roles of zo-1 and zo-2 in establishment of the belt-like adherens and tight junctions with...

MOLECULAR STRUCTURE AND FUNCTION OF THE TIGHT JUNCTION

Roles of ZO-1 and ZO-2 in Establishmentof the Belt-like Adherens and Tight Junctions

with Paracellular PermselectiveBarrier Function

Sachiko Tsukita,a Tatsuya Katsuno,a Yuji Yamazaki,a

Kazuaki Umeda,b Atsushi Tamura,a

and Shoichiro Tsukitaa

aLaboratory of Biological Science, Graduate School of Frontier Biosciences and GraduateSchool of Medicine, Osaka University, Suita, Osaka, Japan

bDepartment of Molecular Pharmacology, Graduate School of Medical Science,Kumamoto University, Honjo, Kumamoto, Japan

Tight junctions (TJs) create the primary permselective barrier to diffusion of solutesand ions through the paracellular pathway. The molecular architecture of TJs has grad-ually been unraveled in recent years, providing the basis for “barriology” (defined byShoichiro Tsukita as the science of the barrier in multicellular organisms). Claudins arenow considered to be the essential basic components of TJ strands, with which other in-tegral membrane proteins, such as occludin, tricellulin, JAMs, and CAR, are associated.Peripherally associated scaffolding proteins are required for the organization of theintegral membrane proteins. Among these, ZO-1, -2, and -3 have attracted a great dealof attention as TJ organizers, since ZO-1 (and in some cases, also ZO-2/3) was reportedto be directly associated with claudins, occludin, and JAMs, as well as with AF-6/afadinand alpha-catenin. Here we summarize recent studies on ZO-1/2/3-deficiency in miceand cells, which have provided clear and important information regarding the functionsof ZO-1/2/3 in vivo. In addition to the respective suppression of ZO-1/2/3 expression,simultaneous suppression of all three proteins has revealed the essential and nonessen-tial in vivo roles of ZO-1/2 and ZO-3, respectively. ZO-3 shows an epithelial-specificTJ localization in a ZO-1/2–dependent fashion. ZO-1 and ZO-2 play pivotal roles in thefinal establishment of the belt-like adherens junctions (zonula adherens), followed bythe formation of the belt-like TJs (zonula occludens) with paracellular barrier function,thereby providing the general basis for selective paracellular permeability in epithelialand endothelial cells.

Key words: adherens junction; tight junction; ZO-1; ZO-2; claudin; cadherin; cell–celladhesion; cell–cell contact; paracellular barrier

Introduction

Tight junctions (TJs) constitute cell–cell ad-hering junctional complexes together with ad-herens junctions (AJs) and desmosomes in the

Address for correspondence: Sachiko Tsukita, Ph.D., Laboratory ofBiological Science, Graduate School of Medicine, Osaka University,Yamadaoka 2-2, Osaka 565-0871, Japan. Voice: +81-6-6879-3320; fax:+81-6-6879-3329. [email protected]

most apical part of epithelial and endothelialcells.1 Evidence has accumulated that the basicarchitecture of belt-like TJs is one composedof TJ strands formed by the polymerizationof claudins, which comprise a multigene fam-ily consisting of at least 24 members in hu-mans and mice.2–4 The other TJ integral mem-brane proteins—occludin, tricellulin, JAMs,and CAR—are associated with TJ strands.5–8

The high organization of the extracellular

Molecular Structure and Function of the Tight Junction: Ann. N.Y. Acad. Sci. 1165: 44–52 (2009).doi: 10.1111/j.1749-6632.2009.04056.x c© 2009 New York Academy of Sciences.

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Tsukita et al.: Roles of ZO-1/2 in Cell–Cell Adhering Junctions 45

Figure 1. Schematic drawings of the ZO-1, -2, and -3 molecules. The three proteins havesimilar domain structures with affinities for various TJ and AJ membrane proteins, as well asfor cytoskeletal and signaling molecules.

domains of integral membrane proteins of TJstrands is essential for the paracellular barrierfunction in the belt-like TJs. An essential ques-tion that relates to the molecular mechanismsis: How are claudins polymerized exclusivelyat TJs in close proximity to AJs in a tem-porally and spatially regulated manner? Theproteins underlying the cytoplasmic surface ofTJs were thought to be important, and threeclosely related proteins designated ZO-1, -2,and -3 were especially noted for their associa-tions with TJ and AJ components to constitutethe plaque structures of TJs,9–11 together withcingulin, Par-3, Par-6, aPKC, MUPP-1, PATJ,ZONAB/dpbA, GEF-H1/Lfc,12–16 and so on.

