Article Figures & Tables
Figures
- Fig. 1.
Deletion of ORM enhances occludin association with the TJ. (A) A part of the sequence (G388-D415) in the C-terminal domain of occludin including ORM (Y398-S408) was deleted from the wild-type human occludin (OCLNWT) to generate a deletion mutant of occludin (OCLNDM). (B) Stable clones of OD-MDCK cells expressing EGFP-OCLNWT, EGFP-OCLNDM and EGFP vector (Vec) were generated. Total protein extracts were immunoblotted for EGFP, occludin (OCLN) and β-actin (β-Act). (C) GFP was immunoprecipitated from the native extracts of OCLNWT, OCLNDM and Vec cells and immunoblotted for ZO-1. Density of ZO-1 was measured and normalized to the corresponding EGFP band density. Values presented in the graph are means±s.e.m. (n=3). (D,E) OCLNWT, OCLNDM and Vec cell monolayers were imaged live for EGFP (D). Junctional fluorescence was evaluated by densitometric analysis (E). Values, in arbitrary units of fluorescence intensity, are means±s.e.m. (n=3). Asterisks indicate that values are significantly (P<0.05) different from the OCLNWT value. (F,G) Z-section images of GFP fluorescence in OLCNWT (WT) and OCLNDM (DM) cell monolayers were captured (F), and Z-profiling of GFP fluorescence in these cell monolayers were analyzed (G). Values are means±s.e.m. (n=4). Asterisks indicate that values are significantly (P<0.05) different from corresponding values for WT cell monolayers. (H,I) Equal numbers of MDCK (blue), OCLNWT (OKD-WT; green), OCLNDM (OKD-DM; brown) and Vec (OKD-VEC; red) cells were seeded onto transwell inserts. TER (H) and FITC-inulin flux (I) were measured at various time points post seeding. Values presented in the graph are means±s.e.m. (n=3; three different clones for each group and the value for each clone is the average of six for 1 h, five for 2 h, four for 3 h and three for 4 h). The experiment was performed twice. Asterisks indicate MDCK and OCLNWT values that are significantly (P<0.05) different from corresponding values for Vec and OCLNDM groups. (K,L) Total protein extracts from OCLNWT, OCLNDM and Vec cells were immunoblotted for claudin-2 (Cldn-2) (K). Immunoblot bands were quantified by densitometric analysis (L). Values are means±s.e.m. (n=3). Asterisks indicate that values are significantly (P<0.05) different from the corresponding OCLNWT value. (J,M) Fixed cell monolayers were stained for Cldn-2 by immunofluorescence method (J). Fluorescence at the intercellular junctions was measured by densitometric analysis (M). Values are means±s.e.m. (n=3). Asterisks indicate that values are significantly (P<0.05) different from the corresponding OCLNWT value. Scale bars: 50 μm.
- Fig. 2.
Deletion of ORM enhances the association of occludin with the actin-rich fraction of cells and decreases its mobility at TJs. (A) Two days after seeding, OCLNWT, OCLNDM and Vec cells were fixed and stained for F-actin (F-Act), β-tubulin (β-Tub) and nucleus (Nuc). Scale bar: 50 µm. (B,C) Triton-soluble and insoluble fractions prepared from OCLNWT, OCLNDM and Vec cells were immunoblotted for GFP and β-actin (β-Act) (B). GFP band densities were measured and normalized to corresponding β-actin band densities (C). Values are means±s.e.m. (n=3). Asterisks indicate the values that are significantly (P<0.05) different from corresponding value for OCLNWT cells. (D–F) FRAP analysis of EGFP was performed in OCLNWT and OCLNDM cell monolayers. Time-lapse images of several ROIs (regions of interest) at intercellular junctions were collected before and after photobleaching (D). Fluorescence intensity in the bleached area was measured (E) and percentage mobile fractions of OCLNWT and OCLNDM were calculated (F). Values in panels E and F are means±s.e.m. (n=8). Asterisks indicate the values that are significantly (P<0.05) different from the corresponding values for OCLNWT cells.
- Fig. 3.
