Characterization of the interaction between protein 4.1R and ZO-2. A possible link between the tight junction and the actin cytoskeleton

Multiple isoforms of the red cell protein 4.1R are expressed in nonerythroid cells, including novel 135-kDa isoforms. Using a yeast two-hybrid system, immunocolocalization, immunoprecipitation, and in vitro binding studies, we found that two 4.1R isoforms of 135 and 150 kDa specifically interact with the protein ZO-2 (zonula occludens-2). 4.1R is colocalized with ZO-2 and occludin at Madin-Darby canine kidney (MDCK) cell tight junctions. Both isoforms of 4.1R coprecipitated with proteins that organize tight junctions such as ZO-2, ZO-1, and occludin. Western blot analysis also revealed the presence of actin and alpha-spectrin in these immunoprecipitates. Association of 4.1R isoforms with these tight junction and cytoskeletal proteins was found to be specific for the tight junction and was not seen in nonconfluent MDCK cells. The amino acid residues that sustain the interaction between 4.1R and ZO-2 reside within the amino acids encoded by exons 19-21 of 4.1R and residues 1054-1118 of ZO-2. Exogenously expressed 4.1R containing the spectrin/actin- and ZO-2-binding domains was recruited to tight junctions in confluent MDCK cells. Taken together, our results suggest that 4.1R might play an important role in organization and function of the tight junction by establishing a link between the tight junction and the actin cytoskeleton.     

Acetaldehyde disrupts tight junctions and adherens junctions in human colonic mucosa: protection by EGF and L-glutamine

Acetaldehyde, a toxic metabolite of ethanol oxidation, is suggested to play a role in the increased risk for gastrointestinal cancers in alcoholics. In the present study, the effect of acetaldehyde on tyrosine phosphorylation, immunofluorescence localization, and detergent-insoluble fractions of the tight junction and the adherens junction proteins was determined in the human colonic mucosa. The role of EGF and L-glutamine in prevention of acetaldehyde-induced effects was also evaluated. Acetaldehyde reduced the protein tyrosine phosphatase activity, thereby increasing the tyrosine phosphorylation of occludin, E-cadherin, and beta-catenin. The levels of occludin, zonula occludens-1, E-cadherin, and beta-catenin in detergent-insoluble fractions were reduced by acetaldehyde, while it increased their levels in detergent-soluble fractions. Pretreatment with EGF or L-glutamine prevented acetaldehyde-induced protein tyrosine phosphorylation, redistribution from intercellular junctions, and reduction in the levels of detergent-insoluble fractions of occludin, zonula occludens-1, E-cadherin, and beta-catenin. These results demonstrate that acetaldehyde induces tyrosine phosphorylation and disrupts tight junction and adherens junction in human colonic mucosa, which can be prevented by EGF and glutamine.     

Ligation of CD24 expressed by oral epithelial cells induces kinase dependent decrease in paracellular permeability mediated by tight junction proteins

In previous studies we demonstrated uniform strong expression of CD24 in the epithelial attachment to the tooth and in the migrating epithelium of the periodontitis lesion. Titers of serum antibodies auto-reactive with CD24 peptide correlated with reduced severity of periodontal disease. In the present study an epithelial culture model with close correspondence for expression patterns for tight junction components in periodontal epithelia was used. Ligation of CD24 expressed by oral epithelial cells with an anti-CD24 antibody induced formation of tight junctions and live-cell imaging confirmed that paracellular diffusion of fluorochrome-labeled dextran was reduced. Expression of mRNA and protein for zona occludens-1, -2, junction adhesion molecule-A (JAM-A), occludin and claudins-1, -4, -8, -15, -18 was significantly increased following ligation of CD24 but only claudins-4 and -15, JAM-A, occludin and zona occludens-1 and -2 were increased at cell contacts. This change in expression patterns reflected that observed between the epithelium of the periodontal lesion and that of the healthy gingival attachment. In the model system, response profiles to kinase inhibitors indicated a key role of c-Src kinase activation in the development of diffusion-limiting tight junction complexes. Activation was confirmed by demonstrating concomitant phosphorylation of the kinase. Pre-incubation with antibodies against JAM-A and claudin-15 prevented barrier-enhancing effects of anti-CD24 antibodies while pre-incubation with antibody to claudin-4 was partially effective. It is concluded that antibodies to CD24 facilitate expression and location of JAM-A, claudins-4 and -15 that mediate enhanced epithelial barrier function in a protective response against bacterial products.     