ZO-1/2/3 as TJ MAGUKFamily Proteins

Cloning and sequencing of cDNAs en-coding ZO-1, -2, and -3 have shown thateach of them contains three PDZ domains(PDZ1 to 3), one SH3 domain, and oneGUK domain in that order starting from theN-terminus,17–20 indicating that they belongto the TJ membrane–associated guanylatekinase-like homologue (MAGUK) family21–23

(Fig. 1). In database analyses of the mouseand human genomes, no other ZO-1–relatedMAGUKs have been found, thus leadingto the conclusion that ZO-1, -2, and -3constitute a subfamily of the TJ MAGUKs.As shown in Figure 1, ZO-1, -2, and -3 showvarious binding characteristics for TJ and AJcomponents and are thus speculated to act asjunctional organizers coordinated by a properswitching mechanism between AJs and TJs.

What Knockout of ZO-1, -2, and -3Tells Us about the Essential Roles of

ZO-1 and ZO-2 for Developmentin Mice

One way to conclusively examine the es-sential roles of ZO-1, -2, and -3 is to knockout their genes (Tjp1, 2, and 3, respectively).This strategy was applied to mice and cul-tured cells. Here, we first summarize studiesinvolving the respective knockout of ZO-1, -2,and -3 in mice, which have been reportedin these 2 years. ZO-3 knockout (Tjp3−/−)mice were described first.24,25 Although ZO-3 exhibited a prominent localization and wasspecifically expressed in epithelial-type cells,

46 Annals of the New York Academy of Sciences

Tjp3−/− mice showed no phenotypes, suggest-ing that ZO-3 was not essential in vivo. In con-trast, the respective knockout mice for ZO-1 and ZO-2 reportedly exhibited embryoniclethality.25,26

In ZO-1 knockout mice, both embryonicand extra-embryonic yolk sac defects wererecognized at around E9.5, leading to em-bryonic lethality at around E11.5. ZO-1 maybe functionally important for cell remodelingand tissue organization in the embryonic andextra-embryonic regions, thus playing essentialroles in embryonic development. Consideringthat TJ formation was significantly delayed inZO-1–deficient epithelial Eph4 cells (see thefollowing section), one possible interpretationwas that the timing of the organization of celladhesion was critical for the developmentalprocess, and that ZO-1 deficiency associatedwith the abnormal time course of the TJ forma-tion might lead to embryonic lethality. Based onthe finding that ZO-2 was not expressed in themesoderm of the yolk sac, another possibilitywas that yolk sac defects owing to ZO-1 defi-ciency caused embryonic lethality with no com-pensatory upregulation of ZO-2 expression.

ZO-2 knockout mice showed a more severephenotype of lethality at around E6.5. The ar-rests of cell proliferation and subsequent pro-grammed cell death between E6.5 and E7.5most likely prevented gastrulation in ZO-2–knockout embryos. Thus, ZO-1 and ZO-2, butnot ZO-3, are likely to be independently es-sential for embryonic development, suggestingnonredundant roles in mice.

What Studies onZO-1/2/3-deficient Cultured Cells

Tell Us about theRequired/Sufficient Roles of ZO-1and ZO-2 in Belt-like TJ Formation

Generation of ZO-1/2/3–deficient Cells

Knockout of the ZO-1/2/3 genes merits ap-plication to cultured cell systems, as well as

knockdown strategies. Recent studies with cul-tured epithelial Eph4 cells (mammary gland–derived cells without ZO-3 expression) haveproduced a reliable series of data sets. The ZO-1 gene was targeted first.27 It was found that TJformation was delayed in ZO-1–deficient Eph4cells [ZO1(ko)/2(wt)/3(−) Eph4 cells]. As an-other phenotype of ZO1(ko)/2(wt)/3(−) Eph4cells, the TJ-localized cingulin was not asso-ciated with TJs. This cingulin-related pheno-type was not detected in ZO-1 knockout mice,suggesting cell-type dependency of the local-ization of cingulin. In contrast, deficiency ofZO-2 and/or ZO-3 did not delay TJ forma-tion in cultured epithelial cell types, suggestingthat ZO-1 plays a major role in the formationof belt-like TJs in epithelial cells compared withZO-2 and ZO-3.

