assembly of c. elegans apical junctions - the hardin labworms.zoology.wisc.edu/pdfs/mcmahon.pdf ·...

INTRODUCTION

During the development of an organism, epithelial tissuesprovide the basis for major morphogenetic changes. Epithelialcells possess particular characteristics that are essential fortheir function, such as specialised cell junctions and a distinctapico-basal polarity. Two major types of subapical junctionscan be distinguished by electron microscopy in epithelial cells(for recent reviews, see Muller, 2000; Tsukita et al., 1999;Yeaman et al., 1999). Tight junctions (TJs) in vertebratesand septate junctions (SJs) in Drosophila are involved incontrolling paracellular solute transport, otherwise known asthe gate function. In addition, TJs maintain apico-basalpolarity. Adherens junctions (AJs) in vertebrates andinvertebrates are essential for cell adhesion. These junctionshave been studied extensively in cell culture and Drosophila,but it is still not fully understood how they are assembled orhow the junctional components contribute to their function.

Cell junctions are specialised domains of the plasmamembrane where transmembrane (TM) proteins are clusteredand linked to the cytoskeleton via cytoplasmic proteins,thereby ensuring tight cell contacts and efficient cell adhesion.Aggregation of proteins at cell junctions is also thought tofacilitate intracellular signalling. Occludin and claudins areTM proteins of TJs (Tsukita et al., 1999), whereas neurexin IV

(NrxIV) is the only known TM protein in Drosophila SJs(Baumgartner et al., 1996). Proteins belonging to themembrane-associated guanylate kinase-like (MAGUK) familyappear to act as central players at the interface betweenjunctional TM proteins and signalling pathway components orthe cytoskeleton (Dimitratos et al., 1999). These proteinstypically have 1 or 3 PDZ (PSD-95/Dlg/ZO-1) domains, anSH3 (src homology 3) domain, and a GUK (guanylate kinase-like) domain, all of which are involved in protein-proteininteractions (Deguchi et al., 1998; Pawson and Scott, 1997;Ponting et al., 1997). Previously characterised MAGUKsinclude Drosophila Lethal, Discs Large (Dlg) and its humanhomologue hDlg/SAP97, as well as vertebrate ZonulaOccludens-1 (ZO-1) and the Drosophila homologue Tamou(Lue et al., 1994; Takahisa et al., 1996; Willott et al., 1993;Woods and Bryant, 1991). Their function has been investigatedextensively in the nervous system where they have beenimplicated in the clustering of TM receptors and ion channelsat synapses, and in facilitating signal transduction (Garner etal., 2000). Genetic studies in Drosophila reveal that, inepithelia, the SJ-associated protein Dlg is required for SJ andAJ formation and the maintenance of epithelial polarity, whilemutations inactivating other SJ components, such as NrxIV andCoracle (Cor), only affect the gate function (Baumgartner etal., 1996; Lamb et al., 1998; Woods et al., 1996). There are

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Specialised subapical junctions play a critical role inmaintaining epithelial cell polarity and tissue integrity, andprovide a platform for intracellular signalling. Here weanalyse the roles of C. elegans genes let-413 and dlg-1, ahomologue of Drosophila lethal discs large, in the assemblyof the C. elegansapical junction (CeAJ), and provide thefirst characterisation of this structure. We have identifieddlg-1 as an essential gene in an RNA interference screenagainst C. eleganshomologues of genes encoding proteinsinvolved in tight or septate junction formation. We showthat DLG-1 colocalises with the junctional protein JAM-1at CeAJs in a unit distinct from HMP-1/α-catenin, andapical to the laterally localised LET-413. Loss of dlg-1activity leads to JAM-1 mislocalisation and thedisappearance of the electron-dense component of theCeAJs, but only mild adhesion and polarity defects. In

contrast, loss of let-413 activity leads to the formation ofbasally extended discontinuous CeAJs and strong adhesionand polarity defects. Interestingly, in LET-413-deficientembryos, CeAJ markers are localised along the lateralmembrane in a manner resembling that observed in wild-type embryos at the onset of epithelial differentiation. Weconclude that the primary function of LET-413 is tocorrectly position CeAJ components at a discrete subapicalposition. Furthermore, we propose that DLG-1 is requiredto aggregate JAM-1 and other proteins forming theelectron-dense CeAJ structure. Our data suggest thatepithelial adhesion is maintained by several redundantsystems in C. elegans.

Key words: C. elegans, epithelial cell polarity, adherens junction,MAGUK protein, α-catenin, cell adhesion, LET-413

SUMMARY

Assembly of C. elegans apical junctions involvespositioning and compaction by LET-413 and proteinaggregation by the MAGUK protein DLG-1Laura McMahon, Renaud Legouis, Jean-Luc Vonesch and Michel Labouesse*Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP163, 1, rue Laurent Fries, 67404 Illkirch, France*Author for correspondence (e-mail: [email protected])

Accepted 20 March 2001 Journal of Cell Science 114, 2265-2277 © The Company of Biologists Ltd

RESEARCH ARTICLE

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other functionally significant TJ/SJ-associated proteins, someof which are conserved between vertebrates and Drosophila.These include the PDZ-containing vertebrate AF-6/afadin orits fly homologue Canoe, the afadin-binding protein ponsin,cortactin/Dcortactin, band 4.1 protein/Coracle and symplekin(Asakura et al., 1999; Ikeda et al., 1999; Katsube et al., 1998;Keon et al., 1996; Lamb et al., 1998; Mandai et al., 1999;Matsuo et al., 1999; Zhadanov et al., 1999).

Within AJs, the TM protein cadherin emerges as the mainadhesion molecule. Cadherins form homophilic bonds throughtheir extracellular domains and interact with the actincytoskeleton via cytoplasmic α- and β-catenins. Geneticstudies in mouse and Drosophila have shown that AJs areessential for the maintenance of cell adhesion and epithelialpolarity (for reviews, see Aberle et al., 1996; Knust and Leptin,1996; Tepass, 1999; Yap et al., 1997). In C. elegans, thecadherin/α-catenin/β-catenin complex encoded by the geneshmr-1, hmp-1 and hmp-2 respectively, is essential foranchoring the actin cytoskeleton to the plasma membrane(Costa et al., 1998). However, it does not appear to support theadhesion and polarity functions that are attributed to thecadherin/catenin complex in other organisms.

Time-lapse imaging and genetic analyses suggest that theassembly of epithelial junctions is a complex and sequentialprocess. In cultured MDCK cells, it has been shown that cell-cell contacts initiate the segregation of proteins into differentdomains of the membrane via Ca2+-dependent cadherin celladhesion (Adams et al., 1998). In Drosophila, the assembly ofAJs depends on the activity of two sets of proteins that act oneither side of the future AJ. Basal to the AJ, Scribble (Scrib)has recently been implicated in maintaining epithelial cellintegrity and apico-basal polarity (Bilder et al., 2000; Bilderand Perrimon, 2000). These functions have also been attributedto the basolateral Scrib homologue, LET-413, in C. elegans(Legouis et al., 2000). Scrib probably functions in a commonpathway with Dlg and Lethal giant larvae (Lgl), as there arestrong genetic interactions between the genes encoding theseproteins, and as Scrib and Dlg colocalise to SJs and overlapwith the membrane domain of Lgl (Bilder et al., 2000). Apicalto the Drosophila AJ, it has been proposed that the TM proteinCrumbs and its interacting partner, the multi-PDZ-domainprotein Discs Lost, form a scaffold that allows the assembly ofproteins leading to mature AJs (Bhat et al., 1999; Klebes andKnust, 2000; Wodarz et al., 1995).

