (B). GFP-positive claudin-4-containing vesicles did not carry EEA1, whereas some vesicles were positive for late endosome markers. The boxed areas in the left panels are enlarged in the right three panels. Bars, 10 µm.

View larger version (91K): [in a new window]   Fig. 9. Internalized NGFP-Cld3 and endogenous TJ proteins. (A) Eph4:NGFP-Cld3 cells were treated by immunofluorescent staining with anti-claudin-4 mAb. NGFP-Cld3 and endogenous claudin-4 were precisely co-concentrated not only at TJs but also on cytoplasmic granular structures (arrows). (B) Parental Eph4 cells were double stained with anti-claudin-3 pAb and anti-claudin-4 mAb. Many claudin-3-positive vesicles were scattered around the cytoplasm, all of which were positive for claudin-4 (arrows). (C) Parental Eph4 cells were double stained with anti-occludin mAb/anti-claudin-3 pAb (upper panels) or anti-ZO-1 mAb/anti-claudin-3 pAb (lower panels). Cytoplasmic granules carrying claudins (arrowheads) were negative for either occludin or ZO-1. Bars, 5 µm.

View larger version (75K): [in a new window]   Fig. 10. Segregation of claudins from other TJ proteins during their internalization from TJs. TCA-fixed parental Eph4 cells were double stained with anti-occludin mAb/anti-claudin-3 pAb (A), anti-JAM pAb/anti-claudin-4 mAb (B) or anti-ZO-1 mAb/anti-claudin-3 pAb (C). Close inspection revealed that occludin/JAM/ZO-1 were co-distributed with claudins in tubular structures invaginated from TJs (arrows), but were absent in claudin-containing vesicles (arrowheads), which appeared to be pinched off from the tubular structures. Bars, 7.5 µm.

View larger version (85K): [in a new window]   Fig. 11. Time-lapse observation of Eph4 cells co-expressing YFP-claudin-3 and ZO-1-CFP. In the first frame, both YFP-claudin-3 (green) and ZO-1-CFP (red) were co-concentrated at TJs. The granule endocytosed from TJs looked green (arrowheads), indicating that they did not contain ZO-1-CFP. Elapsed time is indicated at the bottom of each frame (in minutes). See Movie 5 (http://jcs.biologists.org/supplemental/). Bars, 15 µm (A); 7.5 µm (B).

View larger version (76K): [in a new window]   Fig. 12. Intercellular motility and endocytosis of TJs. (A) Confluent cultures of Eph4:NGFP-Cld3 cells on coverslips were wounded by manually scratching with a needle. After 2 hours culture, the dynamic behavior of GFP-positive TJs in the third row of cells from the front of the wound (lower panel) was compared with that of cells in the nonwounded sheets (upper panel). In the wounded sheet, cells moved upwards (arrow). Elapsed time is indicated at the bottom (in minutes:seconds). (B,C) The number of GFP-positive granules per cell was counted in the nonwounded sheets and in cells in the third row (asterisks in B; phase-contrast image) of the wounded sheet. Bars, 20 µm.

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