Composition and function of PDZ protein complexes during cell polarization - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:45 |只看该作者 |倒序浏览 |打印

作者：Michael H. Roh and Ben Margolis,,作者单位：1 Department of Biological Chemistry, 2 Howard Hughes Medical Institute, and 3 Department of Internal Medicine, University ofMichigan Medical School, Ann Arbor, Michigan 48109-0650
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      【摘要】
   Complexes consisting of PDZ proteins have been implicated in a variety ofcellular processes. In recent years, it has become increasingly clear that PDZproteins play essential roles during the establishment of spatial asymmetry invarious metazoan cell types such as epithelial cells. Epithelial cells possess asymmetry with respect to the apicobasal axis reflected by the differentialdistribution of proteins and lipids in the apical and basolateral surfaces. In Drosophila, three PDZ protein complexes have been shown to playcrucial functions during the establishment of cell-cell adhesions andepithelial cell polarity: Bazooka/Dm-Par6/DaPKC, Crumbs/Stardust/Discs Lost, and Scribble/Discs Large/Lethal Giant Larvae. In this review, we focusprimarily on our current knowledge of the localization and function of thesecomplexes in Drosophila epithelia. We also discuss recent data thatenhance our understanding of the homologous protein complexes and their rolesduring junctional assembly and polarization of mammalian epithelial cells. & G* ~1 }1 a6 y8 x3 F+ Y; ?
      【关键词】 PSD/Discs Large/zonula occludens domain polarity epithelia cell junctions5 v( \2 }% J4 C
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THE ESTABLISHMENT AND MAINTENANCE of cell polarity withinvarious organs are crucial for the proper development of both invertebrates and vertebrates. Two classic cell types for which polarization is essentialfor function are neurons and epithelial cells. The asymmetry of neurons ismanifested by the formation of axon terminals (presynaptic side) and dendrites(postsynaptic side) at opposite aspects( 68 ). Presynaptic andpostsynaptic membranes contain distinct collections of proteins, ultimately allowing for directional transmission of action potentials and nerve impulses.Epithelial cells, which will be the topic of focus throughout this review, arepolarized along the apicobasal axis. The apical surface faces a lumen orexternal environment, and the basolateral membrane contacts the substratum andinternal compartments ( 86 ).The selective targeting and retention of proteins at either the apical orbasolateral surfaces underlie the ability of epithelial monolayers totransport solutes in a vectorial manner( 11 ). Consequently, epitheliacan serve as barriers between distinct physiological compartments and controlthe exchange of molecules betweenthem.

Table 1. Structure and function of mammalian and Drosophila polarityproteins7 r3 y, i- l2 e& j- ?0 F3 U* g

