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First published online August 29, 2005
doi: 10.1242/10.1242/jcs.02528

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PATJ connects and stabilizes apical and lateral components of tight junctions in human intestinal cells

¹ UMR 6156, NMDA, Institute of Developmental Biology of Marseille, Faculté des Sciences de Luminy, case 907, 13288 Marseille Cedex 09, France
² IMVT, Faculté des Sciences de Luminy, case 907, 13288 Marseille Cedex 09, France
³ The Netherlands Ophthalmic Research Institute, Royal Netherlands Academy of Arts and Sciences, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands

View larger version (44K): [in a new window]   Fig. 1. Characterization of PATJ knockdown clones. (A) Cell extracts (50 µg) from control (CT Cl 8) and PATJ KD clones (Cl 4, 5 and 12) of Caco2 cells were analyzed by SDS-PAGE and western blotting with affinity-purified antibodies against human PATJ (top panel) or aPKC (bottom panel). In clones 4 and 12, all the species of PATJ above 130 KDa were barely detectable. The asterisk indicates a band that is not affected by the siRNA used. Clone 5, in contrast to clones 4 and 12 only showed a slight decrease in PATJ expression and is also used in some experiments as a control. (B) Confocal microscopic X-Y sections at the level of tight junctions of control (CT Cl 8) and PATJ KD clones (Cl 4 and 12) of Caco2 cells. Cells grown on filters were labeled with affinity-purified antibodies against human PATJ and secondary antibodies coupled to Texas Red. Note that the monolayer in CT Cl 8 is not flat and PATJ labeling is out of the section plane chosen in some areas (white asterisks). The arrow indicates a small cluster of positive cells in Cl 12 cells. Bars, 10 µm.

View larger version (76K): [in a new window]   Fig. 2. Loss of PATJ expression leads to Pals1 disorganization. (A) Cell extracts (80 µg) from control (CT Cl 8) and PATJ KD clones (Cl 4 and 12) were analyzed by SDS-PAGE and western blotting with affinity-purified antibodies against Pals1. Tubulin was used as an internal control for protein loading. (B,C) Confocal microscopic X-Y (B) or Z (C) sections at the level of tight junctions of control (CT Cl 8) and PATJ KD clone (Cl 12) of Caco2 cells. Cells grown on filters were labeled with affinity-purified antibodies against human PATJ (in red) and Pals1 (in blue). Arrowheads indicate the colocalization of the two proteins. Note that in the absence of PATJ labeling there is no accumulation of Pals1 at the tight junctions. Bars, 10 µm.

View larger version (110K): [in a new window]   Fig. 3. PATJ KD cells are polarized and establish tight junctions. (A) Confocal microscopic Z-sections of Control (CT Cl 8 and Cl 5) and PATJ KD clone (Cl 4 or 12) Caco2 cells grown on filters and labeled with affinity-purified antibodies against aPKC, E-cadherin (E-Cad), DPPIV or ZO-1. All markers are still properly localized on the lateral membrane (arrows) or the apical membrane or tight junctions (arrowheads), respectively. Under our staining conditions ZO-1 is also found in the cytoplasm of Caco2 cells. (B) Control Cl 8 and PATJ KD Cl 4 of Caco2 cells were seeded on filters and the TER was measured after 1, 2, 3 or 4 days of culture. Each point represents the average of five filters (bars indicate s.d.). No significant difference was observed between control and KD PATJ cells. (C) Electron microscopic views of the tight junction region of PATJ KD (a,b) and control cells (c) showing that no morphological differences in the organization of tight junctions (TJ), adherens junctions (AJ) and desmosomes (D) could be detected at this level of resolution. ap, apical cell surface; mv, microvilli. Bars, 10 µm (A,B); 100 nm (C,D).