Differential regulation of junctional complex assembly in renal epithelial cell lines

Several signaling pathways that regulate tight junction and adherens junction assembly are being characterized. Calpeptin activates stress fiber assembly in fibroblasts by inhibiting SH2-containing phosphatase-2 (SHP-2), thereby activating Rho-GTPase signaling. Here, we have examined the effects of calpeptin on stress fiber and junctional complex assembly in Madin-Darby canine kidney (MDCK) and LLC-PK epithelial cells. Calpeptin induced disassembly of stress fibers and inhibition of Rho GTPase activity in MDCK cells. Interestingly, calpeptin augmented stress fiber formation in LLC-PK epithelial cells. Calpeptin treatment of MDCK cells resulted in a displacement of zonula occludens-1 (ZO-1) and occludin from cell-cell junctions and a loss of phosphotyrosine on ZO-1 and ZO-2, without any detectable effect on tight junction permeability. Surprisingly, calpeptin increased paracellular permeability in LLC-PK cells even though it did not affect tight junction assembly. Calpeptin also modulated adherens junction assembly in MDCK cells but not in LLC-PK cells. Calpeptin treatment of MDCK cells induced redistribution of E-cadherin and -catenin from intercellular junctions and reduced the association of p120ctn with the E-cadherin/catenin complex. Together, our studies demonstrate that calpeptin differentially regulates stress fiber and junctional complex assembly in MDCK and LLC-PK epithelial cells, indicating that these pathways may be regulated in a cell line-specific manner. calpeptin; tight junctions; adherens junctions; Rho; cadherin; p120ctn     

Interaction between pericytes and endothelial cells leads to formation of tight junction in hyaloid vessels

The hyaloid vessel is a transient vascular network that nourishes the lens and the primary vitreous in the early developmental periods. In hyaloid vessels devoid of the support of astrocytes, we demonstrate that tight junction proteins, zonula occludens-1 and occludin, are regularly expressed at the junction of endothelial cells. To figure out the factor influencing the formation of tight junctions in hyaloid vessels, we further progress to investigate the interactions between endothelial cells and pericytes, two representative constituent cells in hyaloid vessels. Interestingly, endothelial cells interact with pericytes in the early postnatal periods and the interaction between two cell types provokes the up-regulation of transforming growth factor β1. Further in vitro experiments demonstrate that transforming growth factor β1 induces the activation of Smad2 and Smad3 and the formation of tight junction proteins. Taken together, in hyaloid vessels, pericytes seem to regulate the formation of tight junctions by the interaction with endothelial cells even without the support of astrocytes. Additionally, we suggest that the hyaloid vessel is a valuable system that can be utilized for the investigation of cell-cell interaction in the formation of tight junctions in developing vasculatures.     

Glucocorticoid down-regulation of RhoA is required for the steroid-induced organization of the junctional complex and tight junction formation in rat mammary epithelial tumor cells

In Con8 mammary epithelial tumor cells, we have documented previously that the synthetic glucocorticoid dexamethasone induces the reorganization of the tight junction and adherens junction (apical junction) and stimulates the monolayer transepithelial electrical resistance (TER), which is a reliable in vitro measurement of tight junction sealing. Western blots demonstrated that dexamethasone treatment down-regulated the level of the RhoA small GTPase prior to the stimulation of the monolayer TER. To test the role of RhoA in the steroid regulation of apical junction dynamics functionally, RhoA levels were altered in Con8 cells by transfection of either constitutively active (RhoA.V14) or dominant negative (RhoA.DN19) forms of RhoA. Ectopic expression of constitutively active RhoA disrupted the dexamethasone-stimulated localization of zonula occludens-1 and beta-catenin to sites of cell-cell contact, inhibited tight junction sealing, and prevented the complete formation of the F-actin ring structure at the apical side of the cell monolayer. In a complementary manner, dominant negative RhoA caused a precocious organization of the tight junction, adherens junction, and the F-actin rings in the absence of steroid, whereas the monolayer TER remained glucocorticoid-responsive. Taken together, our results demonstrate that the glucocorticoid down-regulation of RhoA is a required step in the steroid signaling pathway which controls the organization of the apical junctional complex and the actin cytoskeleton in mammary epithelial cells.     