The molecules involved in building epithelial cell architecture have beenintensely investigated during recent years, and a significant number ofstudies have shed light on the diverse protein families responsible fororchestrating epithelial polarization. Cell adhesion molecules mediate theestablishment of distinct cell-cell junctions that are arranged asymmetricallyalong the lateral membrane( 37 ). For instance, theclaudins, occludin, and junctional adhesion molecule (JAM) polymerize to formthe vertebrate tight junction (TJ) localized at the apical-most tip of thelateral membrane ( 41, 79 ). The adherens junction (AJ), the site of cell contact mediated by E-cadherin and nectins, lies justbasal to the TJ ( 74, 86 ). In Drosophila,the zonula adherens (ZA), the invertebrate counterpart to the AJ, represents the most apical site of cell adhesion, and the septate junction (SJ), whichcontains the transmembrane protein neurexin, exists below it( 78 ). Cell adhesion molecules,however, are not sufficient to establish cell-cell junctions; they must bestabilized through cytoplasmic adapter proteins that link the integralmembrane proteins to the actin cytocortex. At the AJ, the catenin complex ( -catenin/ -catenin) and afadin/ponsin complex serve to couple E-cadherin and nectin to the lateral cytoskeleton, respectively ( 2, 74 ). Similarly, zonulaoccludens (ZO)-1, ZO-2, and ZO-3 tether the claudins and occludin to the TJcytocortex ( 20, 23, 82 ). In Drosophila epithelia, the ZA and SJ are associated with F-actin via thearmadillo/ -catenin complex and the band 4.1-like protein coracle,respectively ( 40, 80 ). Collectively, thetargeting and retention of various proteins at distinct junctional complexesalong the lateral membrane represent an important aspect of polarityestablishment and maintenance ( 78, 86 ).  c5 p) G7 C! R7 G
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Recently, an increasing number of studies have emphasized the role ofproteins containing the PSD-95/Discs Large/ZO-1 (PDZ) domain during cellpolarization. PDZ proteins typically associate with the extreme COOH-terminalresidues of their ligands( 71 ). However, this mode ofbinding is not absolute as some PDZ domains heterodimerize with other PDZdomains ( 26, 69 ). PDZ domains are oftenfound in scaffolding proteins along with one or more PDZ and/or other proteininteraction domains. For example, the membrane-associated guanylate kinase(MAGUK) proteins consist of a signature arrangement of at least one PDZ domainplus an Src homology 3 (SH3) and a catalytically inactive guanylate kinase(GUK) domain ( 1 ). Thepreviously mentioned ZO-1, ZO-2, and ZO-3 proteins belong to this proteinfamily. In this review, we will focus on our current understanding of thevariety of PDZ proteins and PDZ protein complexes that are essential during the morphogenesis of polarized epithelia.9 t- V2 V2 E2 s  K
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THE ROLES OF PDZ PROTEIN COMPLEXES IN INVERTEBRATE CELL POLARITY
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Three polarity complexes, each consisting of one or more PDZ proteins in Drosophila, have been topics of intense study during recent years( 6, 78 ). The first complexconsists of Bazooka (Baz), Dm-Par6, and Drosophila atypical PKC(DaPKC). The transmembrane protein Crumbs (Crb), along with its associatedcytoplasmic proteins, Stardust (Sdt) and Discs Lost (Dlt), representscomponents of the second complex. The third consists of Scribble (Scrib), Discs Large (Dlg), and Lethal Giant Larvae (Lgl). Of these nine proteins, sixcontain one or more PDZ domains, reinforcing the notion that PDZ proteins canassemble protein scaffolds that serve essential functions.' o/ O% ?. ?  t" B# Y& ^, Q' X
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The Baz/Dm-Par6/DaPKC complex has been shown to be essential during Drosophila epithelial morphogenesis. In these cells, this complexlocalizes to the subapical region which resides just above the most apicalsite of cell contact, the ZA( 37 ). The position of thesubapical region corresponds to that of the TJ in vertebrate epithelia( Fig. 1 ). Nonetheless, it must be noted that homologues of claudins, occludin, or JAM have yet to beelucidated at the subapical region. Thus whether this region represents aregion of cell-cell adhesion remains unknown. It has been shown that thelocalization of Dm-Par6 and DaPKC to the subapical region is dependent on thatof Baz ( 62, 84 ). The ability of Baz todirectly interact with Dm-Par6 and DaPKC suggests that Baz is responsible forproperly targeting both proteins. Interestingly, Baz is mislocalized in theabsence of either Dm-Par6 or DaPKC, suggesting that only the heterotrimeric complex is stably associated with the subapical region( 62, 84 ). Fly embryos lackingexpression of Baz, Dm-Par6, or DaPKC exhibit a disruption of apicobasalpolarity in epithelia ( 52, 62, 84 ). These epithelia alsoexhibit gross structural defects and lose their regular monolayer arrangement.Furthermore, Baz null flies also exhibit defects in ZA formation. In wild-type embryos, the ZA forms a continuous belt at the apicolateral aspect ofepithelial cells as a result of the fusion of spot adherens junctions (sAJ).In flies missing Baz, sAJ material localizes irregularly along the lateralmembrane and fails to coalesce into a beltlike ZA during early gastrulation( 52 ). Furthermore, the properlocalization of apical polarity markers (e.g, Crb) is compromised. Theseobservations suggest that Baz along with DaPKC and Dm-Par6 are importantplayers during ZA formation and epithelial cell polarization.  M' J8 e4 Q$ X
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Fig. 1. Protein-protein interaction domains involved in stabilizing the polaritycomplexes in vertebrate and Drosophila epithelia. A :vertebrate epithelial cells possess tight junctions (TJs) and adherensjunctions (AJs) at cell-cell adhesions. TJs represent the most apical site ofcell contact. The Crumbs (Crb)/Pals1/Pals1-associated tight junction (PATJ)protein and Par3/Par6/atypical PKC (aPKC) complexes associate via directPar6-Pals1 interaction and colocalize to TJs. B : the zonula adherens(ZA) in Drosophila is the counterpart to the AJ and represents abeltlike junction that encircles the apex of fly epithelial cells. The septatejunctions (SJs) exist at the lateral membrane below ZA. The subapical region(SAR) exists to the apical border of the ZA, reminiscent of the localizationof TJs in epithelia. Strikingly, the Crumbs/Stardust/Discs Lost andBazooka/Dm-Par6/DaPKC complexes colocalize to the subapical region.Protein-protein interaction domains were identified by using the SimpleModular Architecture Research Tool (SMART) database( http://smart.embl-heidelberg.de/ ).PDZ, PSD-95/Discs Large/zonula occludens-1; aPKC, atypical PKC; DaPKC, Drosophila aPKC; GUK, catalytically inactive guanylate kinase; CRIB,CDC42/Rac interactive binding; FERM, band 4.1/ezrin/radixin/moesin.