The generation of epithelial Eph4 cell lineslacking expression of ZO-1, -2, and -3 hasrevealed their definitive functional roles inbelt-like TJ and AJ formation. Eph4 celllines with suppressed ZO-2 expression wereestablished from ZO1(ko)/2(wt)/3(−) Eph4cells by stably expressing short-interferingRNAs for ZO-2. The resulting cell linesmostly lacked expression of ZO-1, -2 and -3[ZO1(ko)/2(kd)/3(−) Eph4 cells]. In a parallelline of experiments, the genes for ZO-1 andZO-2 were targeted by a knockout strategy tosuppress their expression in teratocarcinoma-derived F9 cells. The data obtained with theZO1(ko)/2(kd)/3(−) Eph4 cells and epitheliallydifferentiated ZO1(ko)/2(ko)/3(wt) F9 cells un-equivocally have provided a clear picture of thefunctions of ZO-1, -2, and -3.

ZO-1/2–dependent Formation of theBelt-like TJ (Zonula Occludens)

The ZO1(ko)/2(kd)/3(−) Eph4 cells com-pletely lacked TJs. Specifically, no concen-trations of claudins were detected at cell–cell borders, no TJ strands were observedby freeze-fracture replica electron microscopy,and the barrier function was severely af-fected (Fig. 2). TJ strands were formed in the


Figure 2. The barrier assay for wild-type, ZO1(wt)/2(wt)/3(−) and ZO1(ko)/2(kd)/3(−)Eph4 cultured cells. (A) Schematic drawings of the method for the barrier assay. (B) Im-munofluorescence micrographs (IF) and freeze-fracture replica electron micrographs (FF) ofZO1(wt)/2(wt)/3(−) and ZO1(ko)/2(kd)/3(−) Eph4 cultured cells in barrier assay. ZO-1/2/3 deficiency causes the loss of the TJ-based barrier function in cultured epithelial Eph4cells. Arrows : TJ strands. Bar, 5 μm in IF. Bar, 0.5 μm in FF.

ZO1(ko)/2(kd)/3(−) Eph4 cells by single trans-fection of ZO-1 or ZO-2, suggesting redun-dant roles of ZO-1 and ZO-2 in TJ strandformation. In contrast, ZO-3 could not tar-get junctional areas by itself and required thepreexistence of ZO-1 and/or ZO-2, althoughthe PDZ1 domains of ZO-3 and ZO-1/2 havebeen reported to bind directly to the C-terminiof claudins in vitro.29 Since the expression lev-els of claudins did not differ between wild-typeand ZO1(ko)/2(kd)/3(−) Eph4 cells, and TJstrands were formed after single transfection ofZO-1 or ZO-2, it was concluded that ZO-1 andZO-2 independently play essential roles in thepolymerization of claudins to form TJ strandsaround AJs. Occludin and JAMs were also inte-grated into these TJ strands, possibly through

association with the SH3/GUK and PDZ-3domains of ZO-1/2/3, respectively.30,31 Noneof these proteins were concentrated at the cell–cell borders of ZO1(ko)/2(kd)/3(−) Eph4 cells.Thus, ZO-1, -2, and -3 probably act as a struc-tural cue for TJ constituents.

The ZO1(ko)/2(kd)/3(−) Eph4 cells wereadvantageous for further in-depth functionalanalyses of the domains of ZO-1, -2, and-3. The SH3/GUK domains of ZO-1 (andprobably also ZO-2) were able to inducedimerization of ZO-1 to initiate the poly-merization of claudins for TJ formation andalso determined the correct localization of TJstrands around AJs through their interactionswith afadin/alpha-catenin complexes.32,33 TheSH3/GUK domains of ZO-1 were reported to


be required for the formation of TJ strands.28

Consistently, in the case of a ZO-1 constructlacking the SH3/GUK and C-terminal do-mains, TJ strands were only formed when itsdimerization was enforced. Thus, taking theresults for the ZO1(ko)/2(kd)/3(−) Eph4 cellstogether with those for the epithelially differen-tiated ZO1(ko)/2(ko)/3(wt) F9 cells, it is evidentthat ZO-1 and ZO-2 play essential roles in thepermselective barrier function of the belt-likeTJ (zonula occludens), and that functional belt-like TJs cannot form in the absence of ZO-1/2.