Crosstalk between desmoglein-2/desmocollin-2/Src kinase and coxsackie and adenovirus receptor/ZO-1 protein complexes,regulates blood-testis barrier dynamics

Morphological studies in the testis reported the presence of ‘desmosome-like’ junctions between Sertoli cells at the blood-testis barrier, whose function is also constituted by tight junctions and basal ectoplasmic specializations. Unfortunately, little is known about the role of desmosomes in blood-testis barrier dynamics. This study aims to fill this gap with the functional investigation of two desmosomal cadherins, desmoglein-2 and desmocollin-2, by their specific knockdown in Sertoli cells cultured in vitro. Reminiscent of the blood-testis barrier in vivo, desmosome-like structures were visible by electron microscopy when Sertoli cells were cultured at high density, thereby forming a polarized epithelium with functional cell junctions. At this point, we opted to focus our efforts on desmoglein-2 and desmocollin-2 based on results which illustrated desmosomal mRNAs to be expressed by Sertoli and germ cells, as well as on results which illustrated desmoglein-2 to co-immunoprecipitate with plakoglobin, c-Src and desmocollin-2. Simultaneous knockdown of desmoglein-2 and desmocollin-2 not only led to a reduction in and mislocalization of zonula occludens-1, but also perturbed the localization of c-Src and coxsackie and adenovirus receptor at the cell–cell interface, resulting in disruption of tight junction permeability barrier. We hereby propose a novel regulatory protein complex composed of desmoglein-2, desmocollin-2, c-Src, coxsackie and adenovirus receptor and zonula occludens-1 at the blood-testis barrier.     

Chloroquine and Hydroxychloroquine Increase Retinal Pigment Epithelial Layer Permeability

Antimalarials chloroquine (CQ) and hydroxychloroquine (HCQ) are widely used as antiinflammatory drugs, but side effects include retinopathy and vision loss. The objective of this study was to examine the effect of CQ and HCQ on the barrier integrity of retinal pigment epithelial (RPE) cell monolayers in vitro. Permeability of ARPE‐19 cell monolayers was determined using Fluorescein isothiocyanate (FITC)‐labeled dextran. The influence of CQ and HCQ on cell death and the expression tight junction molecules was examined. CQ and HCQ significantly increased ARPE‐19 monolayer permeability after 3 and 18 h, respectively, and enhanced mRNA levels for claudin‐1 and occludin. Cytotoxicity was only observed after 18 h exposure. Thus, CQ and HCQ rapidly enhance RPE barrier permeability in vitro, independent of cytotoxicity or loss of zonula occludens‐1, claudin‐1, and occludin expression. Our findings suggest that CQ/HCQ‐induced permeability of the RPE layer may contribute to blood–retinal barrier breakdown in case of CQ/HCQ‐induced retinopathy.     

Rho GTPase signaling regulates tight junction assembly and protects tight junctions during ATP depletion

Tight junctions control paracellular permeability and cellpolarity. Rho GTPase regulates tight junction assembly, and ATP depletion of Madin-Darby canine kidney (MDCK) cells (an in vitro modelof renal ischemia) disrupts tight junctions. The relationship between Rho GTPase signaling and ATP depletion was examined. Rho inhibition resulted in decreased localization of zonula occludens-1 (ZO-1) and occludin at cell junctions; conversely, constitutive Rhosignaling caused an accumulation of ZO-1 and occludin at cell junctions. Inhibiting Rho before ATP depletion resulted in more extensive loss of junctional components between transfected cells thancontrol junctions, whereas cells expressing activated Rho bettermaintained junctions during ATP depletion than control cells. ATPdepletion and Rho signaling altered phosphorylation signalingmechanisms. ZO-1 and occludin exhibited rapid decreases in phosphoaminoacid content following ATP depletion, which was restored on recovery.Expression of Rho mutant proteins in MDCK cells also altered levels ofoccludin serine/threonine phosphorylation, indicating that occludin isa target for Rho signaling. We conclude that Rho GTPase signalinginduces posttranslational effects on tight junction components. Ourdata also demonstrate that activating Rho signaling protects tightjunctions from damage during ATP depletion.