The Baz/Dm-Par6/DaPKC complex is also important during neuroblast polarization. Neuroblasts delaminate from the neuroectodermal epithelium andthen proceed to divide asymmetrically, resulting in the generation of aneuroblast and ganglion mother cell ( 5 ). The polarized targeting ofproteins to the apical and basal cortex is a prerequisite for asymmetricalneuroblast division. The Baz/Dm-Par6/DaPKC complex is localized to the apicalpole and is excluded from the basal cortex( 5 ). As in the case ofepithelia, the localization of one member of the complex relies on the correcttargeting of the other two proteins. Ectopically expressing one of theseproteins at the basal cortex causes mistargeting of the other components( 62, 84 ). Furthermore, a deficiencyin any one of these three proteins leads to abnormal mitotic spindleorientation during cell division. Asymmetrical neuroblast division isreminiscent of asymmetrical cell division in the Caenorhabditiselegans zygote, where the Baz/Dm-Par6/DaPKC complex is conserved( 16 ). The homologous Par3/Par6/PKC3 complex is essential in orchestrating asymmetrical celldivision in the worm zygote( 60 ). Finally, Baz and DaPKC have been shown to be crucial during the polarization and differentiation of Drosophila oocytes( 13, 18 ). Thus Baz/Dm-Par6/DaPKCfunctions in establishing asymmetry in a variety of cellular contexts.4 s+ c1 k/ y" A$ C; e2 S

In epithelia, the Crb/Sdt/Dlt complex colocalizes with Baz/Dm-Par6/DaPKC atthe subapical region ( Fig. 1 )and is also important for the establishment of epithelial cell polarity( 37, 49, 77 ). Sdt likely represents anadapter protein that mediates the indirect interaction between Crb and Dlt( 66 ). In the absence of Sdt,Crb and Dlt are mislocalized, suggesting that the intact complex is stablyretained at the subapical region( 3, 27 ). Overexpression of Crbleads to apical surface expansion, ZA disruption, and multilayering ofepithelia; however, overall apicobasal polarity seems to be preserved( 36, 83 ). Thus Crb is an importantapical surface determinant( 31, 37, 77 ), and this correlates withits ability to organize the apical spectrin/actin cytocortex through D -moesin, a member of the band 4.1 superfamily of actin-associatedproteins ( 50, 61 ). Drosophila embryos lacking expression of Crb, Sdt, or Dlt exhibit apicobasal polaritydefects ( 4, 38 ). Furthermore, in the absence of Crb or Sdt, a continuous ZA also does not form from sAJs, similarto the case in baz null embryos( 24, 52 ). Interestingly, comparedwith Baz null flies, ZA defects in crb or sdt null mutantsare observed later during gastrulation( 9, 52 ). This is consistent withthe observation that Crb is required to maintain Baz at the subapical regionbut is dispensable for the initial localization of Baz in early gastrulae( 9 ). Furthermore, the early ZAdefects seen in baz sdt double-mutant embryos resemble those observedin baz null flies, suggesting that the Baz complex functions upstreamof the Crb complex ( 52 ). However, in all of these mutants, the overall architecture of ectodermallyderived epithelia is disrupted by the end of gastrulation. Collectively, theseresults suggest that the Baz/Dm-Par6/DaPKC and Crb/Sdt/Dlt complexes functioncooperatively during ZA formation. It should be noted that whereas the formerorchestrates polarization in various cellular contexts, the function of the latter could be restricted to epithelial cells. This is evident in sdt mutant embryos, where the polarity and asymmetrical cell divisionof neuroblasts remain unaffected( 27 ).

The third PDZ protein polarity complex consists of Scrib, Dlg, and Lgl. Ithas been shown that all three proteins colocalize in epithelia and that theyfunction in a common pathway( 7 ). In the absence of Lgl orDlg, Scrib is mislocalized. In dlg or scrib mutantepithelia, Lgl is mistargeted as well, suggesting that Scrib, Dlg, and Lglcould exist in a complex. Nonetheless, there is a deficiency in biochemicaldata to confirm this notion. Loss of Scrib, Dlg, or Lgl leads to apicalsurface expansion and mislocalization of ZA proteins to more lateral positions along the basolateral membrane during late gastrulation, which is reminiscentof the Crb overexpression phenotype( 7, 8 ). The opposite but commontemporal characteristics of the Crb/Sdt and Scrib/Dlg/Lgl mutant phenotypessuggest that the functions of these complexes are delicately balanced,allowing for the proper positioning of the ZA and determination of apical and basolateral membranes ( 9, 76 ). The functional importanceof Scrib and Dlg is supported by studies performed in C. elegans. Inworms, Dlg and Let-413, the Scrib homologue, are also essential for epithelialmorphogenesis and formation of C. elegans apical junctions( 10, 39, 43, 48 ). However, adhesion andpolarity defects seem to be more severe in Let-413-deficient embryos thanDlg-deficient embryos( 48 ).& V: X1 b$ w8 [- F1 |