We have been using C. elegansas a model system toinvestigate the epithelial cell characteristics of apico-basalpolarity and specialised cell junctions. In contrast to otherorganisms, C. elegansepithelia possess only one type ofapical junction, which has been referred to as a beltdesmosome (Priess and Hirsh, 1986) or an AJ (Costa et al.,1998; Mohler et al., 1998; Podbilewicz and White, 1994;Raich et al., 1999) due to its ultrastructure, but which we willcall the C. elegans apical junction (CeAJ). Two sets ofproteins, the above-mentioned cadherin/catenin complex(Costa et al., 1998) and a protein called JAM-1 (Priess andHirsh, 1986; Francis and Waterston, 1991; Podbilewicz andWhite, 1994; Mohler et al., 1998), have been reported tolocalise to CeAJs, based on their subapical localisation andanalogy with other systems (for the cadherin/catenincomplex; Costa et al., 1998), or on immunogold experiments(for JAM-1; D. Hall, personal communication). LET-413 is

the only protein implicated to date in CeAJ assembly andapico-basal polarity maintenance in C. elegansepithelial cells(Legouis et al., 2000). C. eleganspossesses homologues ofproteins associated with TJ/SJs or involved in their assembly,although a structure resembling these junctions has not beendescribed. We thus decided to investigate whether thesehomologues were involved in generating functional epithelialcells using an RNA interference (RNAi) approach (Fire et al.,1998). We found that the only gene encoding an essentialepithelial protein is the C. eleganshomologue of Drosophiladlg, which we have named dlg-1. As the phenotype of DLG-1-deficient embryos resembled that of let-413 mutants, weinvestigated whether these genes act in the same pathway.Employing newly developed tools, we analysed thecomposition of CeAJs in LET-413- and DLG-1-deficientembryos compared with the wild-type situation. We show thatDLG-1 is required for CeAJ assembly, but not to maintaincell polarity in the same way as LET-413, and does notcolocalise with LET-413. We propose a model for CeAJassembly and function.

MATERIALS AND METHODS

Strains and GFP markersAnimals were maintained as described (Brenner, 1974). The wild-type reference strain is Bristol N2 (Brenner, 1974). The followingmarkers were used: jcIs1 [unc-29 (+) – rol-6 (su1006) – jam-1::gfp];the pML624 plasmid, which is a che-14::gfptranslational fusion; andthe pML801 plasmid, which is a functional let-413::gfptranslationalfusion (Legouis et al., 2000; Michaux et al., 2000; Mohler et al.,1998). To obtain LET-413-deficient embryos we performed RNAiagainst let-413, which has been shown to result in the samephenotype as strong loss of function or null let-413alleles (Legouiset al., 2000).

Molecular biologyWe carried out Blast searches to identify possible homologues ofproteins involved in TJ/SJ formation in the C. elegansgenome (TheC. eleganssequencing Consortium, 1998). Specific regions of genesencoding these homologues (Table 1) were PCR-amplified usingprimers carrying a T3 promoter sequence (ATTAACCCTCACTAAA-GG) at their 5′ ends. Double-stranded RNA (dsRNA), generated usingthe Ambion mMessage mMachine kit, was injected into the syncytialgonad arms of N2 and jam-1::gfptransgenic animals. Injected animalswere generally left for 8-12 hours to recover and then transferred tonew plates every 8-12 hours. The plates were checked 16-24 hoursafter adult removal to score for the presence of dead embryos, andsubsequently every 24 hours to monitor the presence of later visiblephenotypes. We concentrated on the predicted ORF C25F6.2(EMBLaccession number U39742), which we named dlg-1. The cDNAsyk481c12, yk481e2, yk333g2 and yk435h12, isolated by Y. Kohara,were sequenced. The longest of these, yk435h12 (EMBL accessionnumber AJ295228) starts with the last six nucleotides of SL1,suggesting that dlg-1 is SL1 trans-spliced. To obtain a dlg-1::gfptranslational fusion (GFP, green fluorescent protein; Chalfie et al.,1994) we cloned almost all the dlg-1 coding sequence and 7 kb ofpromoter sequence up to the preceding gene C25F6.3 (position 27630to 38785 in C25F6) between the BamHI and XmaI sites of the GFP-containing vector pPD95.75 (a kind gift from A. Fire). The resultingplasmid, pML902, contains 3′UTR sequence from the unc-54geneand not from dlg-1. dsRNA against the dlg-1 3′UTR was synthesisedin the same way as dsRNA against dlg-1coding sequence; the progenyof animals injected with dsRNA against the dlg-13′UTR were nameddlg-1(3′UTR RNAi)embryos.

JOURNAL OF CELL SCIENCE 114 (12)

2267Assembly of C. elegans apical junctions

Staining and microscopyTo assess the difference in DLG-1::GFP levels between uninjectedand dlg-1(RNAi) dlg-1::gfptransgenic animals, fluorescent imagesof expressed GFP were captured using a Zeiss Axiophot microscopeand the Coolsnap aquisition system (version 1.2).Immunocytochemistry was performed either as described(Labouesse et al., 1996) or using the freeze-crack, methanol/acetonefixation method (Miller and Shakes, 1995). Images were capturedon a Leica TCS3 C confocal microscope (immunostaining) or on aZeiss Axiophot microscope (Nomarski, che-14::gfp). To obtaintransverse sections in the Z-axis starting from confocal images, wecaptured 25 or 36 lateral sections spaced 0.25 µm apart, covering onaverage one third to one half of the embryonic thickness.Subsequently, these images were processed using the TCSTKprogram, an interactive tool for processing and viewing confocalmicroscopy data (J.L.V., unpublished). One of its functions is the3D-projection mode, which allowed us to view orthogonal slices atdifferent positions within the embryo. Each transverse slice alongthe apico-basal axis had a depth of 25 or 36 sections and a width of1 pixel (each confocal section represents 512×512 pixels). In orderto score the spread (extent) and the position of JAM-1 and HMP-1(as viewed with specific antibodies; Costa et al., 1998; Francis andWaterston, 1991) along the lateral membranes of epidermal cells,transverse sections resulting from Z-axis rotations were viewed inAdobe Photoshop. Anti-UNC-70 (C. elegans β-G spectrin) staining(Moorthy et al., 2000) was used to denote the basal extent of thelateral membrane for each cell. The spread of JAM-1 or HMP-1 wascalculated as a percentage of the total membrane length. To assessposition, each lateral membrane was divided into threecompartments along its height (top, middle and bottom) and thedetected proteins were scored as present or absent in each section.