     

Oxidized low density lipoprotein-induced senescence of retinal pigment epithelial cells is followed by outer blood-retinal barrier dysfunction

Age-related macular degeneration is the most common cause of vision loss in the elderly, which starts from aging processes of retinal pigment epithelial cells. Among variable risk factors in occurrence and progression of age-related macular degeneration, oxidized low density lipoprotein could be causally involved in pathobiological changes of RPE cells. Herein we showed that oxidized low density lipoprotein-induced senescence of retinal pigment epithelial cells is followed by outer blood-retinal barrier dysfunction. Under sub-lethal concentration, oxidized low density lipoprotein could promote advanced senescence of retinal pigment epithelial cells. Interestingly expression of CRALBP and RPE 65, indicators of retinal pigment epithelial cell differentiation, was decreased by oxidized low density lipoprotein. In addition, oxidized low density lipoprotein induced reactive oxygen species production and up-regulated inflammatory factors such as tumor necrosis factor-α and vascular endothelial growth factor, when β-catenin, a critical mediator of the canonical Wnt pathway, was also elevated. Oxidized low density lipoprotein increased paracellular permeability of retinal pigment epithelial cells, when zonula occludens-1 at intercellular junctions markedly decreased as well. Furthermore, in retinal pigment epithelial cells and choriocapillaris of human apolipoprotein E2 transgenic mouse eye, increased vascular endothelial growth factor and decreased zonula occludens-1 expression was observed. Therefore, our results suggest that oxidized low density lipoprotein could promote senescence of retinal pigment epithelial cells which leads to induce outer blood-retinal barrier dysfunction as an early pathogenesis of age-related macular degeneration.     

sPAR-3, a splicing variant of PAR-3, shows cellular localization and an expression pattern different from that of PAR-3 during enterocyte polarization

PAR-3 (partitioning-defective) is a scaffold-like PDZ (postsynaptic density-95/discs large/zonula occludens-1) domain-containing protein that forms a complex with PAR-6 and atypical PKC, localizes to tight junctions, and contributes to the formation of functional tight junctions. There are several alternatively spliced isoforms of PAR-3, although their physiological significance remains unknown. In this study, we show that one of the major isoforms of PAR-3, sPAR-3, is predominantly expressed in the Caco-2 cells derived from colon carcinoma and is used as a model to investigate the events involved in the epithelial cell differentiation and cell polarity development. During the polarization of Caco-2 cells, the expression of PAR-3 increases as do those of other cell-cell junction proteins, whereas the expression of sPAR-3 decreases. Biochemical characterization revealed that sPAR-3 associates with atypical PKC, as does PAR-3. On the other hand, immunofluorescence microscopy revealed that sPAR-3 does not concentrate at the cell-cell contact region in fully polarized cells, whereas it concentrates at premature cell-cell junctions. This makes a contrast to PAR-3, which concentrates at tight junctions in fully polarized cells. These results provide evidence suggesting the difference in the role between sPAR-3 and PAR-3 in epithelial cells.     

Suppression of the transformed phenotype of breast cancer by tropomyosin-1

Gap junctional intercellular communication (GJIC) is thought to play a crucial role in cell differentiation. Small gap junction plaques are frequently associated with tight junction strands in hepatocytes, suggesting that gap junctions may be closely related to the role of tight junctions in the establishment of cell polarity. To examine the exact role of gap junctions in regulating tight junctions, we transfected connexin 32 (Cx32), Cx26, or Cx43 cDNAs into immortalized mouse hepatocytes derived from Cx32-deficient mice and examined the expression and function of the endogenous tight junction molecules. In transient wild-type Cx32 transfectants, immunocytochemistry revealed that endogenous occludin was in part localized at cell borders, where it was colocalized with Cx32, whereas neither was detected in parental cells. In Cx32 null hepatocytes transfected with Cx32 truncated at position 220 (R220stop), wild-type Cx26, or wild-type Cx43 cDNAs, occludin was not detected at cell borders. In stable wild-type Cx32 transfectants, occludin, claudin-1, and ZO-1 mRNAs and proteins were significantly increased compared to parental cells and all of the proteins were colocalized with Cx32 at cell borders. Treatment with a GJIC blocker, 18 beta-glycyrrhetinic acid, resulted in decreases of occludin and claudin-1 at cell borders in the stable transfectants. The induction of tight junction proteins in the stable transfectants was accompanied by an increase in both fence and barrier functions of tight junctions. Furthermore, in the stable transfectants, circumferencial actin filaments were also increased without a change of actin protein. These results indicate that Cx32 formation and/or Cx32-mediated intercellular communication may participate in the formation of functional tight junctions and actin organization.     

PATJ regulates tight junction formation and polarity in mammalian epithelial cells

Recent studies have revealed an important role for tight junction protein complexes in epithelial cell polarity. One of these complexes contains the apical transmembrane protein, Crumbs, and two PSD95/discs large/zonula occludens domain proteins, protein associated with Lin seven 1 (PALS1)/Stardust and PALS1-associated tight junction protein (PATJ). Although Crumbs and PALS1/Stardust are known to be important for cell polarization, recent studies have suggested that Drosophila PATJ is not essential and its function is unclear. Here, we find that PATJ is targeted to the apical region and tight junctions once cell polarization is initiated. We show using RNAi techniques that reduction in PATJ expression leads to delayed tight junction formation as well as defects in cell polarization. These effects are reversed by reintroduction of PATJ into these RNAi cells. This study provides new functional information on PATJ as a polarity protein and increases our understanding of the Crumbs-PALS1-PATJ complex function in epithelial polarity.     

Connexin 26 expression prevents down-regulation of barrier and fence functions of tight junctions by Na+/K+-ATPase inhibitor ouabain in human airway epithelial cell line Calu-3

Gap junctions are considered to play a crucial role in differentiation of epithelial cells and to be associated with tight junction proteins. In this study, to investigate the role of gap junctions in regulation of the barrier function and fence function on the tight junctions, we introduced the Cx26 gene into human airway epithelial cell line Clau-3 and used a disruption model of tight junctions employing the Na(+)/K(+)-ATPase inhibitor ouabain. In parental Calu-3 cells, gap junction proteins Cx32 and Cx43, but not Cx26, and tight junction proteins occludin, JAM-1, ZO-1, claudin-1, -2, -3, -4, -5, -6, -7, -8, -9, and -14 were detected by RT-PCR. The barrier function and fence function of tight junctions were well maintained, whereas the GJIC was low level. Treatment with ouabain caused disruption of the barrier function and fence function of tight junctions together with down-regulation of occludin, JAM-1, claudin-2, and -4 and up-regulation of ZO-1 and claudin-14. In Cx26 transfectants, Cx26 protein was detected by Western blotting and immunocytochemistry, and many gap junction plaques were observed with well-developed tight junction strands. Expression of claudin-14 was significantly increased in Cx26 transfectants compared to parental cells, and in some cells, Cx26 was co-localized with claudin-14. Interestingly, transfection with Cx26 prevented disruption of both functions of tight junctions by treatment with ouabain without changes in the tight junction proteins. Pretreatment with the GJIC blockers 18beta-glycyrrhetinic acid and oleamide did not affect the changes induced by Cx26 transfection. These results suggest that Cx26 expression, but not the mediated intercellular communication, may regulate tight junction barrier and fence functions in human airway epithelial cell line Calu-3.     

Zonula occludens-1 (ZO-1) is involved in morula to blastocyst transformation in the mouse

It is unknown whether or not tight junction formation plays any role in morula to blastocyst transformation that is associated with development of polarized trophoblast cells and fluid accumulation. Tight junctions are a hallmark of polarized epithelial cells and zonula occludens-1 (ZO-1) is a known key regulator of tight junction formation. Here we show that ZO-1 protein is first expressed during compaction of 8-cell embryos. This stage-specific appearance of ZO-1 suggests its participation in morula to blastocyst transition. Consistent with this idea, we demonstrate that ZO-1 siRNA delivery inside the blastomeres of zona-weakened embryos using electroporation not only knocks down ZO-1 gene and protein expressions, but also inhibits morula to blastocyst transformation in a concentration-dependent manner. In addition, ZO-1 inactivation reduced the expression of Cdx2 and Oct-4, but not ZO-2 and F-actin. These results provide the first evidence that ZO-1 is involved in blastocyst formation from the morula by regulating accumulation of fluid and differentiation of nonpolar blastomeres to polar trophoblast cells.     