The majority of the Drosophila genetic studies on cell polarity have focused on the activities of individual members of a single PDZ polaritycomplex at a time. However, recent work from the Bilder( 9 ) and Tepass( 76 ) laboratories hasaddressed the important issue of how the functions of these individualcomplexes are coordinated to yield polarized epithelia by analyzing double mutant embryos, respectively. For instance, these studies have confirmed theantagonistic relationship between the Crb and Scrib complexes. Specifically, dlg or scrib mutations exaggerate the Crb overexpressionphenotype and suppress the crb null phenotype( 76 ). Interestingly, in theabsence of the activities of both the Crb and Scrib complexes, ZA and polaritydefects are rescued to an extent. This suggests that Crb is not absolutely required for epithelial polarity( 51 ). It has been speculated that the Baz complex could compensate for the lack of the Crb complex in crb scrib and sdt dlg double mutant embryos( 76 ).

Analysis of double mutants also facilitates the elucidation of epistaticrelationships between the three polarity complexes. These studies support thenotion that the Baz/Dm-Par6/DaPKC complex is epistatic to both the Crb andScrib complexes. This is consistent with the fact that Baz seems to functionearlier in development ( 9, 76 ). Furthermore,Scrib/Dlg/Lgl is epistatic to the Crb complex, in agreement with theobservation that Scrib localization is unaffected in the absence of Crb. Given these epistatic relationships, the nature of polarity defects exhibited by themutants, and the temporal differences in phenotypic onsets, a hierarchicalmodel has been proposed ( 9 ).The initial event of epithelial polarization involves the assembly of sAJ material and establishment of apical membrane identity by Baz/Dm-Par6/DaPKC. This complex subsequently targets Crb/Sdt/Dlt to the apical surface.Meanwhile, Scrib/Dlg/Lgl is localized to the lateral membrane and counteractsthe apicalizing effects of the Crb complex at the basolateral membrane domain.Because Crb is required for the maintenance of Baz localization, it seems likely that Crb/Sdt/Dlt and Baz/Dm-Par6/DaPKC function cooperatively inestablishing apical surface identity. As the activities of these complexescoordinate with that of the laterally localized Scrib complex, sAJs fuse atthe apical aspect of cell contacts to form a continuous, beltlike ZA,ultimately leading to the proper establishment and maintenance of apicobasalpolarity.4 C$ o6 r7 M: Q5 `
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THE ROLES OF EVOLUTIONARILY CONSERVED B AZ, C RB,AND S CRIB COMPLEXES IN VERTEBRATE EPITHELIA2 B, ^/ \. l" s: K/ [5 X# n$ g
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The Baz/Dm-Par6/DaPKC, Crb/Sdt/Dlt, and Scrib/Dlg/Lgl polarity complexesare conserved in vertebrate epithelial cells: Par3/Par6/aPKC, Crb/Pals1/Pals1-associated tight junction protein (PATJ), and Scrib(Vartul)/mammalian Dlg (mDlg)/mammalian Lgl (mLgl), respectively. Thissuggests that these homologues also serve important polarity functions invertebrates ( 49, 78 ). The majority of studies that examined the localization, protein-protein interactions, and functions ofthese complexes have been performed using mammalian epithelial cell lines.Studies utilizing zebrafish mutants have also clarified the importance of someof these polarity proteins during the development of epithelium-rich tissuesduring embryogenesis. In contrast to Drosophila epithelia, the TJrepresents the most apical site of cell contact in mammalian epithelial cells;it is at the TJ where the Par3/Par6/aPKC and Crb/Pals1/PATJ complexes reside( Fig. 1 ). On the other hand,mDlg and mLgl have been demonstrated to localize along the lateral membranebelow the TJ ( 42, 53 ). Scrib/Vartul also localizes to the lateral membrane; however, the precise localization withrespect to the TJ and AJ has yet to be determined( 55 ).5 @* K' t$ Z4 k- Y  @  [! r
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Par3/Par6/aPKC has been shown by several groups to be important during TJformation and epithelial polarization. Biochemical data suggest that aPKCserves as an adapter protein as Par3 and Par6 bind to the kinase domain andNH 2 terminus of aPKC, respectively( 32, 33, 45, 73 ). Par6 contains a singlePDZ domain, whereas Par3 bears three PDZ domains, the first of which binds tothe extreme COOH terminus of JAM( 17, 30 ). The localization of Par3,Par6, and aPKC to TJs is interdependent( 59 ). At least in mammalianepithelia, members of the small GTPase family associate with this complex viaPar6 ( 33, 45 ). Specifically, activatedCDC42 and Rac directly interact with the CDC42/Rac interactive binding domainof Par6, which lies just NH 2 terminal to the PDZ domain.Overexpression of Par3, Par6, aPKC, and CDC42 mutants negatively regulates theinitial formation of TJs but does not affect TJ maintenance( 22, 33, 54, 67, 73, 85 ). For instance,overexpressing the Par6 N protein, which lacks the ability to bind aPKC,delays TJ formation. Conversely, overexpression of a kinase null aPKC mutantnegatively regulates TJ biogenesis and causes basolateral markers tomislocalize to the apical surface. Furthermore, expression of a truncated Par3(1-371), which is able to bind JAM but not aPKC or Par6, delays TJ formation.This suggests that aPKC is an important player during assembly of TJ strandsand apicobasal polarization ( 59 ). The importance of aPKCactivity has also been demonstrated by analyzing zebrafish that carrymutations in heart and soul (has), the gene encoding an aPKC( 28 ). Clearly, the regulated phosphorylation of proteins by aPKC plays significant roles in these processesas protein phosphatase 2A has been recently shown to regulate aPKC activityand also TJ assembly ( 56 ). Currently, Par3 is the only known substrate for aPKC at the TJ. Interestingly,aPKC phosphorylates Ser827 of Par3, which lies within the aPKC binding site,and this modification destabilizes the Par3/aPKC association( 54 ). The dynamic nature ofthis interaction plays a role in TJ formation as the Par3S827A mutant, but notwild-type Par3, acts as a dominant negative protein that disrupts TJ assembly.The identification of other downstream targets of aPKC should shed more lighton the mechanisms by which Par3/Par6/aPKC orchestrates TJ biogenesis andepithelial cell polarization. It should also be noted that, as ininvertebrates, the function of this complex is not confined to epithelia. Par3/Par6/aPKC is involved in establishing polarity in astrocytes andhippocampal neurons ( 19, 70 ).