ORM deletion attenuates Ca2+-depletion-mediated disruption of the AJC and barrier dysfunction. (A–C) OCLNWT and OCLNDM cell monolayers were incubated with low-Ca2+ medium (LCM) or normal-Ca2+ medium (NCM) for 16 h. Live-cell images for EGFP fluorescence were captured before and after incubation (A). Fixed cell monolayers were stained for EGFP-occludin (GFP-OCLN), ZO-1, E-cadherin (E-Cad) and β-catenin (β-Cat) (B) or cytoskeletal proteins F-actin and β-tubulin (C). (D,E) MDCK (blue), OCLNWT (OKD-WT; green), OCLNDM (OKD-DM; brown) and Vec (OKD-VEC; red) cell monolayers on transwell inserts were incubated with LCM for 16 h followed by incubation with NCM for up to 3 h. TER (D) and FITC-inulin flux (E) were measured at various time points. Values are mean±s.e.m. (n=6). Asterisks indicate the values for MDCK and OCLNWT cell monolayers that are significantly (P<0.05) different from corresponding values for OCLNDM and Vec-cell monolayers. (F,G) MDCK (blue), Vec (red), OCLNWT (green) and OCLNDM (brown) cell monolayers on transwell inserts were incubated with (circles) or without (squares) 4 mM EGTA. TER (F) and FITC-inulin flux (G) were measured at various time points. Values are means±s.e.m. (n=6). Asterisks indicate the values for MDCK and OCLNDM monolayers that are significantly (P<0.05) different from corresponding values for Vec and OCLNWT monolayers. (H–J) Cell monolayers after EGTA treatment were co-stained for EGFP-occludin and ZO-1 (H), E-cadherin and β-catenin (I) or F-actin and β-tubulin (J). (K–M) Cell monolayers were incubated with LCM (L) or NCM (N) for 6 h. Phospho-threonine (p-Thr) was immunoprecipitated from denatured protein extracts. Immunoprecipitates and original extracts (Load) were immunoblotted for ZO-1 (K), E-cadherin (L), β-catenin and β-actin (M). Scale bars: 50 µm.
- Fig. 4.
ORM deletion attenuates disruption of the AJC and barrier dysfunction caused by osmotic stress and hydrogen peroxide. (A) MDCK (blue), OCLNWT (green), OCLNDM (brown) and Vec (red) cell monolayers on transwell inserts were incubated in DMEM (Control; data in Fig. S4A) or DMEM containing 0.3 M mannitol to induce osmotic stress (OS). FITC-inulin flux (A) was measured at various time points. Values are means±s.e.m. (n=6). Asterisks indicate the values for OCLNWT cell monolayers that are significantly (P<0.05) different from corresponding values for OCLNDM and Vec cell monolayers. (B) Fixed cell monolayers were stained for EGFP-occludin (GFP-OCLN) and ZO-1. (C) OCLNWT and OCLNDM cell monolayers were treated with hydrogen peroxide (100 µM) and live-cell images for EGFP fluorescence were collected at various time points. (D) MDCK (blue), OCLNWT (green), OCLNDM (brown) and Vec (red) cell monolayers on transwell inserts were treated with hydrogen peroxide (100 µM) in DMEM. FITC-inulin flux was measured at various time points. Values are means±s.e.m. (n=6). Asterisks indicate the values for OCLNWT cell monolayers that are significantly (P<0.05) different from corresponding values for OCLNDM and/or Vec cell monolayers. Control values are in Fig. S6A. (E,F) Fixed cell monolayers at different time points were stained for EGFP-occludin and ZO-1 (E) or E-cadherin and β-catenin (F).
- Fig. 5.
Phosphorylation of ORM on T403/T404 and Y398/402 determines the dynamic properties of TJs. (A,B) OCLNWT (WT), OCLNDM (DM), OCLNT403/404A (T403/404A), OCLNT403/404D (T403/404D), OCLNY398/402A (Y398/402A) and OCLNY398/402D (Y398/402D) cell monolayers were incubated with low-Ca2+ medium (LCM) or normal-Ca2+ medium (NCM) for 1–24 h. Live-cell images for EGFP fluorescence were collected before and after incubation (A). Cell monolayers fixed at 1 h and 16 h were stained for EGFP-Occludin and ZO-1 (B). Scale bars: 50 µm. (C,D) OCLNWT, OCLNDM, OCLNT403/404A, OCLNT403/404D, OCLNY398/402A and OCLNY398/402D cell monolayers on transwell inserts were incubated with LCM for 16 h and TER (C) and FITC-inulin flux (D) were measured. Values are means± s.e.m. (n=6). Asterisks indicate the values that are significantly (P<0.05) different from corresponding values for OCLNWT cell monolayers. (E–G) FRAP analysis of EGFP was performed in OCLNWT (WT), OCLNDM (DM), OCLNT403/404A (T2A), OCLNT403/404D (T2D), OCLNY398/402A (Y2A) and OCLNY398/402D (Y2D) cell monolayers. Time-lapse images were collected before and after photobleaching of several ROIs at intercellular junctions. Fluorescence intensity was measured (E) and % mobile fraction (F) and t1/2 values (G) were calculated. Values are means±s.e.m. (n=4). Asterisks indicate the values that are significantly (P<0.05) different from the value for OCLNWT cell monolayers.
- Fig. 6.
Inhibition of tyrosine kinase activity blocks Ca2+-depletion-mediated disruption of TJs and barrier function. (A,B) MDCK cell monolayers on transwell inserts were treated with 100 µM genistein (Gen) 30 min prior to LCM treatment. TER (A) and FITC-inulin flux (B) were measured after 1 h. Values are means±s.e.m. (n=6). Asterisks indicate the values that are significantly (P<0.05) different from corresponding value for OCLNWT cell monolayers and hash signs indicate the values that are significantly (P<0.05) different from that of OCLNDM cell monolayers. (C,D) Cell monolayers were co-stained for GFP-occludin (C) and ZO-1 (D). Merged images are presented in panel E. Scale bar: 50 µm.
- Fig. 7.
Deletion of ORM attenuates occludin mobility and Ca2+-depletion-mediated disruption of the AJC in the intestinal epithelium. (A,B) Total protein extracts from IEC-6 cells expressing EGFP-OCLNWT, EGFP-OCLNDM and EGFP vector (Vec) were immunoblotted for EGFP, occludin and β-actin (β-Act) (A). OCLNWT, OCLNDM and Vec cell monolayers were imaged live for EGFP (B). (C–E) FRAP analysis of EGFP was performed in OCLNWT (WT) and OCLNDM (DM) cell monolayers. Time-lapse images of several ROIs at intercellular junctions were collected before and after photobleaching (C). Fluorescence intensity in the bleached area was measured (D) and percentage mobile fractions of OCLNWT and OCLNDM were calculated (E). Values are means±s.e.m. (n=8). Asterisks indicate values that are significantly (P<0.05) different from the value for OCLNWT cells. (F,G) OCLNWT and OCLNDM cell monolayers on transwell inserts were incubated in LCM, and TER (F) and FITC-inulin flux (G) were measured at various time points. Values presented in the graph are means±s.e.m. (n=6). Asterisks indicate the values that are significantly (P<0.05) different from corresponding values for the OCLNWT group. (H,I) Fixed cell monolayers were stained for TJ proteins EGFP-occludin and ZO-1 (H) or AJ proteins E-cadherin and β-catenin (I). (J-L) Intestinal loops (∼2 cm long) from the mid small intestine of wild-type (WT) and occludin-deficient (OCLN−/−) mice were prepared and filled with saline containing FITC-inulin with or without 1 mM EGTA. Loss of inulin from the loops was measured after 30 min incubation in DMEM (J). Values are means±s.e.m. (n=3). The asterisk indicates the value for the OCLN−/− group that is significantly (P<0.05) different from the value for the WT group. Cryosections of the loops were immunostained for ZO-1, F-actin and nucleus (K) or E-cadherin and β-catenin (L). Scale bars: 50 µm.
- Fig. 8.
Absence of ORM impairs directional cell migration in renal and intestinal epithelia. (A,B) OD-MDCK cells expressing EGFP-OCLNWT (WT), EGFP-OCLNDM (DM) and EGFP vector (Vec) were grown to confluence, and cell migration assay was performed by scrape wounding. Phase-contrast images were captured at various time points (A); the purple lines indicate the origin of migration. Area of migration was measured using ImageJ and presented in arbitrary units (B). Values are means±s.e.m. (n=5; each value is an average of five images from the same monolayer). Asterisks indicate the values that are significantly (P<0.05) different from corresponding values for Vec cells, and the hash signs indicate the values that are different from corresponding values for EGFP-OCLNWT cells. (C,D) IEC-6 cells expressing EGFP-OCLNWT (WT), EGFP-OCLNDM (DM) and EGFP vector (Vec) were grown to confluence, and cell migration assay was performed by scratch wounding. Phase-contrast images were captured at 12 h or 24 h (C). The wound area was measured using ImageJ and presented in arbitrary units (D). Values are means±s.e.m. (n=5; each value is an average of five images from the same monolayer). Asterisks indicate the values that are significantly (P<0.05) different from corresponding values for Vec cells, and the hash signs indicate the values that are different from corresponding values for EGFP-OCLNWT cells. (E,F) OD-MDCK cells (E) and IEC-6 cells (F) expressing EGFP vector (Vec), EGFP-OCLNWT (WT) or EGFP-OCLNDM (DM) (105 cells /well), and MDCK cells (E) were seeded at low density on to transwells. Cells that migrated to the bottom surface of transwells were counted (E). Similar to MDCK cells, IEC-6 cells transfected with EGFP vector (Vec), EGFP-OCLNWT (WT) or EGFP-OCLNDM (DM) were seeded on to transwells to measure transmigration (F). Values are means±s.e.m., and the values within the bars are the number of samples per group. Asterisks indicate significant differences (P<0.05) between the groups.
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