Amyloid-β(1-42) alters structure and function of retinal pigmented epithelial cells

Age-related macular degeneration (AMD) is characterized by the formation of drusen, extracellular deposits associated with atrophy of the retinal pigmented epithelium (RPE), disturbance of the transepithelial barrier and photoreceptor death. Amyloid-β (Aβ) is present in drusen but its role during AMD remains unknown. This study investigated the in vitro and in vivo effects of the oligomeric form of Aβ(1-42) – OAβ(1-42) – on RPE and found that it reduced mitochondrial redox potential and increased the production of reactive oxygen species, but did not induce apoptosis in RPE cell cultures. It also disorganized the actin cytoskeleton and halved occludin expression, markedly decreasing attachment capacity and abolishing the selectivity of RPE cell transepithelial permeability. Antioxidant pretreatment partially reversed the effects of OAβ(1-42) on mitochondrial redox potential and transepithelial permeability. Subretinally injected OAβ(1-42) induced pigmentation loss and RPE hypertrophy but not RPE cell apoptosis in C57BL/6 J mice. Rapid OAβ(1-42)-induced disorganization of cytoskeletal actin filaments was accompanied by decreased RPE expression of the tight junction proteins occludin and zonula occludens-1 and of the visual cycle proteins cellular retinaldehyde-binding protein and RPE65. The number of photoreceptors decreased by half within a few days. Our study pinpoints the role of Aβ in RPE alterations and dysfunctions leading to retinal degeneration and suggests that targeting Aβ may help develop selective methods for treating diseases involving retinal degeneration, such as AMD.     

AMP-18 protects barrier function of colonic epithelial cells: role of tight junction proteins

Antrum mucosal protein (AMP)-18 and a synthetic peptide of amino acids 77-97 have mitogenic and motogenic properties for epithelial cells. The possibility that AMP-18 is also protective was evaluated in the colonic mucosa of mice and monolayer cultures of human colonic epithelial Caco-2/bbe (C2) cells. Administration of AMP peptide to mice with dextran sulfate sodium (DSS)-induced colonic injury delayed the onset of bloody diarrhea and reduced weight loss. Treatment of C2 cells with AMP peptide protected monolayers against decreases in transepithelial electrical resistance induced by the oxidant monochloramine, indomethacin, or DSS. A molecular mechanism for these barrier-protective effects was sought by asking whether AMP peptide acted on specific tight junction (TJ) proteins. Immunoblots of detergent-insoluble fractions of C2 cells treated with AMP peptide exhibited increased accumulation of specific TJ proteins. Occludin immunoreactivity was also increased in detergent-insoluble fractions obtained from colonic mucosal cells of mice injected with AMP peptide. Observations using laser scanning confocal (CF) microscopy supported the capacity of AMP peptide to enhance accumulation of occludin and zonula occludens-1 in TJ domains of C2 cell monolayers and together with immunoblot analysis showed that the peptide protected against loss of these TJ proteins following oxidant injury. AMP peptide also protected against a fall in TER during disruption of actin filaments by cytochalasin D and stabilized perijunctional actin during oxidant injury when assessed by CF. These findings suggest that AMP-18 could protect the intestinal mucosal barrier by acting on specific TJ proteins and stabilizing perijunctional actin.     

ZO-1- and ZO-2-dependent integration of myosin-2 to epithelial zonula adherens

For the zonula adherens (ZA) to be established by linear arrangement of adherens junctions (AJs) in epithelial sheet cells, critical for the epithelial cell sheet formation and intercellular barrier function, myosin-2 is supposedly integrated into the ZA with the result of overlapping localization of E-cadherin/actin/myosin-2. Here, we immunofluorescently showed that myosin-2 failed to be integrated into the ZA in cultured epithelial-type ZO1(ko)/2(kd) Eph4 cells lacking ZO-1 and -2 (zonula occludens-1 and -2) by knockout and knockdown, respectively. Instead, a linearized but fragmented arrangement of AJs was formed in the way that it was positive for E-cadherin/actin, but negative for myosin-2 (designated prezonula-AJ). Transfection of full-length ZO-1 or ZO-2, or ZO-1 lacking its PDZ (PSD-95/discs large/zonula occludens-1)-1/2 domains (but not one lacking PDZ-1/2/3) into ZO1(ko)/2(kd) Eph4 cells restored the junctional integration of myosin-2 with prezonula-AJ to establish the ZA. Transfection of dominant-active RhoA or Rho-kinase (ROCK), as well as administration of lysophosphatidic acid or Y27632, which activates RhoA or inhibits ROCK, respectively, suggested that RhoA regulated the junctional integration of myosin-2 into ZA in a manner such that ROCK played a necessary but not-sufficient role. Fluorescence resonance energy transfer analyses revealed that spatiotemporal Rho-activation occurred in a ZO-1/2–dependent way to establish ZA from primordial forms in epithelial cells.