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In Drosophila epithelia, Baz/Dm-Par6/DaPKC and Crb/Sdt/Dlt colocalize to the subapical region. Similarly, the Par3/Par6/aPKC andCrb/Pals1/PATJ complexes target to TJs in mammalian epithelial cells( Fig. 1 ). It should be notedthat whereas only one Crb isoform is expressed in Drosophila, threeCrb isoforms (CRB1, CRB2, and CRB3) are predicted to exist based on therecently sequenced human genome( 44, 78 ). CRB1 is primarilyexpressed in the brain and retina and, when exogenously expressed in Madin-Darby canine kidney (MDCK) cells, localizes to TJs( 14, 15, 66 ). However, attempts todetect endogenous CRB1 protein in epithelia have not been successful,suggesting that CRB1 is not the predominant Crb protein in epithelia. Incontrast, CRB3 protein is found predominantly in epithelium-rich tissues andin the MDCK epithelial cell line( 46 ). Unlike CRB1, however, CRB3 targets not only to TJs but also to the apical surface. The expressionpattern of CRB2 remains unknown. While CRB1 and CRB2 contain numerousextracellular EGF-like repeats and laminin A G-like domains, CRB3 bears ashort extracellular region that is N-glycosylated( 46 ). Nevertheless, all Crb proteins contain a conserved transmembrane segment and an intracellular 37-amino acid tail ( 49 ).$ x7 F2 ~9 T' y/ f4 ~  `% X. h
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There are two recognized domains contained within the cytoplasmic tail ofCrb isoforms: a juxtamembrane consensus motif predicted to bind proteins ofthe band 4.1/ezrin/radixin/moesin (FERM) superfamily and a PDZ binding motifat the extreme COOH terminus ( 31, 49 ). Drosophila Crbbinds to D -moesin via the FERM binding motif and the PDZ domain ofSdt via the COOH-terminal ERLI sequence( 3, 27, 50 ). Similarly, we have shownthat the ERLI motifs of CRB1 and CRB3 are capable of binding to Pals1, the PDZdomain of the mammalian Sdt homologue( 46, 66 ). Pals1 consists ofmultiple protein-protein interaction domains besides its PDZ domain( 34, 66 ). TheNH 2 -terminal unknown 1 and two L27 domains precede the PDZ domain,whereas the SH3, 4.1B, and GUK domains follow it. The NH 2 -terminalL27 (L27N) domain of Pals1 interacts with the NH 2 -terminal MAGUKrecruitment (MRE) domain of PATJ ( Fig.1 ). We have also observed that Pals1 and multiple PDZ domainprotein 1 (MUPP1), the PATJ paralogue, also associate in a similar manner( 66 ). The L27 and MRE domains are also conserved in Sdt and Dlt, respectively. We were able to demonstratethat the L27-MRE mode of interaction could mediate the direct associationbetween Sdt and Dlt ( 66 ).Interestingly, on closer examination of the sequences and predicted secondary structures of the MRE domains, it has become clear that MRE domains belong tothe L27 family of protein interaction domains ( 12 ). In fact, according tothe Simple Modular Architecture Research Tool database( http://smart.embl-heidelberg.de/ ), MRE domains are considered to be L27 domains.8 p5 _. Y. z$ i% T  r& r; V
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The Pals1/PATJ interaction is required for localizing Pals1 to TJs as Pals1missing its L27N domain mistargets apically ( 66 ). On the other hand, thisinteraction is not sufficient for the targeting of PATJ itself( 65 ). These data areconsistent with our observations that an NH 2 -terminal fragment ofPATJ (residues 1-238) displays a diffuse localization pattern, and endogenousPals1 is mislocalized in these cells( 66 ). Furthermore, the extremeCOOH termini of ZO-3 and claudin 1 associate with the sixth and eighth PDZdomains of PATJ, respectively. However, this ZO-3-PATJ interaction is crucialfor targeting PATJ to TJs, suggesting that ZO-3 could serve to tether theCRB3/Pals1/PATJ complex to TJs( 65 ). Coincidentally, MUPP1, aPATJ paralogue that can also bind Pals1, has also been shown to localize to TJs ( 25, 63 ). The ninth and tenth PDZdomains of MUPP1 bind to the COOH termini of JAM and claudin 1, respectively( 25 ). Thus it is conceivablethat a CRB3/Pals1/MUPP1 complex could exist at TJs; however, this has yet tobe directly examined to date.+ \: m, P9 d: B5 t& j0 s
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The role of the Crb complex in Drosophila epithelial polarization has been intensively studied during the past decade. The similarities oflocalization patterns between the fly Crb/Sdt/Dlt and mammalian CRB3/Pals1/PATJ complexes suggest that they serve similar functions. However,what exactly are the functions of the recently identified CRB3/Pals1/PATJcomplex in vertebrate epithelia? Studies using zebrafish demonstrate theimportance of Pals1-containing complexes during epithelial polarization.Recently, the gene product of nagie oko (nok) has been identified asa Pals1 homologue that plays an essential role during the morphogenesis of two polarized cell types in the retina, neuroepithelial and photoreceptor cells( 81 ). Polarization andjunctional assembly can also be conveniently monitored in mammalian cell linesusing the calcium-switch method( 64 ). When grown inlow-calcium media, MDCK cells do not form adhesive contacts. However, whencalcium is replenished in the culture medium, AJs start to assemble and TJsbecome visible soon thereafter. We have observed that MDCK cells expressingPATJ (1-238) exhibit delayed TJ biogenesis although continuous TJs eventuallyform. This is likely attributable to the mislocalization of Pals1 and membersof the Par3/Par6/aPKC complex in these cells( 29 ). The importance of Pals1in TJ assembly has also been investigated with respect to CRB3. Specifically, overexpression of CRB3 at the plasma membrane negatively regulates TJformation in a manner dependent on the Pals1 PDZ-binding ERLI motif (Roh andMargolis, unpublished observations). Furthermore, disruption of endogenousPals1-CRB3 interaction also leads to similar effects on TJ assembly (Fan S andMargolis B, unpublished observations). Thus our data seem to suggest that thiscomplex, similar to Par3/Par6/aPKC, not only resides at TJs but also regulatesTJ assembly at the apicolateral membrane. This is reinforced by our recentfinding that the CRB3/Pals1/PATJ and Par3/Par6/aPKC complexes physicallyinteract via the direct association of Par6 with Pals1( 29 ).8 d# W5 W7 r5 i8 U! B& C0 |: j6 V
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MDCK monolayers composed of cells expressing PATJ (1-238) or overexpressingCRB3 exhibit delayed TJ formation. Furthermore, in CRB3-overexpressing MDCKcells subjected to the calcium-switch protocol, the localization of CRB3 andtwo apical markers, ezrin and gp135, is seen to extend into the lateral plasma membrane domain during early polarization (Roh and Margolis, unpublishedobservations). Nonetheless, there were no significant disruptions in theoverall apicobasal polarity axis in these monolayers. Monolayers of cellsexpressing PATJ (1-238) also exhibit normal polarity as CRB3 and E-cadherinare localized to the apical and lateral membranes, respectively( 29 ). This may be attributedto the presence of a free surface and cell contacts representing cuessufficient to establish polarity in monolayers.' q* F3 G3 F3 j8 I4 R0 j