Specificities of the Epithelial ParacellularPermselective Barrier

It should be noted that the function ofthe epithelial paracellular permselective bar-rier varies according to the different parts of thebody. Therefore, paracellular permeability andtranscellular transport show enormous variety.The characteristics of the epithelial paracellu-lar permselective barrier may be determinedby varying combinations of TJ components,such as claudins, occludin and tricellulin; thus,this is an important issue in “barriology” (thescience of barriers in multicellular organisms).Although ZO-1, -2, and -3 regulate the poly-merization of these components, they are notlikely to determine the specific polymerizationof each component. However, it remains to beelucidated whether they determine the charac-teristics of the barrier-based paracellular per-meability in any situation.

ZO-1/2–independent Fence Functionand Apico-basal Cell Polarization

in Epithelial Cells

In addition to their barrier function, it haslong been assumed that the belt-like TJs act as afence to prevent membrane proteins from freelydiffusing between apical and basolateral mem-branes.34–36 However, in ZO1(ko)/2(kd)/3(−)Eph4 cells lacking TJs, normal apicobasal seg-regation of membrane proteins took place.28

Therefore, consistent with the previous simi-

lar claim,37 it has been convincingly revealedthat TJ strands are not vital to the asymmet-ric distribution of membrane proteins on cellmembranes. Epithelial polarity-related factorssuch as Par3, aPKC and Pals1 exhibited nor-mal apico-basal polarity but had broad lat-eral distributions in ZO1(ko)/2(kd)/3(−) Eph4cells as compared to wild-type epithelial Eph4cells. Thus, TJs are thought to play a tun-ing role, though not an essential one, in theestablishment of epithelial cell polarity. Stillmore studies are required to prove the roleof the belt-like arrangement of TJs in cellpolarization.

What Studies onZO-1/2/3-deficient Cells Tell Us

about the Critical Roles of ZO-1/2in the Final Establishment of theBelt-like AJ (Zonula Adherens)

Detailed analyses of ZO-1/2/3–deficientepithelial Eph4 cells [ZO1(ko)/2(kd)/3(−)Eph4 cells] have revealed that ZO-1, -2, and-3 play critical roles not only in the formationof TJ strands but also in the final establishmentof the belt-like AJs. In epithelial cells, ZO-1 andZO-2 were exclusively localized at belt-like TJs,and in the absence of belt-like TJs, ZO-1 andZO-2 were enriched at cell–cell AJs.18,38 Takentogether with the previous reports that ZO-1 and ZO-2 bound to AJ and TJ constitutiveproteins,29–33 the functional aspects of ZO-1and ZO-2 in belt-like TJs and AJs were be-lieved to be coordinated by opposite switchingmechanisms. The linear belt-like arrangementof E-cadherin–based cell–cell AJs was also sig-nificantly delayed by ZO-1/2/3 deficiency.39

Further analyses of ZO1(ko)/2(kd)/3(−) Eph4cells suggested a prominent defect in con-stitution of the E-cadherin-based AJs, whichis associated with actin, but abnormally notwith myosin-2.40 This type of AJ with an in-complete belt-like arrangement represented apreviously undescribed junctional state, desig-nated the “prezonula-AJ,” which we observed


Figure 3. Schematic drawing of a tentative model for ZO-1/2–based formation of thebelt-like adherens and tight junctions [zonula adherens (ZA) and zonula occludens]. (I) Whenconfluent epithelial cells initiate cell–cell adhesion to form epithelial sheets by raising calciumconcentrations from zero-level to a normal level (2 mM), the punctate adherens junctions (AJs)are initially formed at cell–cell adhesion sites. (II) Punctate AJs develop into prezonula-AJs,which are partially linearized and associated with E-cadherin and actin, but not with myosin-2integrated. Myosin-2 is distributed in a ring-like arrangement with actin, along prezonula-AJs.In the absence of ZO-1/2, prezonula-AJs do not develop to the completely belt-like AJs(zonula adherens). (III) In ZO1(wt)/2(wt)/3(−) Eph4 cells in which ZO-1/2 are expressed,the formation of prezonula-AJs and the integration of myosin-2 with them to establish belt-likeAJs [zonula adherens (ZAs)] are likely to proceed simultaneously in such a way that punctateAJs appear to be directly organized into a belt-like arrangement. (IV) The belt-like TJs (zonulaoccludens) are synergistically formed just above the belt-like AJs and associated with ZO-1and ZO-2, thus establishing epithelial cell sheets with a TJ paracellular permselective barrierfunction.

for the first time in cells lacking both ZO-1 and ZO-2. In contrast, the belt-like AJs inZO1(wt)/2(wt)/3(−) Eph4 cells were positivefor E-cadherin, actin and myosin-2.

The junctional integration of myosin-2 withprezonula-AJs is likely to be a critical step to-ward the final establishment of the belt-likeAJs, and it has been suggested that this pro-

cess is dependent on ZO-1/2 and RhoA. Inthis respect, extensive activation of RhoA wasdetected at cell–cell contacts by FRET imag-ing during the formation of cell–cell junctionsin wild-type Eph4 cells, and ZO-1/2 deficiencyprevented this activation. Taken together withthe previous findings that Rac1 plays a rolein the conversion of AJ arrangements from


fibroblastic to linear epithelial types in a ZO-1/2-dependent manner, it is likely that ZO-1and ZO-2 act as signaling cues as well as cy-toskeletal cues to establish the belt-like arrange-ments of cell–cell junctions in epithelial celltypes.

Tentative Model for theZO-1/2–based Establishment

of the Belt-like Arrangement of AJsand TJs with the ParacellularPermselective Barrier Function

Based on the findings described above, wehave developed a model for the synergetic for-mation of the belt-like arrangement of AJsand TJs, as shown schematically in Figure 3.In this model, the process unfolds as follows:(1) E-cadherin–based cell–cell adhesion ini-tially occurs as described previously,41,42 andthen punctate AJs form complexes with as-sociated components, including catenins andactin. (2) Punctate AJs develop into prezonula-AJs, which are partially linearized and asso-ciated with E-cadherin and actin, but not withmyosin-2 integrated. In the absence of ZO-1/2,prezonula-AJs do not develop into the com-pletely belt-like AJs (zonula adherens). (3) InZO1(wt)/2(wt)/3(−) Eph4 cells in which ZO-1/2 are expressed, the formation of prezonula-AJs and the integration of myosin-2 with themto establish a belt-like arrangement of AJs arelikely to proceed simultaneously in such a waythat punctate AJs appear to be directly orga-nized into a belt-like arrangement. (4) The belt-like TJs are synergistically formed just abovethe belt-like AJs, thus establishing epithelialcell sheets with a TJ paracellular permselec-tive barrier function.3,4,43 As shown here, stud-ies on ZO1(ko)/2(kd)/3(−)Eph4 cells have ledus to a better understanding of the ZO-1/2–dependent formation of belt-like AJs and TJs.Further studies on ZO1(ko)/2(kd)/3(−)Eph4cells will contribute to reveal the molecularmechanism of switching of ZO-1/2 betweenAJs and TJs and of formation of cell polarity.

Summary

In conclusion, studies of ZO-1/2/3–deficient cells have revealed that ZO-1 andZO-2 are required and sufficient for the finalestablishment of the belt-like AJ (zonula ad-herens) as well as the formation of the belt-likeTJ (zonula occludens), under the condition thatprezonula AJs are arranged around the apicalside of epithelial sheet cells and that TJ integralmembrane proteins, such as claudins, are ex-pressed in cells. Thus, ZO-1 and ZO-2 are crit-ical signaling and scaffolding organizers of theparacellular barrier and barrier-based perms-electivity. These findings will help resolve animportant issue in “barriology.” The furtherdetailed studies on signaling and scaffoldingfactors and on the TJ integral membrane pro-teins are required for providing a clear pictureon the molecular architecture and function ofthe tight junction–based permselective barrier.“Barriology” is quite important for general ep-ithelial physiology and also for the permselec-tive barrier–related diseases.

Conflicts of Interest

The authors declare no conflicts of interest.

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