View larger version (59K): [in a new window]   Fig. 4. Loss of PATJ expression leads to mislocalization of Occludin and ZO-3. (A) Confocal microscopic Z-sections of control (Cl 8) and PATJ KD clone 12 (Cl 12) Caco2 cells grown on filters and labeled with affinity-purified antibodies against PATJ (in red) and occludin (Occ) (in green). In PATJ KD cells, occludin is delocalized on the lateral membrane (arrows) instead of being restricted to tight junctions (arrowheads). (B) Confocal microscopic X-Y (top panels) and Z-sections (bottom panels) of Control (CT Cl 8) and PATJ KD clone (Cl 4) of Caco2 cells grown on filters and labeled with affinity-purified antibodies against ZO-3 (in red) and occludin (Occ) (in green). In PATJ KD cells, both occludin and ZO-3 are accumulated in thick spots in the lateral membrane (arrows). (C) PATJ KD cells (Cl 4) were prepared for low-temperature embedding in K4M Lowicryl resin. Occludin (left panel) and ZO-3 (right panel) are found along the lateral membrane (arrowheads) whereas ZO-3 also occasionally accumulated (arrow) in desmosome-like structures (D). ap, apical membrane; mv, microvilli; TJ, tight junctions. Bars, 10 µm (A,B); 100 nm (C).

View larger version (99K): [in a new window]   Fig. 5. Crb3 is accumulated in a sub-apical compartment in PATJ KD cells. Confocal microscopic X-Y (top panels) and Z-sections (bottom panels) of Control (CT Cl 8) and PATJ KD clone (Cl 12) of Caco2 cells grown on filters and labeled with antibodies against SI (in red) and affinity-purified Crb3 antibodies (in blue). SI is at the apical membrane (arrowheads) in both clones whereas Crb3 is also accumulated in a dense sub-apical compartment in clone 12 (arrows). Bars, 10 µm.

View larger version (77K): [in a new window]   Fig. 6. Crb3 is present in an EEA-1 positive compartment in PATJ KD cells. Confocal microscopic Z-sections (A) and microscopic X-Y sections (B) of control (CT Cl 8) and PATJ KD clones (Cl 12 or 4) of Caco2 cells grown on filters and labeled with antibodies against EEA-1 (in red) and affinity-purified antibodies Crb3 (in blue). EEA-1 is found in early endosomes mostly accumulated below the apical membrane (arrowheads) and overlaps strongly with Crb3 in Cl 4 or 12 (arrows). In control cells, Crb3 is also found in small vesicular structures in the cytoplasm. (C) Immunogold labeling of PATJ KD Cl 4 cells prepared by cryosubstitution. Ultra-thin sections were labeled with antibodies against EEA-1 (15 nm gold particles) and affinity-purified Crb3 antibodies (6 nm gold particles). Crb3 labeling (arrowheads) is found at the plasma membrane and in sub-apical vesicles, some of which are also labeled with EEA-1 (arrows). ap, apical membrane; mv, microvilli. Bars, 10 µm (A,B); 100 nm (C).

View larger version (28K): [in a new window]   Fig. 7. Overexpression of myc-Pals1 or VSV-G Crb3 does not redistribute PATJ. (A) Caco2 cells were transfected by electroporation with a plasmid containing the cDNA of Pals1 tagged with a myc epitope and grown on filters for 3 days. Images are confocal microscopic Z-sections of cells processed for immunofluorescence as described in Materials and Methods and labeled with affinity-purified antibodies against PATJ (in blue) and myc (9E10) (in red). Myc-Pals1 is accumulated on the apical membrane (arrow) whereas PATJ is mainly at the level of tight junctions (arrowheads). (B,C) Overexpression of VSV-G-Crb3 leads to mislocalization of aPKC (B) but not of PATJ (C). Caco2 cells were transfected by electroporation with a plasmid containing the cDNA of hCrb3 tagged with a VSV-G epitope on the extracellular domain and grown on filters for 3 days. Images are confocal microscopic Z-sections of cells processed for immunofluorescence and labeled with affinity-purified antibodies against PATJ or aPKC (in blue) and VSV-G (P5D4) (in red). VSV-G-Crb3 is distributed in an unpolarized fashion when overexpressed and leads to mislocalization of aPKC (arrows) but not of PATJ (arrowheads). Bars, 10 µm.

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