An alternative method used to examine MDCK cell polarization involvesculturing cells in collagen matrix. Under these conditions, the entire cellsurface is in contact with an extracellular matrix. However, as successiverounds of cell division ensue, polarity is established in a stepwise fashionthat results in the de novo formation of a continuous apical membrane( 58 ). Specifically, parentalMDCK cells develop into three-dimensional cysts, in which cells surround acentral lumen surrounded by the apical surface( Fig. 2 ). Meanwhile, thebasolateral membrane is engaged in cell-cell and cell-matrix contacts. MDCKcells overexpressing PATJ (residues 1-238) do not develop into cysts butinstead form multicellular aggregates where apical markers exhibit an abnormaldistribution ( Fig. 2 ).Similarly, cysts arising from CRB3 overexpressing cells also exhibit abnormal polarity; this phenotype requires the presence of the Pals1 PDZ binding motif(M. Roh. and B. Margolis, unpublished observations). Therefore, the mammalianCrb complex represents an evolutionarily conserved complex that plays anessential role during epithelial polarization.1 M' C) J6 ]1 d- `8 ^# E
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Fig. 2. Expression of a PATJ (residues 1-238) dominant negative protein disruptsthe apicobasal polarity of 3-dimensional Madin-Darby canine kidney (MDCK)cysts cultured in collagen gels. Parental MDCK cells ( left ) and MDCKcells expressing the Myc-PATJ (1-238) protein ( right ) were grown incollagen gels and immunostained with the indicated antibodies as describedpreviously ( 57 ). E-cadherinand gp135 are markers of the lateral and apical surfaces, respectively.9 ^0 b' K$ m5 {  G
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Finally, homologues of Scrib, Dlg, and Lgl are also expressed in mammalianepithelia: Scrib/Vartul, mDlg (also referred to as SAP97), and mLgl,respectively. Recently, it was shown that mDlg/SAP97 directly associates withmLin-2/CASK, Dlg2, and Dlg3 and that these interactions influence thetargeting of mDlg/SAP97 to the lateral membrane( 35, 42 ). Furthermore, mLgllocalizes in a similar manner( 53 ). Interestingly, mLgl iscapable of being serine phosphorylated, and this seems to be essential forpreventing its localization to the apical surface( 53 ). Consistent with this,mLgl coimmunoprecipitates with syntaxin-4, a N -ethylmaleimide-sensitive factor attachment receptor proteininvolved in trafficking of vesicles to the basolateral surface. However, it isnot currently known whether mLgl interacts directly with either Scrib/Vartulor SAP97. Regardless, the evolutionarily conservation of these proteins inmammalian epithelia suggest that these proteins function cooperatively duringthe establishment of basolateral membrane identity during apicobasalpolarization.# `8 n0 n8 e4 V! R" o0 G

PERSPECTIVES4 ]. \  p" v* V

Here, we have discussed the genetic studies highlighting the functionalimportance of three complexes, each containing PDZ proteins during thepolarization of invertebrate cells: Baz/Dm-Par6/DaPKC, Crb/Sdt/Dlt, andScrib/Dlg/Lgl. Homologues of all nine proteins have been identified invertebrate epithelial cells. Studies using the mammalian MDCK cell line as amodel system have facilitated the biochemical characterization of theprotein-protein interactions that stabilize the Par3/Par6/aPKC andCRB3/Pals1/PATJ complexes. Furthermore, recent work from our laboratory hasdemonstrated that these two protein complexes are conserved in regard to theirsubcellular localization and that, at least in mammalian epithelia, they canexist as a single complex( 29 ). Scrib, Dlg, and Lgl aswell as their homologues colocalize to the lateral membrane in invertebrateand vertebrate epithelial cells, suggesting that these respective groups ofproteins may form ternary complexes.
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Studies using Drosophila as a model organism have shed light onthe coordinated function of these three polarity complexes during invertebrateepithelial polarization ( 9, 76 ). In parallel, experimentsusing MDCK cells have provided insight into the functions of these complexesduring the establishment of polarity in mammalian epithelia. The majority ofthese reports have focused on the role of Par3/Par6/aPKC during TJ assembly at the apical pole of the lateral surface. We have also demonstrated that theCRB3/Pals1/PATJ complex also regulates TJ assembly. This is highly reminiscentof the situation in Drosophila, where Baz/Dm-Par6/DaPKC andCrb/Sdt/Dlt are essential for the biogenesis of the ZA at the apex of cellcontacts.
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Models describing junction formation and apicobasal polarization in flyepithelia have been recently reported( 9, 76 ). Based on those and ourcurrent knowledge of the three conserved PDZ protein polarity complexes inmammals, we now propose a model regarding TJ assembly and mammalian epithelialpolarization ( Fig. 3 ).Establishment of polarity commences when two epithelial cells form initialcell contacts via E-cadherin and nectin ( 74 ). Nectin is involved inthe recruitment of JAM to these early adhesive contacts( 21 ). In turn, thePar3/Par6/aPKC can interact with JAM and be subsequently recruited to these early AJs ( 17, 30, 47, 72, 75 ). We have observed thatPals1 and PATJ are recruited to these premature AJs (Roh M, Fan S, andMargolis B, unpublished observations), consistent with our recent finding thatPals1 can bind directly to Par6. Therefore, the initial phase of polarizationlikely involves the recruitment of a variety of polarity proteins to initialsites of epithelial cell contacts. Intriguingly, CRB3 is predominantlyintracellular during the formation of premature AJs and starts to appear later at the apical surface as Par3/Par6/aPKC and Pals1/PATJ are being recruited toearly cell contacts in MDCK cells (Roh M, Fan S, and Margolis B, unpublishedobservations). This is consistent with Drosophila studiesdemonstrating that the Baz complex recruits Crb to the apical membrane.

Fig. 3. Model depicting the role of PDZ polarity complexes during junctionalassembly and apicobasal polarization of mammalian epithelial cells. Theestablishment of mammalian epithelial polarity commences when initialnectin/E-cadherin-based cell contacts (red) are formed between cells.Subsequently, JAM (green) is recruited to these sites of cell adhesion. JAMrecruits Par3 and its associated proteins, Par-6 and aPKC; in turn, thiscomplex recruits Pals1 and its binding partners as TJs start to becomeestablished. Meanwhile, a Crb isoform (CRB3) is being trafficked to the apicalplasma membrane. CRB3 could serve as an apical cue allowing for the completepolymerization of tight junctions at a plane subjacent to the apical surface.In fully polarized epithelial monolayers, TJs exist at the apical tip of thelateral membrane and contribute to the maintenance of apicobasal polarity bypreventing the intermixing of apical and basolateral membraneconstituents., d3 V0 R9 ^: I8 @' a4 T
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As premature AJs become beltlike AJs, TJ proteins polymerize to form acontinuous TJ at the apicolateral plane of the monolayer. This suggests thatsome molecular cue exists to ensure that TJs lie subjacent to the apicalmembrane. CRB3 could serve as a possible candidate because it binds directlyto Pals1, which, in turn, associates with Par3/Par6/aPKC. The resulting supramolecular complex could then function to drive assembly of TJ strands ina single plane under the apical membrane. In support of this hypothesis, Drosophila geneticists have observed that the Baz and Crb complexesare important for the formation of a continuous ZA at the apicolateral aspectof epithelial cell contacts. Furthermore, we have observed that disrupting theendogenous CRB3/Pals1 association significantly delays the formation ofcontinuous TJs (Fan S and Margolis B, unpublished observations). Thus it istempting to speculate about the role of CRB3 as a guide for the polymerizationof TJ strands to form TJs at the apicolateral plane of the epithelial monolayer. Finally, the formation and stabilization of TJs are essential forthe maintenance of apicobasal polarity by preventing the intermixing of apicaland basolateral membrane proteins and lipids.

In summary, significant progress has been made during the past severalyears in identifying the molecules essential for establishment of cellularasymmetry. However, this increased knowledge has spurred more questions,especially those centering on the actual mechanisms by which these proteinsorchestrate cell polarization. Epithelial cells represent one system where theintricate mechanisms of PDZ protein function still remain largely elusive.Future studies will hopefully provide more insight into numerous issues, suchas the signaling events that occur during the various phases of polarization;the way in which signaling influences the dynamic nature of protein-proteininteractions within these polarity complexes; the identities of additionalmolecules that associate with these polarity complexes; and the exact roles ofmammalian homologues of Scrib, Dlg, and Lgl and their role during junctionalassembly and establishment of epithelial polarity. Answering some of thesequestions will make possible a more refined understanding of cellpolarization.5 p* {( S1 e) F! [/ j* Y
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DISCLOSURES

This work was partially supported by National Institute of Diabetes andDigestive and Kidney Diseases Grant DK-58208. M. Roh was supported by MedicalScientist Training Program Grant T32 GM-07863 and Genetics PredoctoralTraining Program Grant T32 GM-07544024 to the University of Michigan. B.Margolis is an investigator of the Howard Hughes Medical Institute.3 ?* q& ^) Q. o

ACKNOWLEDGMENTS

We thank members of the Margolis laboratory for helpful discussions on thiswork. We apologize to those whose work is not described here due to spacelimitations.% X4 k2 ^4 W9 U, v' h! c8 z
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Address for reprint requests and other correspondence: B. Margolis, HowardHughes Medical Institute, Univ. of Michigan Medical Ctr, 4570 MSRB II, Box0650, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0650 (E-mail: bmargoli{at}umich.edu
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