1. Volume 121, Issue 3, Pages 566-579 (September 2001)
Rho kinase regulates tight junction function and is necessary for tight junction assembly in polarized intestinal epithelia 
Shaun V. Walsh, Ann M. Hopkins, Jason Chen, Shuh Narumiya, Charles A. Parkos, Asma Nusrat 
Gastroenterology 
Volume 121, Issue 3, Pages (September 2001)
DOI: /gast
Copyright © 2001 American Gastroenterological Association Terms and Conditions

2. Fig. 1
ROCK inhibitor Y induces a decrease in TER in a dose-dependent manner. Confluent T84 intestinal epithelial monolayers were incubated with Y (10–100 μmol/L) or with vehicle alone. Control monolayers maintained high TER of ~1500 Ω · cm2. Y induced a dose-dependent decrease in TER in T84 monolayers (P < at 3 hours). Results shown represent the mean and SEM from 5 independent experiments.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

3. Fig. 2
ROCK inhibition enhances paracellular permeability to an inert tracer molecule. Confluent T84 epithelial monolayers were incubated with Y (50–100 μmol/L) or vehicle alone for 1 hour. FD-3, 1 mg/mL, was added to the apical compartment, and the accumulation of FD-3 in the basolateral compartment was quantitated spectrofluorometrically at 30-minute intervals over 2 hours. There was a slow, steady flux of the fluorescent tracer across control monolayers over time, reflecting passive paracellular transport when normalized with respect to controls at each time point. Results shown represent the mean and SEM of data pooled from 3 separate experiments.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

4. Fig. 3
(Panel I) ROCK inhibition induces reorganization of F-actin filaments and the actin-regulating protein villin in the apical pole of intestinal epithelial cells. Confluent T84 and Caco-2 intestinal epithelial monolayers were incubated for 1 hour with the ROCK inhibitor Y (100 μmol/L) or vehicle alone (CON). Cells were fixed and stained with rhodamine phalloidin (red) or antivillin antibodies (green) and analyzed by confocal laser scanning microscopy. Shown here are en face (xy) images taken at the level of the apical pole of the epithelial monolayer and computer-reconstructed vertical section (xz) images taken through the full thickness of the monolayer (scale bar = 10 μm). The supporting filter in the xz computer-reconstructed images is shown as a blue line, and the apical and basolateral compartments are annotated (a and bl, respectively). (A) In control T84 cell monolayers, F-actin is organized as a dotlike apical microvillous staining pattern and as prominent perijunctional F-actin rings (arrowheads). (B) ROCK inhibition using Y (100 μmol/L) induced reorganization of apical F-actin with formation of F-actin condensations in the apical plane (arrows). This reorganization was accompanied by a loss of the normal apical microvillous staining pattern. In addition, the intensity of the perijunctional F-actin ring was reduced after incubation with Y In the xz plane, the apical dense aggregates of F-actin (arrows) induced by Y (B, xz) are seen extending down from the apical membrane through the level of the terminal web but are not detected in control monolayers (A, xz). (D) Analogous reorganization of F-actin was observed in another intestinal epithelial cell line, Caco-2, after incubation with Y (C) Normal F-actin organization in the apical region of this cell type. (E, G) Villin distribution in the apical poles of control (E) T84 and (G) Caco-2 monolayers. (F, H) Incubation with Y (100 μmol/L) induced dramatic changes in villin expression with loss of the fine brush border pattern and formation of large apical aggregates (arrows). (Panel II) Epithelial cells. Confluent T84 intestinal epithelial monolayers were incubated for 1 hour with the ROCK inhibitor Y (100 μmol/L) or vehicle alone (CON). Cells were fixed and stained with rhodamine phalloidin and analyzed by confocal laser scanning microscopy. Shown here are en face (xy) images taken at the level of the basal pole of the epithelial monolayer. Incubation with Y reduced the number of F-actin–rich stress fibers at the basal pole of cells. (Panel III) Ultrastructural examination shows unusual organization of microfilament-rich microvillous core rootlets after ROCK inhibition. Confluent T84 monolayers, incubated with vehicle alone or with the ROCK inhibitor Y (100 μmol/L), were examined by transmission electron microscopy. (A) Low-power views of control monolayers (CON) showed intact monolayers composed of well-polarized, tall columnar cells. (B) No change in cell polarity or length was observed after treatment with Y (C, E) In control monolayers (CON), F-actin filaments were observed as aggregates in short microvilli and as irregularly dispersed core rootlets (arrow). (D, F) Incubation with Y induced elongated microvillous F-actin core rootlets that were organized in clusters (arrows) extending downward from the apical region of cells.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