Electron microscopyPreparation of embryos and electron microscopy analysis were carriedout as described (Legouis et al., 2000). All embryos were harvestedbetween the 1.5- and two-fold stages. Blocks of four embryos weresectioned and analysed for wild-type and RNAi embryos, with sectionscontaining between one and four embryos. Sections were taken eithertransversely or longitudinally. The presence or absence of electron-dense apical junctions was scored only where apposing cellmembranes were clearly visible between neighbouring epidermalcells.

RESULTS

C25F6.2, a putative homologue of Drosophila Dlg, isnecessary for epithelial cell junction integrity andmorphogenesisThere are three main classes of epithelia in the C. elegansembryo: epidermal, intestinal and pharyngeal marginal cells.The epidermis is essential for morphogenetic changes duringembryogenesis. Soon after terminal differentiation, epidermalcells extend around the developing embryo forming new cellcontacts at the ventral surface, a process known as enclosure(Sulston et al., 1983; Williams-Masson et al., 1997). This isfollowed by a fourfold elongation of the embryo, which isdriven by contraction of circumferentially aligned actinbundles in the epidermis (Priess and Hirsh, 1986; Sulston etal., 1983). The developmental stage of a wild-type embryo isdetermined by its shape, indicating the stage of elongation ithas achieved, thus, comma stage (end of enclosure), 1.5-foldstage (beginning of elongation; Fig. 1A), two-fold and three-fold stages (mid-elongation) and pretzel stage (end ofelongation; Fig. 1B).

We used RNAi (Fire et al., 1998) to screen predicted genesencoding homologues of proteins involved in TJ/SJ formationin order to investigate their functions in C. elegans (Table 1).Distribution of the subapical protein JAM-1 was used to assessepithelial integrity (Mohler et al., 1998). RNAi produced aphenotype for two out of 16 genes tested, F25G6.2 andC25F6.2, which are homologues of genes encoding symplekinand Dlg/hDlg respectively. F25G6.2(RNAi) embryos (i.e.embryos that lack F25G6.2 function as a result of RNAi)arrested prior to the 150-cell stage with rounded cells,indicating a possible cell adhesion problem, and no visibletissue differentiation (data not shown).

Due to the early embryonic arrest phenotype ofF25G6.2(RNAi) embryos, we decided to concentrate instead onC25F6.2, which we have named dlg-1. RNAi against dlg-1resulted in lethality during embryonic morphogenesis. dlg-1(RNAi) embryos arrested at the two-fold stage of elongation

Table 1. RNAi screen for C. elegans homologues of proteins involved in TJ/SJ formationVertebrate/ C. elegans Position of dsRNA RNAi Drosophilaprotein homologue E-value* LG‡ RNAi primers§ length phenotype

hDlg/SAP97; Dlg C25F6.2 5e−142 X 35540-36916 1159 >99% Emb¶ZO-1; Tamou Y105E8B.Z e−100 I 21133-21901 783 nop**Discs lost C52A11.4 1e−56 II 22918-23993 917 nopMAGUK (ZO-1; Dlg) F44D12.1 5e−32 IV 6051-7079 891 nop

K01A6.1 8e−83 IV 20335-21339 955 nopK01A6.2 1e−16 IV 9443-9847 359 nopC01B7.4 2e−58 V 12177-13083 707 nop

Band 4.1/ Coracle‡‡ ZK270.2 6e−81 I 1573-2647 1074 nopC48D5.2A 4e−63 III 34001-34997 499 nopT04C9.6 5e−66 III 5434-6937 648 nopH05G16.1 7e−68 X 6459-7326 867 nop

Afadin W03F11.6 9e−09 I 33289-22804 514 nopC43E11.6 8e−13 I 5034-5842 638 nop

Ponsin Y45F10D.13 2e−22 IV 9102-9892 545 nopSymplekin F25G6.2 1e−102 V 30699-31604 905 >99% EmbCortactin/D-cortactin K08E3.4 4e−18 III 19531-20460 796 nop

*Expectation value, indicating the number of Blast hits one would expect to find by chance in a Blast search carried out with each C. eleganspredicted ORF;‡C. eleganslinkage group; §Refers to the position of primers on the corresponding cosmid sequence used to generate dsRNA; ¶embryonic lethality; **noobvious phenotype; ‡‡pooled dsRNAs for ZK270.2, C48D5.2A, T04C9.6 and H05G16.1 injected together gave no phenotype either.

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with vacuoles in epithelial tissues and leakage of a smallamount of cytoplasm from the tip of the tail and ventral surface(Fig. 1C,D). Immunostaining with the monoclonal antibodyMH27 (which recognises JAM-1; Francis and Waterston, 1991;Mohler et al., 1998) showed a completely discontinuouspattern in dlg-1(RNAi)embryos (Fig. 1G,H), in contrast to thebelt-like staining observed in wild-type embryos (Fig. 1E,F).This punctate MH27 pattern is strikingly similar to that of let-413mutant embryos, which display a more severe phenotype,either arresting around the 1.7-fold stage of elongation orrupturing from the ventral surface (Legouis et al., 2000). Theseresults suggest that epithelial integrity is compromised due toabnormal CeAJs in dlg-1(RNAi) embryos.

dlg-1 encodes a member of the MAGUK family ofproteinsACeDB (A C. elegans DataBase) predictions, cDNAsequencing and RT-PCR reveal that dlg-1 (Fig. 2Ai) encodesa protein of 967 amino acids with closest homology tothe Drosophila and human MAGUK proteins Dlg andhDlg/SAP97 respectively (Fig. 2Aii). Pfam database analysisindicated that DLG-1 possesses three PDZ domains (of 86, 85and 80 amino acids respectively), an SH3 domain of 62 aminoacids, and a GUK domain of 102 amino acids. In severalMAGUK proteins, the region between the SH3 and GUKdomains, known as the HOOK domain, is required for thelocalisation of Dlg to the SJs in Drosophila and interactionwith the cytoskeleton band 4.1 protein in vertebrates (Houghet al., 1997; Lue et al., 1994; Marfatia et al., 1996). This regionin DLG-1 shows only 32% similarity/43% identity with Dlgand hDlg/SAP97 isoform 2 and the configuration of lysineresidues thought to bind protein 4.1 is not conserved, but itincludes a particularly conserved FSRKFPF motif of unknownfunction (Lue et al., 1994; Marfatia et al., 1996).

DLG-1 is localised to the subapical membrane ofepithelial cellsTo determine the expression pattern and subcellularlocalisation of DLG-1, we constructed a translational fusionbetween dlg-1and a cDNA coding for green fluorescent protein(GFP), which was inserted in the penultimate exon of the dlg-1 coding sequence (Fig. 2Ai, large arrows). As the dlg-1::gfpconstruct is 106 bases shorter than the full coding sequence,and there were no available dlg-1 mutants, we determined thefunctionality of the DLG-1::GFP using an RNAi approach. Wedesigned dsRNA against the 3′UTR sequence of dlg-1 (Fig.2Ai, small arrows) and verified that the dlg-1(3′UTR RNAi)phenotype was as described above for dlg-1(RNAi). The dlg-1::gfp plasmid does not possess the dlg-13′UTR sequence andtherefore should not be affected by the 3′UTR dsRNA. Inthis way we specifically inactivated endogenous DLG-1 andexamined whether DLG-1::GFP was able to ‘rescue’ the dlg-1(3′UTR RNAi)phenotype. The results are shown in Table 2and clearly establish that the DLG-1::GFP is functional.