5. Fig. 3
(Panel I) ROCK inhibition induces reorganization of F-actin filaments and the actin-regulating protein villin in the apical pole of intestinal epithelial cells. Confluent T84 and Caco-2 intestinal epithelial monolayers were incubated for 1 hour with the ROCK inhibitor Y (100 μmol/L) or vehicle alone (CON). Cells were fixed and stained with rhodamine phalloidin (red) or antivillin antibodies (green) and analyzed by confocal laser scanning microscopy. Shown here are en face (xy) images taken at the level of the apical pole of the epithelial monolayer and computer-reconstructed vertical section (xz) images taken through the full thickness of the monolayer (scale bar = 10 μm). The supporting filter in the xz computer-reconstructed images is shown as a blue line, and the apical and basolateral compartments are annotated (a and bl, respectively). (A) In control T84 cell monolayers, F-actin is organized as a dotlike apical microvillous staining pattern and as prominent perijunctional F-actin rings (arrowheads). (B) ROCK inhibition using Y (100 μmol/L) induced reorganization of apical F-actin with formation of F-actin condensations in the apical plane (arrows). This reorganization was accompanied by a loss of the normal apical microvillous staining pattern. In addition, the intensity of the perijunctional F-actin ring was reduced after incubation with Y In the xz plane, the apical dense aggregates of F-actin (arrows) induced by Y (B, xz) are seen extending down from the apical membrane through the level of the terminal web but are not detected in control monolayers (A, xz). (D) Analogous reorganization of F-actin was observed in another intestinal epithelial cell line, Caco-2, after incubation with Y (C) Normal F-actin organization in the apical region of this cell type. (E, G) Villin distribution in the apical poles of control (E) T84 and (G) Caco-2 monolayers. (F, H) Incubation with Y (100 μmol/L) induced dramatic changes in villin expression with loss of the fine brush border pattern and formation of large apical aggregates (arrows). (Panel II) Epithelial cells. Confluent T84 intestinal epithelial monolayers were incubated for 1 hour with the ROCK inhibitor Y (100 μmol/L) or vehicle alone (CON). Cells were fixed and stained with rhodamine phalloidin and analyzed by confocal laser scanning microscopy. Shown here are en face (xy) images taken at the level of the basal pole of the epithelial monolayer. Incubation with Y reduced the number of F-actin–rich stress fibers at the basal pole of cells. (Panel III) Ultrastructural examination shows unusual organization of microfilament-rich microvillous core rootlets after ROCK inhibition. Confluent T84 monolayers, incubated with vehicle alone or with the ROCK inhibitor Y (100 μmol/L), were examined by transmission electron microscopy. (A) Low-power views of control monolayers (CON) showed intact monolayers composed of well-polarized, tall columnar cells. (B) No change in cell polarity or length was observed after treatment with Y (C, E) In control monolayers (CON), F-actin filaments were observed as aggregates in short microvilli and as irregularly dispersed core rootlets (arrow). (D, F) Incubation with Y induced elongated microvillous F-actin core rootlets that were organized in clusters (arrows) extending downward from the apical region of cells.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

6. Fig. 4
(Panel I) ROCK inhibition does not influence distribution of TJ and AJ proteins. Confluent T84 monolayers were incubated with vehicle alone (CON), Y (100 μmol/L, 1 hour), or the Rho-inactivating toxin, DC3B (3.2 μg/mL basolaterally for 6 hours). The en face (XY) confocal images shown in this figure were taken at the level of (A–C, F–H, K–M) TJ and (D, E, I, J) AJ. In control monolayers (CON), ZO-1, occludin (OCC), and claudin-1 (CLD-1) were distributed in a chicken-wire pattern consistent with their localization in TJs. A large intracellular pool of CLD-1 was also identified. Incubation with Y did not influence the distribution of TJ proteins. In contrast, Rho inactivation with DC3B toxin completely disrupted the normal localization of all 3 TJ proteins. In an analogous manner, localization of E-cadherin and β-catenin in AJs was not influenced by incubation with Y Incubation with Y (20–50 μg/mL, 1–24 hours) did not change the localization of TJ and AJ proteins. (Panel II) Differential detergent solubility of TJ proteins is not influenced by ROCK inhibition. T84 monolayers were incubated with 100 μmol/L Y for 1 hour or with vehicle alone (CON). Monolayers were subsequently incubated at 4°C with extraction buffer containing 1% TX-100. The TX-100–soluble and –insoluble fractions were analyzed by SDS-PAGE and immunoblotted for occludin (OCC), ZO-1, claudin-1 (CLD-1), E-cadherin (E-cad), or β-catenin (β-cat). T, S, and I represent proteins in whole-cell lysate, TX-100–soluble, and TX-100–insoluble pools, respectively. High-molecular-weight occludin (72–79 kilodaltons) and ZO-1 were identified predominantly in the TX-100–insoluble pool of cells incubated with vehicle alone. In an analogous manner, a large pool of the AJ proteins E-cadherin and β-catenin were TX-100 insoluble. Incubation with Y (20–50 μmol/L, 1–24 hours) did not change the TX-100 solubility profiles of these TJ and AJ proteins (data not shown).
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

7. Fig. 4
(Panel I) ROCK inhibition does not influence distribution of TJ and AJ proteins. Confluent T84 monolayers were incubated with vehicle alone (CON), Y (100 μmol/L, 1 hour), or the Rho-inactivating toxin, DC3B (3.2 μg/mL basolaterally for 6 hours). The en face (XY) confocal images shown in this figure were taken at the level of (A–C, F–H, K–M) TJ and (D, E, I, J) AJ. In control monolayers (CON), ZO-1, occludin (OCC), and claudin-1 (CLD-1) were distributed in a chicken-wire pattern consistent with their localization in TJs. A large intracellular pool of CLD-1 was also identified. Incubation with Y did not influence the distribution of TJ proteins. In contrast, Rho inactivation with DC3B toxin completely disrupted the normal localization of all 3 TJ proteins. In an analogous manner, localization of E-cadherin and β-catenin in AJs was not influenced by incubation with Y Incubation with Y (20–50 μg/mL, 1–24 hours) did not change the localization of TJ and AJ proteins. (Panel II) Differential detergent solubility of TJ proteins is not influenced by ROCK inhibition. T84 monolayers were incubated with 100 μmol/L Y for 1 hour or with vehicle alone (CON). Monolayers were subsequently incubated at 4°C with extraction buffer containing 1% TX-100. The TX-100–soluble and –insoluble fractions were analyzed by SDS-PAGE and immunoblotted for occludin (OCC), ZO-1, claudin-1 (CLD-1), E-cadherin (E-cad), or β-catenin (β-cat). T, S, and I represent proteins in whole-cell lysate, TX-100–soluble, and TX-100–insoluble pools, respectively. High-molecular-weight occludin (72–79 kilodaltons) and ZO-1 were identified predominantly in the TX-100–insoluble pool of cells incubated with vehicle alone. In an analogous manner, a large pool of the AJ proteins E-cadherin and β-catenin were TX-100 insoluble. Incubation with Y (20–50 μmol/L, 1–24 hours) did not change the TX-100 solubility profiles of these TJ and AJ proteins (data not shown).
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

8. Fig. 5
Localization of ROCK in TJs of epithelial monolayers. Confluent T84 epithelial monolayers were immunostained for ROCK (green) and ZO-1 (red) and imaged by confocal microscopy. (A–C) En face (xy) confocal images taken in the apical plane of epithelial cells at the level of TJs. Corresponding computer-reconstructed vertical sections in the xz plane are shown in the bottom panels. The supporting filter in these xz images is rendered as a white line and the apical and basolateral compartments are annotated (a and bl, respectively). (A) Distribution of ROCK in a chicken-wire pattern consistent with its localization in TJs. In addition, the diffuse staining represents an intracytoplasmic pool of ROCK. (B) Distribution of ZO-1 in TJs. (C) Double labeling confirmed colocalization of ZO-1 and ROCK in TJs of epithelial monolayers (yellow).
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

9. Fig. 6
Transfection with a dominant negative mutant of ROCK inhibits formation of the perijunctional F-actin ring and apical actin-rich structures in subconfluent Caco-2 cells. Caco-2 cells were transfected with a dominant negative mutant of ROCK termed ROCK-KDIA and stained for cMyc and F-actin or occludin. Colonies of Caco-2 cells showed focal loss of apical F-actin structures (*) including the perijunctional actin ring (arrows) and apical brush border (arrows). (A, C) When examined for expression of the Myc-tagged dominant negative mutant of ROCK (green; arrows), the cells lacking apical F-actin structures strongly expressed the dominant negative mutant ROCK protein. (B, D) Neighboring nontransfected cells clearly maintained apical actin structures serving as excellent internal controls. (C) and (D) show higher-power views of another colony. No loss of occludin staining was detected in transfected cells double stained for occludin and Myc. (E) Occludin only (arrows); (F) occludin and Myc colocalization (arrows).
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