To assess the efficiency of dlg-1(RNAi), we injected dsRNAagainst the dlg-1coding region (Fig. 2Ai, arrowheads) into dlg-1::gfp transgenic animals and examined GFP expression in theprogeny. We found that DLG-1::GFP expression was greatlyreduced in the progeny of injected animals compared tothe progeny of uninjected controls and that a dlg-1(RNAi)phenotype was produced, which indicates that dsRNA against


Fig. 1. dlg-1(RNAi)embryos show abnormal morphogenesis andepithelial defects. (A-D) Nomarski interference microscopy of wild-type and dlg-1(RNAi) embryos. (E-H) Confocal images showingimmunostaining with the monoclonal antibody MH27, whichrecognises CeAJs in the epidermis, pharynx (arrows) and intestine(arrowheads). (C,E,G) External focal plane showing the epidermis.(A,B,D,F,H) Internal focal plane showing the pharynx and intestine.(A,E,F) 1.5-fold stage wild-type embryos (6-7 hours development).(B) Wild-type pretzel stage embryo (9-10 hours development).(C,G,H)dlg-1(RNAi)embryos at 6-7 hours development. (D)dlg-1(RNAi)embryos at 9-10 hours development. In dlg-1(RNAi)embryos, CeAJ staining is punctate and worsens with age; epidermalCeAJs are more abnormal than in the pharynx or intestine. In C andD, white arrows indicate epithelial vacuoles. Also in C a blackarrowhead indicates abnormal bulges at the embryo surface and in Da white arrowhead indicates loose cells that have leaked out of theembryo. In this and all following figures unless otherwise stated, theembryos are orientated with the anterior to the left and dorsal to thetop of the image. Scale bar,10 µm.

Table 2. ‘Rescue’ of dlg-13′UTR RNAi phenotype bytransgenic DLG-1::GFP

Percentage embryonic lethality*

3′UTR RNAi (n) Exonic RNAi (n)Wild-type 99.4 (942) 98.9 (726)dlg-1::gfp transgenic 4.8 (1290)‡ 99.0 (869)

worms

*The embryonic lethal phenotype in all cases is that described as the dlg-1(RNAi)phenotype

‡95.2% of these dlg-1::gfp transgenic worms grew normally to adulthoodand were fertile.


dlg-1specifically and significantly reduces DLG-1 levels in theembryo (Fig. 2B,C).

DLG-1::GFP was first detected at the 350-cell stage indifferentiating epithelial cells and neuroblasts (data notshown). The neuronal expression was transient and itsfunctional significance was not investigated. After ventralenclosure, DLG-1::GFP was detected in the epidermis,pharynx and intestine (Fig. 2D,E), forming a continuous beltaround epithelial cells at a subapical position, which persiststhroughout embryonic, larval and adult development (Fig.2F,H). In adults, the DLG-1::GFP protein was also detected inepithelial cells contributing to the reproductive system: thevulva, uterus and spermatheca (Fig. 2G).

Our results indicate that the observed DLG-1::GFP expressionpattern reflects that of the endogenous protein, although dlg-1may also be expressed at very low levels in other cells.

Epithelial cell polarity is affected in LET-413- but notin DLG-1-deficient embryos To determine whether the epithelial defects seen in dlg-

1(RNAi) embryos resulted from apico-basal polarityabnormalities, we examined the localisation of both basal andapical proteins. The basal marker, MH46, which recognises theTM protein myotactin (Francis and Waterston, 1991; Hreskoet al., 1999), showed a normal distribution (data not shown).To mark the apical surface of the embryonic pharynx andintestine, we used antibodies against the PDZ-containingproteins PAR-3 and PAR-6 (Fig. 3A,A′,D,D′; Leung et al.,1999). Similarly, a GFP reporter construct of the multi-TMprotein CHE-14 (Michaux et al., 2000) was used to mark theapical surface of a subset of epidermal cells (Fig. 3G). Weobserved that these proteins all remained at the apical surfacein dlg-1(RNAi) embryos (Fig. 3B,B′,E,E′,H), although PAR-3appeared to move to a position coincident with, or just apicalto, the CeAJ (marked by HMP-1) in the intestine (Fig. 3B′),and small areas of subapical localisation were also detected forCHE-14::GFP in the epidermis (arrows in Fig. 3H). In contrast,PAR-3 and PAR-6 became progressively mislocalised basallyalong the lateral membrane of intestinal cells in let-413(RNAi)embryos (Fig. 3C,F,C′,F′). Similarly, CHE-14::GFP was

Fig. 2. DLG-1, a member of theMAGUK family of proteins, is localisedat the subapical membrane of epithelialcells. (Ai) The genomic structure of thedlg-1gene is shown underneath the scalebar, which corresponds to thecoordinates in cosmid C25F6. Whiteboxes correspond to the 12 coding exonsand grey boxes at either end of the geneto the 5′ and 3′ untranslated regions(UTR), respectively. Note that the ATGis 291 nucleotides downstream of theprevious ACeDB prediction, that exon12 is 86 nucleotides longer at the 5′endthan the previous ACeDB prediction,and that the stop site is 766 nucleotidesupstream of the previous ACeDBprediction. Large black arrows show thelocalisation of primers used to generate along-range PCR product that was taggedwith a green fluorescent protein (GFP)cDNA. Arrowheads and small arrowsshow the localisation of T3-taggedprimers used to generate dsRNA, againstcoding and 3′UTR sequence,respectively, for RNA interference(RNAi). (Aii) Schematic structure of theC. elegans DLG-1 protein andhomologous proteins, DrosophilaLethal(1)Discs Large (accession no:P31007) and human presynaptic proteinhDlg/SAP97 (accession no:NP_004078). Each protein possessesthree PDZ domains (red boxes), 1 SH3(green box) and 1 GUK (blue box) domain; percentages of amino acid identity/similarity between the individual domains of DLG-1 and itshomologues are shown above each domain. The vertical black arrowhead indicates the position of GFP fusion. (B,C) Fluorescence and Nomarskiviews of uninjected and dlg-1(RNAi) dlg-1::gfp transgenic embryos demonstrating the strong reduction in DLG-1::GFP expression after injectionwith dsRNA against dlg-1. Both fluorescent images were taken using the same exposure time (1 second) after 8 hours of development. Confocalimages showing an external (D) and internal (E) focal plane of a 1.5-fold dlg-1::gfp transgenic embryo after immunostaining with anti-GFPantibody. Weak background fluorescence could often be seen around the pharyngeal lumen and rectum (arrowheads in E; these expressing cellshave not been identified). (F-H) GFP fluorescence of a young dlg-1::gfp transgenic adult showing expression in epithelial cells of the pharynx(arrow in F), intestine (arrowhead in F and large arrow in H), vulva (large arrow in G), uterus (arrowheads in G), spermatheca (long arrows in G),rectum (long arrow in H) and epidermis (arrowhead in H). Scale bars, 50 µm (B,C,F-H); 10 µm (D,E).

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progressively mislocalised to the lateral cell membrane in LET-413-deficient embryos (Fig. 3I).

In conclusion, these results show that DLG-1 does not playa major role in localising apical proteins in epithelia andconfirm that LET-413 is required for the maintenance of apico-basal polarity (Fig. 3J).

DLG-1 is required for the maintenance of the CeAJelectron-dense structureTo further examine the dlg-1(RNAi) phenotype, we performedelectron microscopic analysis of dlg-1(RNAi) embryos. Inwild-type embryos, the visible component of CeAJs is denotedby a precisely defined, electron-dense subapical structure in theepidermis (Fig. 4A) and intestine (Fig. 4F). Among 30 sectionscontaining single wild-type embryos, 86 electron-densejunctions were observed in the epidermis. In contrast, among44 sections containing 2-4 dlg-1(RNAi) embryos, only 4slightly extended electron-dense structures were observed (Fig.4C). In these embryos the majority of cell-cell contacts weredevoid of any electron-dense structure (Fig. 4B), although theepidermal cell membranes remained correctly apposed, as inwild-type embryos. Intestinal cells did not appear to be asbadly affected as epidermal cells, although intestinal electron-dense structures were often missing (Fig. 4G,H). Thesestructural defects differ from the CeAJ defects seen in let-413(RNAi) embryos, which show that the electron-densestructure is either discontinuous and greatly extended along thelateral membrane (38 junctions observed in 21 sectionscontaining single embryos; Fig. 4D,I) or absent (Legouis et al.,

2000). In double let-413(RNAi) dlg-1(RNAi) embryos, nolaterally extended electron-dense structures were observed in12 sections containing single embryos (Fig. 4E).

The ultrastructural analysis demonstrates that DLG-1function is necessary for the formation or maintenance of theCeAJ electron-dense component while LET-413 appears tofunction in the compaction and positioning of these junctions.

CeAJ-associated proteins are distributed along thelateral membrane of differentiating epidermal cellsIn order to understand the CeAJ defects resulting from theabsence of DLG-1 and LET-413, we investigated the processof CeAJ formation in wild-type embryos, focusing on theepidermis, which is the most severely affected tissue. Weexamined the subcellular distribution of three CeAJ-associated proteins (henceforth referred to as CeAJ proteins)during epidermal cell differentiation by producing transversesections of projected confocal images along the Z-axis asschematised in Fig. 5A (see Materials and Methods). HMP-1/α-catenin is expressed ubiquitously from the beginning ofembryogenesis and is concentrated at regions of contactbetween blastomeres but, by the end of enclosure, it isrestricted to a subapical belt in epidermal, intestinal andpharyngeal cells (Costa et al., 1998). We observed a punctateHMP-1 distribution along the lateral membrane of most cells,including all LIN-26-expressing cells, at stages preceding(Fig. 5B,B′) and coincidental with (Fig. 5C,C′) epidermaldifferentiation.

JAM-1 expression begins at the time of epithelial


Fig. 3. Apico-basal polarity is affected in let-413(RNAi) but not dlg-1(RNAi) embryos. (A-F) Confocal analysis showing the internal focalplane of embryos after immunostaining with anti-PAR-3 (A-C) or anti-PAR-6 (D-F) antibodies alone, or along with anti-HMP-1 antibodies(A′-C′ and D′-F′, respectively). (A′-F′) Regions within the dotted box in A-F, respectively, at over 2× magnification. (G,H) Most external focalplane, (I) more internal focal plane within epidermis, showing GFP fluorescence of che-14::gfptransgenic embryos. (A,D,G) Wild-typeembryos; (B,E,H) dlg-1(RNAi)embryos; (C,F,I)let-413(RNAi)embryos. In this and all following figures, dlg will refer to dlg-1(RNAi)embryosand let-413 will indicate let-413(RNAi)embryos. Apical markers of the epidermis and intestine are mislocalised in let-413(RNAi)embryoswhile the same markers remain apical to or coincidental with HMP-1 in dlg-1(RNAi)embryos; note that there are regions of lateral CHE-14::GFP expression in dlg-1(RNAi) embryos (arrows in H) and that CHE-14::GFP was not visible in the most external focal plane of let-413(RNAi)embryos. The intestinal lumen is wider in dlg-1(RNAi)embryos than in wild-type embryos (compare A,D with B,E). (J) Diagramillustrating the general localisation of PAR-3, PAR-6 and CHE-14 (green) in a hypothetical epithelial cell in wild-type, dlg-1(RNAi)and let-413(RNAi)embryos. Scale bars, 10 µm (A-I); 5 µm (A′-F′).


differentiation, showing punctate localisation aroundepidermal cells (Podbilewicz and White, 1994). Using UNC-70, the C. elegansβ-G spectrin, as a lateral membrane markerto indicate the extent of the epidermal cell layer (Moorthy etal., 2000), we found that JAM-1 was localised towards the baseof the lateral membrane when it was initially expressed (Fig.5D,D′). Using the dlg-1::gfp transgene as a marker, weestablished that the onset of DLG-1 expression was temporallyand spatially coincident with JAM-1 expression withinepithelial cells (Fig. 5E,E′). In contrast, we observed thatHMP-1 and DLG-1, were distributed in a mutually exclusivemanner (Fig. 5C,C′). Small patches of yellow staining wereobserved (Fig. 5C′), which may indicate areas of overlap, butthis was impossible to resolve within the limits of confocalmicroscopy.

These results indicate that CeAJ proteins are initially

distributed along the lateral membrane rather than in a discretesubapical region (Fig. 5F). In addition, these proteins formdistinct membrane domains, containing either DLG-1 andJAM-1, or HMP-1.

CeAJ proteins localise to distinct components ofmature epidermal CeAJs To continue this CeAJ analysis, we investigated the localisationof CeAJ proteins and LET-413 in mature (fully differentiated)wild-type epidermal cells using Z-axis projections, asschematised in Fig. 6A,B. At this stage, DLG-1 and JAM-1remained colocalised at the mature junction in a subapical belt(Fig. 6C) and HMP-1 formed a slightly punctate belt separatefrom, and generally apical to, the DLG-1/JAM-1 belt (Fig. 6D).Further evidence that HMP-1 is distinct from JAM-1/DLG-1comes from observations of HMP-1 and JAM-1 localisation in

Fig. 4. DLG-1 is necessary for the assembly of the CeAJ electron-dense structure. Electron microscopic analysis of CeAJs in the epidermis(A-E) and intestine (F-I) of wild-type (A,F), dlg-1(RNAi) (B,C,G,H), let-413(RNAi)(D,I) and double dlg-1(RNAi) let-413(RNAi) embryos (E).The electron-dense structure of CeAJs (arrowhead in A) was absent in most epidermal cells of dlg-1(RNAi)embryos (see text), whileneighbouring cell membranes remained correctly apposed (B); where this structure was present it was slightly more elongated than in wild-typeembryos (arrowhead in C). Where epidermal junctions are present in let-413(RNAi)embryos, they are discontinuous and largely extendedfurther basally along the lateral membrane (arrowheads in D). In double dlg-1(RNAi) let-413(RNAi)embryos, the basolaterally mislocalisedelectron-dense junctions are no longer detectable (E). In the intestine of dlg-1(RNAi)embryo (G,H), CeAJs are also abnormal compared towild-type (arrowheads in F) but not affected as badly as in the epidermis. In many cases, the electron-dense junctions are clearly visible(arrowhead in G) while others are absent or faint (arrows in G and H). (I) Shows a typical intestinal section in let-413(RNAi)embryos whereboth CeAJs are present (arrow and arrowhead), although one is elongated with respect to wild-type junctions (arrowhead). Scale bar, 300 nm.

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dlg-1(RNAi)and let-413(RNAi)embryos. The JAM-1 belt wascompletely punctate and disorganised in both dlg-1(RNAi)andlet-413(RNAi)embryos (Fig. 6E-G). However, the HMP-1 beltwas similar to wild-type in most dlg-1(RNAi) embryos,specifically 86% of wild-type (n=50) and 50% of dlg-1(RNAi)(n=50) embryos displayed a slightly punctate HMP-1 beltwhile this belt was more interrupted in remaining embryos.

HMP-1 was more interrupted, but still much less abnormal thanJAM-1, in let-413(RNAi)embryos (Fig. 6H-J).

In conclusion, mature CeAJs in epidermal cells areheterogeneous, with JAM-1 and DLG-1 (and associatedproteins) forming a compartment distinct to that of HMP-1(and associated proteins), which can be independentlyaffected.


Fig. 5. CeAJ proteins are spread along the lateralmembrane in differentiating epidermal cells ofwild-type embryos. (A) Diagram of an early wild-type embryo (at the time of epidermaldifferentiation) showing a dorsal viewrepresenting embryos in images B-E.(A′) Schematic transverse section across anepidermal cell corresponding to the blue bracketin A and representing images B′-E′.(B-E) Confocal analysis showing external focalplane projections of wild-type (B,D) and dlg-1::gfp transgenic (C,E) embryos at a stage ofdevelopment prior to (B), and coincidental with(C-E), epidermal cell differentiation, afterimmunostaining with anti-HMP-1 (B,C), anti-LIN-26 (B), MH27 (anti-JAM-1) (D,E), anti-GFP(C,E) and anti-UNC-70 (D) antibodies and DAPI(D). UNC-70 is used here and elsewhere as ageneral membrane marker. (B′-E′) Z-axis rotationsof a single transverse slice through a section ofthe embryo as denoted by the corresponding linesin embryos B-E, showing CeAJ markersdistributed along the lateral membranes. Apical isto the right in all Z-axis rotations here andelsewhere. (C′) Reveals the mutually exclusivenature of DLG-1 and HMP-1 localisation and (E′)illustrates that DLG-1 and JAM-1 exhibitcomplete co-localisation. Asterisks in B′ and D′ indicate the position of epidermal nuclei. (F) A cartoon showing the position of membrane-associated proteins in immature and mature epidermal cells. CeAJ proteins are positioned along the membrane in immature epidermal cells andrelocate to a compact subapical junction as the cells mature (see Fig. 6). Scale bars, 10 µm.

Fig. 6. Epidermal CeAJs are heterogeneous structures inwild-type embryos. (A) Schematic diagram of part of anembryo at the beginning of elongation indicating thepositions of the projected transverse sections (blue and tanbrackets) displayed in (B) and in Fig. 7C. (C,D)Z-axisrotations of transverse sections through the epidermis alongthe membrane of two connecting epidermal cells where theCeAJ is visible as a subapical belt, as illustrated in B. In B-D, asterisks denote epithelial nuclei on either side of themembrane in question. (C)dlg-1::gfp transgenic embryoafter immunostaining with MH27 (anti-JAM-1), anti-GFPand anti-LIN-26 antibodies; (D) jam-1::gfp transgenicembryo after immunostaining with anti-HMP-1, anti-GFPand anti-LIN-26 antibodies. DLG-1 and JAM-1 appear tocolocalise completely (C) while HMP-1 and JAM-1 appearto be mutually exclusive in their localisation (D; yellowregions may result from limited resolution). (E-G) Wild-type, dlg-1(RNAi) and let-413(RNAi)embryos,respectively, at 8-11 hours development afterimmunostaining with MH27. (H-J) Wild-type, dlg-1(RNAi)and let-413(RNAi)embryos, respectively, at 8-11 hoursdevelopment after immunostaining with anti-HMP-1antibodies. It can be seen that the JAM-1 belt aroundepidermal cells can be altered independently of the HMP-1belt. Scale bars, 5 µm (C,D); 10 µm (E-J).


CeAJ proteins are mislocalised differently in dlg-1(RNAi) and let-413(RNAi) embryos To investigate the possible functions of DLG-1 and LET-413further, we analysed the distribution of either JAM-1 or HMP-1 in DLG-1- or LET-413-deficient embryos and scored boththe spread (extent) of each detected protein and its positionalong the cell membrane (Fig. 7A,B; and see Materials andMethods). We found that the spread of JAM-1 (where present)and HMP-1 was increased slightly in dlg-1(RNAi)embryos (by1/6 for JAM-1 and 1/3 for HMP-1), and markedly in let-413(RNAi)embryos (by 1/2 for JAM-1 and 4/5 for HMP-1)compared to mature wild-type embryos (Fig. 7A). The spreadof JAM-1 in early wild-type epidermal cells was comparableto that seen in the mature wild-type epidermis (Fig. 7A, eWTand mWT).

To assess the subcellular position of these proteins in adifferent manner, we also scored the fraction of lateralmembranes where HMP-1 or JAM-1 could be detected in thetop, middle or bottom part of the membrane (Fig. 7B,C). In themature wild-type epidermis, both HMP-1 and JAM-1 stainingwas observed at the top and middle of the lateral membrane,but never at the bottom (Fig. 7B, green bars, and D,G). Indlg-1(RNAi) embryos, although the HMP-1 position wasessentially as wild type, there was also a small percentage ofmembranes where the protein was detected at the bottom of thelateral membrane (Fig. 7B, red bars, and H). Where present,the distribution of JAM-1 was more abnormal in these

embryos, detected at the middle section in the majority ofmembranes and at the bottom section in a small percentage(Fig. 7B, red bars, and E). In let-413(RNAi) embryos, bothHMP-1 and JAM-1 positions were strongly altered (Fig. 7B,blue bars, and F,I). HMP-1 was detected at the top, middle andbottom sections in similar percentages of membranes, andJAM-1 was also found at each membrane section but detectedat the middle section in most membranes. Interestingly, thisdistribution of JAM-1 along the membrane in let-413(RNAi)embryos is particularly similar to its localisation in immatureepidermal cells (Fig. 7B, yellow bars).

In order to determine whether DLG-1 function is necessaryfor LET-413 localisation, or vice versa, we carried out dlg-1(RNAi) in let-413::gfptransgenic embryos and let-413(RNAi)in dlg-1::gfp transgenic embryos. In let-413(RNAi) embryos,DLG-1 localisation was very discontinuous in the epidermisand intestine, yet remained coincident with JAM-1 (Fig. 7J,K).In wild-type embryos, LET-413 is expressed ubiquitously allaround cell membranes from an early stage until just prior toenclosure, when it is restricted to the basolateral surfaces ofepithelia and the nervous system (Fig. 7L) (Legouis et al.,2000). In contrast to the requirement for LET-413 in DLG-1localisation, LET-413 remained normally localised in dlg-1(RNAi)embryos (Fig. 7M).

In conclusion, LET-413 is required to position CeAJ proteinssubapically and it is striking that the distribution of JAM-1 inlet-413(RNAi)embryos resembles the situation in the early

Fig. 7. CeAJ proteins are mislocaliseddifferently in epidermal cells of let-413(RNAi)and dlg-1(RNAi)embryos. Embryos werestained with anti-HMP-1 or anti-JAM-1 alongwith anti-UNC-70 antibodies and DAPI.Confocal sections were captured and analysedas described in Materials and Methods. (A) Bargraph showing the spread of CeAJ proteins(JAM-1, pink bars, and HMP-1, orange bars)along the lateral membrane of epidermal cellsin mature wild-type (mWT), dlg-1(RNAi), let-413(RNAi)and early wild-type (eWT)embryos. Values are means ± s.e.m. (B) Bargraphs showing the position of the same CeAJproteins along the lateral membranes ofepidermal cells. The graphs show thepercentage of membranes where JAM-1 orHMP-1 were detected at the top (T), middle(M) or bottom (B) of the lateral membrane (seeC). Proteins were often distributed over morethan one membrane section and so the totalpercentage for each category is greater than100%. Note that the spread and position ofHMP-1 was not determined in early wild-type embryos (ND in graphs). The numbers of lateral membranes scored for each set of embryoswere as follows. Wild-type: 71 (JAM-1), 88 (HMP-1); dlg-1(RNAi): 57 (JAM-1), 80 (HMP-1); let-413(RNAi): 78 (JAM-1), 85 (HMP-1); earlywild-type: 85 (JAM-1). (C) Schematic diagram of a transverse section through the mature epidermal layer of a wild-type embryo (see tanbracket, Fig. 6A), indicating how a lateral membrane was sectioned to score protein position. Nuclei are shown in blue, CeAJs in pink andlateral membranes in green. (D-I) Z-axis rotations showing transverse sections through the epidermal layer of mature wild-type (D,G), dlg-1(RNAi)(E, H), and let-413(RNAi)(F,I) embryos after immunostaining with MH27 (red) (D-F) or anti-HMP-1 (red) (G-I), along with anti-UNC-70 (green) antibodies and DAPI (blue). The basal extent of UNC-70 denotes the position of the epidermal basal membrane and thenucleus of the cell under investigation is indicated with an asterisk, as in C. (J,K) Internal view of a let-413(RNAi) dlg-1::gfp transgenicembryo after immunostaining with anti-GFP antibody alone (J), and both anti-GFP and MH27 antibodies (K), showing that DLG-1 ismislocalised in the absence of LET-413 but remains colocalised with JAM-1. (L,M) Internal view of a wild-type (L) and a dlg-1(RNAi) (M)let-413::gfp transgenic embryo after immunostaining with an anti-GFP antibody, illustrating that LET-413 is not mislocalised in the absenceof DLG-1. Scale bars, 5 µm (D-I); 10 µm (J-M).

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wild-type embryo. DLG-1 has a role in forming andmaintaining a continuous JAM-1 belt at the CeAJ, but does notappear to be necessary for HMP-1 or LET-413 localisation.

DISCUSSION

This work has identified a second locus, dlg-1, required for theassembly of C. elegansapical junctions (CeAJs) in addition tolet-413 (Legouis et al., 2000). We show that the MAGUKprotein DLG-1 colocalises with JAM-1, a marker of the CeAJelectron-dense structure (Mohler et al., 1998; D. Hall, personalcommunication), but not with HMP-1 or LET-413. DLG-1 isalso required for JAM-1, but not HMP-1 or LET-413,localisation. Our data suggest that DLG-1 probably actsdownstream of LET-413 in junction assembly and is notessential in maintaining epithelial polarity or adhesion.

Few homologues of tight junction (TJ) or septatejunction (SJ) components are essential for C.elegans embryogenesis Among the 16 predicted genes that we tested encodinghomologues of proteins involved in TJ/SJ formation, only twoproved essential for embryogenesis, one of which, dlg-1, wasrequired for normal epithelial development and morphogenesisof the embryo. There are several possible reasons to explainwhy silencing most of these genes produced no obvious effect.The genes may function in the nervous system, which is knownto be partially resistant to RNAi (for a review, see Bosher andLabouesse, 2000). In addition, redundancy between proteinsmay mask further phenotypes. Our findings are consistent,however, with a deficiency screen covering at least 75% of theC. elegansgenome (Labouesse, 1997), not including thedlg-1locus (data not shown). This deficiency screen uncovered onlytwo deficiencies which disrupted the JAM-1 staining pattern,one that deletes let-413, and the other that deletes the jam-1locus.

CeAJ function and the role of DLG-1 in CeAJformation Our observations provide an assessment of the role andprobable molecular composition of CeAJs, and possiblefunctions of DLG-1. We will deal in turn with the CeAJstructure and with DLG-1 itself. The CeAJ constitutes thesingle subapical junction visible in the epithelia of C. elegansembryos, which has ultrastructural characteristics of a beltdesmosome or adherens junction (AJ) (Priess and Hirsh, 1986;Leung et al., 1999) and yet is associated with a protein (DLG-1) homologous to a septate junction (SJ)-associated protein(this work). Our most striking, and yet unexpected, results canbe summarised as follows. Firstly, HMP-1/α-catenin defines amembrane domain which remains distinct from that defined byJAM-1 and DLG-1, both in wild-type and LET-413- or DLG-1-deficient embryos. Secondly, in the absence of dlg-1function, the electron-dense junction disappears and JAM-1localisation becomes very punctate and slightly more basal,whereas HMP-1 localisation remains largely unaffected.Finally, despite the disappearance of the electron-densejunction, dlg-1(RNAi) embryos can elongate to the twofoldstage and do not rupture. If the electron-dense structure wereessential for adhesion, one would expect embryos lacking this

structure to rupture and fail to elongate beyond the 1.5-foldstage. For comparison, let-413embryos elongate to around the1.7-fold stage or rupture, and hmp-1mutants do not elongateat all. From this, we infer that CeAJs comprise at least twocomponents, one which includes JAM-1/DLG-1 and probablyother proteins (the JAM-1/DLG-1 unit), and a second thatincludes HMP-1, presumably HMR-1 (cadherin) and HMP-2(β-catenin), and possibly other proteins (the HMP-1 unit). Boththese components may contribute to the electron-densestructure, although in the absence of DLG-1, when the HMP-1 unit remains at the CeAJ, the dense structure is absent.Ultrastructural analysis has not been reported forhmr-1, hmp-1 or hmp-2mutants. One could formulate several hypothesesconcerning the respective functions of these components. Onepossibility is that the HMP-1 unit mainly contributes to celladhesion while other CeAJ components play only a minor role.Alternatively, the JAM-1/DLG-1 unit could contain the maincell adhesion proteins, which may function adequately in theabsence of DLG-1. A third possibility could be that there areseveral partially redundant adhesion systems. The phenotypesof hmp-1, hmp-2and hmr-1 mutant embryos lend support tothis third model. When the maternal and zygotic functions ofthese genes is absent, only the cells that drive ventral enclosurefail to attach to each other, while other cells can still adhere atthe ventral midline, general epithelial integrity is intact, andlateral cells maintain a continuous JAM-1 belt (Costa et al.,1998). This suggests that the HMP-1 unit is dispensable foradhesion and cell polarity (Costa et al., 1998; Raich et al.,1999). Similarly, the JAM-1/DLG-1 unit alone does not appearto play a major role in establishing or maintaining membranepolarity as the removal of DLG-1 does not result in amislocalisation of apical proteins. The localisation of JAM-1is affected, but this is more likely to be due to the absence ofan aggregating protein rather than a loss of polarity.

MAGUK proteins are linked with a role in organisingvarious proteins at cell membranes and facilitating signaltransduction, although these functions have been confirmed bymutational analysis in only a few cases (Garner et al., 2000).In Drosophila, Dlg is localised to the SJ and its absenceinduces the mislocalisation of many proteins (includingScribble, Coracle, Expanded and Fasciclin III) along the entirelateral membrane (Bilder et al., 2000; Woods and Bryant, 1991;Woods et al., 1996). Dlg is also required at the neuromuscularjunction to cluster the Shaker potassium channel and the celladhesion molecule Fasciclin II (Tejedor et al., 1997; Thomaset al., 1997). In mice lacking the MAGUK PSD-95, NR2-NMDA receptors still normally localise to synapses butneuronal transmission is impaired (Migaud et al., 1998). Sinceour results show that the electron-dense structure of CeAJs isabsent in dlg-1(RNAi) embryos, we propose that one of thefunctions of DLG-1 is to cluster JAM-1 and other CeAJproteins (excluding those in the HMP-1 unit) and in doing so,primarily contributes to the formation of the electron-densestructure. In addition, as numerous vacuoles were observed inepithelial tissues in the absence of DLG-1, we postulate thatsome of the proteins clustered by DLG-1 are required forsignalling, and thus that DLG-1 is required to facilitate cellsignalling. dlg-1 is not essential for the maintenance ofepithelial membrane polarity, in contrast to C. elegans let-413and Drosophila dlg and scrib. This may be due to severaldifferences between Drosophila Dlg/Scrib and C. elegans



DLG-1/LET-413 including localisation and protein structure(Bilder and Perrimon, 2000; Legouis et al., 2000).

A pathway for the assembly of CeAJsOur previous electron microscopic analysis showed that in theabsence of LET-413, the electron-dense part of the CeAJ iseither missing or extends more basally along the lateralmembrane (Legouis et al., 2000). In addition, we now showthat JAM-1/DLG-1 and HMP-1 are also positioned or extendmore basally in let-413(RNAi) embryos. Strikingly, the CeAJprotein distributions observed in LET-413-deficient embryosclosely resemble their localisation in immature wild-typeepidermal cells. We have also shown that apical proteins, PAR-3, PAR-6 and CHE-14, become progressively more basal in theabsence of LET-413. One of our previous models of LET-413function, also proposed by Bilder and Perrimon for Scrib(Bilder and Perrimon, 2000; Legouis et al., 2000), was thatLET-413/Scrib may be involved in protein trafficking, and mayprevent the docking of apical membrane- and junction-destined proteins at the basolateral membrane. However, in thiswork we have shown that CeAJ proteins are initially distributedalong the lateral membrane, long after LET-413 expression has

begun, and that apical proteins are initially targeted correctlyand only progressively mislocalise along the lateral membrane.Therefore, we propose an alternative model in which LET-413would be primarily involved in condensing and positioning allCeAJ components, thus forming a mature subapical CeAJ,which may subsequently prevent the lateral diffusion of apicalproteins. Interestingly, it has recently been reported inDrosophila that AJs are also found at a basal position duringcellularisation and move apically, in the absence of the novelprotein Nullo, before becoming condensed into a maturesubapical junction (Hunter and Wieschaus, 2000). Thisapically directed movement and coalescence is coincidentalwith the microtubule-dependent migration of the Golgiapparatus from the basal to the apical pole (Sisson et al., 2000).

We raise the possibility that, during epithelial differentiation,LET-413 facilitates the dynamic apical compaction of CeAJcomponents by mediating a direct or indirect interaction witha motor complex (Fig. 8). Subsequently, we suggest that DLG-1 acts to aggregate several proteins, contributing to theformation of an electron-dense structure. How the HMP-1 unitremains distinct from the JAM-1/DLG-1 unit is not clear. Onepossibility is that specific repulsive interactions betweenproteins in the different components segregate them intodistinct membrane domains. Some key objectives for the futureinclude identifying the system which controls the movement ofHMP-1/JAM-1/DLG-1 along the membrane, and discoveringbinding partners for LET-413 and DLG-1. We think thatthe investigation of C. elegans epithelia can contributeconsiderably to the understanding of junction assembly andfunction.

We are grateful to Jim Priess for HMP-1 antibodies, Ken Kemphuesfor PAR antibodies, Vann Bennett for UNC-70 antibodies, YujiKohara for cDNAs, Andy Fire for vectors, Alan Coulson for cosmidsand Grégoire Michaux for the che-14::gfptransgene. We thank AnneGansmuller for assistance with electron microscopy, Marcel Boeglinand Didier Hentsch for help at the confocal microscope. We thankJulia Bosher, Bettina Hermann and Elisabeth Georges-Labouesse forcritical reading of the manuscript. This work was supported by grantsfrom the EEC-TMR program and the Association pour la Recherchecontre le Cancer to M.L. and by funds from the CNRS, INSERM andHôpital Universitaire de Strasbourg.

Note added in proofWhile this work was being reviewed, Bassinger et al. alsodescribed the function of dlg-1. In agreement with our results,they show that loss of DLG-1 affects AJ assembly but not PARprotein localisation. In addition, they show that DLG-1 isrequired for proper distribution of the apical marker CRB-1, aCrumbs homologue. (Bassinger, O. et al.(2001). Dev. Biol.230, 29-42.)

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