10. Fig. 7
(Panel I) ROCK is necessary for the recovery of monolayer barrier function after calcium depletion. Confluent epithelial monolayers were incubated with HBSS− and EGTA to induce TJ disassembly. Monolayers were allowed to recover in calcium-containing medium in the presence and absence of Y (20 μmol/L). Monolayer TER was followed up for 24 hours after calcium repletion. TER of control monolayers recovered to >1000 Ω · cm2 within 24 hours. In contrast, in the presence of Y-27632, monolayers failed to recover TER. (Panel II) ROCK is necessary for the reassembly of TJs, AJs, and the apical actin cytoskeleton in intestinal epithelial cells after calcium depletion. Confluent epithelial monolayers were incubated with HBSS− and EGTA to induce TJ disassembly. Monolayers were allowed to recover in calcium-containing medium in the presence and absence of Y (20 μmol/L). ZO-1, occludin (OCC), E-cadherin (Ecad), and F-actin were immunolocalized by confocal microscopy before calcium depletion (Cont 0 h), after calcium depletion (EGTA), after 24 hours of recovery in medium alone (Cont, 24 h), or after 24 hours in medium containing 20 μmol/L Y (Y-27632/24 h). The en face (xy) confocal images shown in this figure were taken in the apical plane of epithelial cells at the level of TJ and AJ. (A, E, M) In control monolayers (CONT 0 h), ZO-1, occludin, and E-cadherin are distributed in the normal chicken-wire pattern, consistent with their localization in TJ and AJ, respectively. (I) Rhodamine phalloidin labeling of F-actin in the apical plane showed the expected dotlike pattern of microvillous F-actin and its organization in peri-junctional rings. (B, F, J, N) After calcium depletion (EGTA), junctional proteins (occ, ZO-1, E-cad) and F-actin were redistributed to a perinuclear compartment. When allowed to recover in media alone (Control 24 h), (C, G, O) junctional proteins relocalized to the apical aspect of the lateral membrane within 24 hours and (K) the F-actin staining pattern returned to normal. (D, H, P) In contrast, ZO-1, occludin, and E-cadherin failed to redistribute in their corresponding intercellular junctions in the presence of Y ROCK inhibition also prevented the recovery of a normal F-actin staining pattern. Results shown are representative of 3 independent experiments.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

11. Fig. 7
(Panel I) ROCK is necessary for the recovery of monolayer barrier function after calcium depletion. Confluent epithelial monolayers were incubated with HBSS− and EGTA to induce TJ disassembly. Monolayers were allowed to recover in calcium-containing medium in the presence and absence of Y (20 μmol/L). Monolayer TER was followed up for 24 hours after calcium repletion. TER of control monolayers recovered to >1000 Ω · cm2 within 24 hours. In contrast, in the presence of Y-27632, monolayers failed to recover TER. (Panel II) ROCK is necessary for the reassembly of TJs, AJs, and the apical actin cytoskeleton in intestinal epithelial cells after calcium depletion. Confluent epithelial monolayers were incubated with HBSS− and EGTA to induce TJ disassembly. Monolayers were allowed to recover in calcium-containing medium in the presence and absence of Y (20 μmol/L). ZO-1, occludin (OCC), E-cadherin (Ecad), and F-actin were immunolocalized by confocal microscopy before calcium depletion (Cont 0 h), after calcium depletion (EGTA), after 24 hours of recovery in medium alone (Cont, 24 h), or after 24 hours in medium containing 20 μmol/L Y (Y-27632/24 h). The en face (xy) confocal images shown in this figure were taken in the apical plane of epithelial cells at the level of TJ and AJ. (A, E, M) In control monolayers (CONT 0 h), ZO-1, occludin, and E-cadherin are distributed in the normal chicken-wire pattern, consistent with their localization in TJ and AJ, respectively. (I) Rhodamine phalloidin labeling of F-actin in the apical plane showed the expected dotlike pattern of microvillous F-actin and its organization in peri-junctional rings. (B, F, J, N) After calcium depletion (EGTA), junctional proteins (occ, ZO-1, E-cad) and F-actin were redistributed to a perinuclear compartment. When allowed to recover in media alone (Control 24 h), (C, G, O) junctional proteins relocalized to the apical aspect of the lateral membrane within 24 hours and (K) the F-actin staining pattern returned to normal. (D, H, P) In contrast, ZO-1, occludin, and E-cadherin failed to redistribute in their corresponding intercellular junctions in the presence of Y ROCK inhibition also prevented the recovery of a normal F-actin staining pattern. Results shown are representative of 3 independent experiments.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions