D uring animal development, specialized junctional domains are

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1 Coopertive regultion of AJM-1 ontrols juntionl integrity in Cenorhitis elegns epitheli rtiles Mthis Köppen*, Jeffrey S. Simske, Pul A. Sims, Bonnie L. Firestein, Dvi H. Hll, Anthony D. Rie, Christopher Rongo# n Jeffrey D. Hrin* ** *Progrm in Cellulr n Moleulr Biology, University of Wisonsin-Mison, 1117 West Johnson Street, Mison, Wisonsin 53706, USA Deprtment of Zoology, University of Wisonsin-Mison, 1117 West Johnson Street, Mison, Wisonsin 53706, USA Deprtment of Cell Biology n Neurosiene, Rutgers University, Pistwy, New Jersey 08854, USA Center for C. elegns Antomy, Deprtment of Neurosiene, Alert Einstein College of Meiine, 1410 Pelhm Prkwy, Bronx, New York 10461, USA Deprtment of Stem Cell Biology, Linsley F. Kimll Reserh Institute, New York Bloo Center, 310 Est 67th Street, New York, New York 10021, USA #Deprtment of Genetis, The Wksmn Institute, Rutgers University, Pistwy, New Jersey 08854, USA **e-mil: jhrin@fstff.wis.eu The funtion of epithelil ell sheets epens on the integrity of speilize ell ell juntions tht onnet neighouring ells. We hve hrterize the novel oile-oil protein AJM-1, whih lolizes to n pil juntionl omin of Cenorhitis elegns epitheli sl to the HMR HMP (herin tenin) omplex. In the sene of AJM-1, the integrity of this omin is ompromise. Proper AJM-1 loliztion requires LET-413 n DLG-1, homologues of the Drosophil tumour suppressors Srile n Diss lrge, respetively. DLG-1 physilly interts with AJM-1 n is require for its norml pil istriution, n LET-413 meites the rpi umultion of oth DLG- 1 n AJM-1 in the pil omin. In the sene of oth lg-1 n let-413 funtion AJM-1 is lmost ompletely lost from pil juntions in emryos, wheres HMP-1 (α-tenin) loliztion is only milly ffete. We onlue tht LET-413 n DLG-1 oopertively ontrol AJM-1 loliztion n tht AJM-1 ontrols the integrity of istint pil juntionl omin in C. elegns. D uring niml evelopment, speilize juntionl omins re ruil for the funtion of epithelil ell sheets. In oth vertertes n invertertes, herens juntions re thought to regulte ell ell hesion n ynmi hnges in ell morphology 1,2. Verterte tight juntions n inverterte septte juntions hve een implite in ontrolling the piosl polrity of epithelil ells (the fene funtion) s well s regulting the pssge of fluis n solutes through the prellulr spe (the gte funtion) 3 5. Reently, proteins tht regulte the polrity n juntionl integrity of epitheli hve een hrterize. Drosophil Diss lrge, memer of the protein fmily of memrne-ssoite gunylte kinses (MAGUKs), lolizes to n mintins septte juntions 6,7. Furthermore, Diss lrge n the LAP4 (for leuinerih repets n four PDZ omins ) protein Srile lolize together t septte juntions n t in ommon pthwy with the ortil protein Lethl gint lrve in ontrolling the loliztion of pil n herens juntion proteins n in regulting ell prolifertion 8. Similrly, the Cenorhitis elegns LAP1 protein LET- 413 lolizes to solterl omins of epitheli n hs een shown to e ruil for herens juntion ssemly n for the loliztion of pil proteins 9. Among the juntionl omponents whose loliztion is ffete in lg n sr mutnts in Drosophil re proteins speifilly require for the gte funtion of septte juntions suh s Neurexin IV n Bn 4.1-Corle 6, However, the proper loliztion of these n other juntionl omponents proly requires norml epithelil polrity, whih is lost in lg n sr mutnts. Therefore, speifi role for Srile n Diss lrge in regulting proteins iretly require to mintin the integrity of juntionl omins hs een iffiult to emonstrte. Here we show tht the novel oile-oil protein AJM-1 (for pil juntion moleule ) is require for the integrity of epithelil juntions of C. elegns n tht it lolizes to n pil juntionl omin. (AJM-1 ws originlly lle JAM-1 (refs 13, 14) ut hs een renme to voi onfusion with the verterte trnsmemrne tight juntion protein, JAM-1.) This omin is sl to the HMR HMP(herin tenin) omplex; on the sis of the loliztion of the Diss lrge homologue DLG-1 to the sme omin, it might e require for mintining tight pil sel etween epithelil ells t pil juntions. Furthermore, we show tht AJM- 1 iretly ins DLG-1, whih is require for the proper istriution of AJM-1 roun the juntionl elt ut not for generl ell polrity. In ition, we show tht in emryos lking LET-413 the ptterns of oth DLG-1 n AJM-1 re eqully isrupte, inluing ely in onentrtion of these proteins t nrrow pil omin. Almost omplete loss of juntionl AJM-1 is oserve in the sene of oth LET-413 n DLG-1, wheres HMP-1 (α-tenin) loliztion is reue ut juntionl. We propose moel in whih LET- 413 n DLG-1 ontrol the integrity of istint pil suomin y oopertively regulting the loliztion of AJM-1. Results AJM-1 enoes novel oile-oil protein lolizing to C. elegns pil juntions. As n initil step in unerstning the moleulr omposition of pil juntions in C. elegns, we hrterize the ntigen reognize y the ntioy. The ntioy h een previously shown to stin pil orers of C. elegns epitheli 15,16. We nme the ntigen AJM-1 n ientifie the jm-1 gene y proing C. elegns expression lirry with the ntioy. One omplementry DNA (DNA), mh-1, ws ientifie; sequening revele tht jm-1 orrespons to the preite gene C25A11.4, whih spns the genomi lones H08J11 n C25A11 (Fig. 1). The jm-1 trnsript ws nlyse y reverse-trnsriptse-meite polymerse hin retion (RT PCR), northern lot nlysis n sequening of expresse sequene tgs (ESTs) from the C. elegns DNA projet. Five oneptul splie forms were ientifie. A NATURE CELL BIOLOGY VOL 3 NOVEMBER

2 0 LGX k HO8J11 C25A11 C25A11.4 g (1) j18 ok (2) (3) AJM-1 loliztion Funtion sp AJM-1 GFP vl ut sp (4) Yes Yes (5) k Yes No e f h Figure 1 jm-1 enoes novel oile-oil protein., jm-1 mps to the entre of LGX n orrespons to the preite gene C25A11.4, whih is overe y the osmis H08J11 n C25A11., jm-1 splie vrints. Fille oxes inite exons; white oxes inite 5 n 3 UTRs. Exons were numere Five oneptul splie forms enoing the mino is () inite were eue from the sequening of ESTs, RT PCR n northern lot nlysis. Seonry struture nlysis preits mino is to form oile-oil motif (shown in grey). The j18 n ok160 lesions re inite. ok160 lmost ompletely eletes exons 6 9. j18 eletes exons 4 6; however, its preise 3 n 5 rekpoints hve not een ientifie. Domins require for AJM-1 loliztion n funtion were etermine y resue experiments with speifi splie forms (see Methos). Amino is were shown to e suffiient for funtion. Amino is were suffiient for juntionl loliztion of AJM-1 (t not shown), ut not for funtion. BLAST serh of the tses revele no ovious homologues of AJM-1. Amino is , whih re ommon to ll splie vrints of AJM-1, re preite to form oile-oil motif (Fig. 1). Hyrophoiity nlysis suggests tht AJM-1 is ompletely hyrophili n is therefore proly ytoplsmi protein (t not shown). Resue of jm-1 mutnts ws otine with 12-kilose (k) genomi frgment tht enoes the two smllest splie forms; these enoe mino is n , respetively. We therefore onlue tht mino is re suffiient for AJM-1 funtion (Fig. 1). To ress the requirement of speifi omins of AJM-1 for loliztion n funtion of the protein, we nlyse the expression pttern n resuing ility of ifferent green-fluoresent-protein (GFP)-tgge forms of the protein (see Methos). We oserve tht mino is , enoing minly the oile-oil omin, re suffiient to lolize AJM-1 to pil juntions, yet nnot resue jm-1 mutnts (Fig. 1). This suggests tht AJM-1 mino is re n essentil omin of AJM-1 tht is not require for loliztion. The AJM-1 expression pttern ws etermine y stining with n expression of n jm-1::gfp onstrut ontining exons 2 9 n le to resue jm-1 mutnts. AJM-1 lolizes to the pil orers of ll C. elegns epitheli. Expression ours in the emryoni hypoermis, phrynx n intestine (Fig. 2,, e, f), s well s in post-emryoni epitheli, inluing the vulv, uterus, spermthee (Fig. 2, ), phrynx, intestine, hingut, hypoermis n mle til (t not shown). stining of emryos expressing HMP- 1 GFP revels tht AJM-1 oupies n pil omin tht is sl to the HMR HMP(herin tenin) omplex in ll epithelil tissues exmine (Fig. 2e, f). Trnsmission eletron mirosopy (TEM) nlysis of emryoni hypoerml ell juntions revele ph it Figure 2 AJM-1 lolizes to istint pil omin of epithelil juntions.,, Emryoni AJM-1 expression. Emryos in this n ll susequent figures re presente with nterior to the left n orsl t the top., stining outlines the pil orers of hypoerml ells., A Nomrski imge of omprle emryo.,, AJM-1 expression in lrvl epitheli., AJM-1 GFP loliztion to pil ell orers of the spermthee (sp), vulv (vl) n uterus (ut) of n L4 lrv., A Nomrski imge of the sme niml. e, f, Comprison of the (in re) n (in green) ptterns in n emryo. e, Both mrkers outline the pil orers of the phrynx (ph) n the intestine (it). f, Mgnifition of the phrynx region revels tht is loser to the lumen n is flnke y, initing tht stins more sl omin. The sme reltive positions of AJM-1 n HMP-1 were lso oserve in the hypoermis (t not shown). g, A TEM imge of hypoerml ell juntion of wil-type emryo. An eletron-ense pil omin (rrow) links the ells, n lerly efine septe in the juntion re not present. h, Immunogol lelling with revels speifi lelling t the pil juntion. Sle rs, 10 µm (,), 5 µm (f),100 nm (g) n 50 nm (h). n pil juntionl omin resemling herens juntions of flies n vertertes, wheres no morphologilly istint septte or tight juntions re oserve. The piosl extent of this omin is 100 nm (Fig. 2g). Immuno-TEM nlysis of hypoerml ells of lrve with the ntioy revele tht AJM-1 speifilly lolizes to the pil omin (Fig. 2h). jm-1 mutnt emryos re rreste uring lte emryoni elongtion. To ress the funtion of AJM-1, we generte jm-1 mutnt lleles. jm-1(j18) ws isolte in linke-lethl sreen n jm- 1(ok160) in PCR-se sreen (t not shown). ok160 eletes lmost the entire preite oile-oil omin. j18 eletes exons 4 6, lthough the preise 3 n 5 rekpoints re unientifie (Fig. 1). Both lleles re preite nulls n show the sme phenotype s jm-1(rnai) emryos (t not shown). ok160 ws use for ll susequent nlysis. During wil-type emryogenesis, the niml is enlose y n epithelil sheet (hypoermis) n susequently elongtes to out four times its originl length efore hthing (Fig. 3 ) 17,18. jm-1 emryos enlose normlly n initite elongtion without ovious morphologil normlities (Fig. 3, e), yet t slower rte thn the wil type. Wil-type emryos reh the 2-fol stge 90 ± 0.4 min (men ± SEM, n = 12) fter ompletion of enlosure, wheres jm-1 emryos require 124 ± 3.8 min (n = 13). In jm-1 emryos, 984 NATURE CELL BIOLOGY VOL 3 NOVEMBER

3 Wil type jm-1(ok160) 50 min 120 min e f 90 min g ok min elongtion is onsistently rreste t the 2 3-fol stge, ompnie y the formtion of lrge vuole in the posterior region (Fig. 3e). jm-1 emryos lk stining (Fig. 3f). We onlue tht AJM-1 is require for the orret rte n ompletion of elongtion of the C. elegns emryo. The proess of emryoni elongtion hs een shown to epen on the HMR HMP(herin tenin) omplex, whih meites the juntionl tthment of irumferentil mirofilments proviing the ontrtile fore tht is thought to rive elongtion 17,19. In ition, elongtion pst the 2-fol stge epens on the proper tthment of oy wll musle to the hypoermis 20,21. We therefore nlyse jm-1 emryos for possile efets in the integrity of the HMR HMP omplex, mirofilment lignment n the pttern of oy wll musle. Mutnts for omponents of the HMR HMP omplex typilly isply inomplete enlosure or severe efets uring initil elongtion, ut show unltere AJM-1 loliztion 14,19. We nlyse the pttern of HMP-1 (α-tenin) in jm-1 mutnts y using n nti-hmp-1 expression n oserve ptterns ientil to the wil type in oth ses. Furthermore, jm-1 emryos isply norml lignment of irumferentil mirofilments (t not shown). We nlyse the pttern n funtion of oy wll musle in jm-1 emryos y h i ok160 Figure 3 jm-1(ok160) emryos re rreste t the twofol stge n isply ompromise integrity of pil juntions., Wil-type emryos. Two time points of time-lpse reoring show tht uring elongtion the wil-type emryo rehes the 1.5-fol stge 50 min fter ompletion of enlosure (), n the 3-fol stge is rehe 120 min fter enlosure (). stining shows the typil juntionl pttern (). f, jm-1(ok160) emryos. The emryo rehes the 1.5-fol stge without pprent morphologil normlities, yet is signifintly elye, rehing 1.5-fol 90 min fter enlosure (). After rehing the 2-fol stge t 130 min fter enlosure (t not shown) elongtion stops n lrge vuole forms in the oy vity ner the posterior en of the tively twithing emryo (rrow) (e), followe y evelopmentl rrest. stining is ompletely olishe in jm-1(ok160) emryos (f). g i, TEM nlysis of pil juntions of n jm-1(ok160) emryo n wil-type emryo. (g) A glning setion through the pil surfe of hypoerml ell of n jm-1(ok160) emryo, permitting the ssessment of juntionl integrity long the entire ell orer. As is onsistently oserve in jm-1(ok160) emryos, seprtions within the pil juntion re visile (rrowhes n rrow). A higher mgnifition of the lrgest seprtion (rrow) revels tht the juntion is evenly split open, reting prellulr gp of 190 nm in with (h). The juntionl res flnking the seprtion seem properly sele n 15 nm wie, s in the wil-type (WT) juntion in (i), with prellulr gps rnging etween 0 n 2 nm. Sle rs, 10 µm (), 0.5 µm (g) n 100 nm (h). WT using the musle-speifi ntioy R224 (ref. 22) n oserve wil-type pttern. In ition, jm-1 emryos egin twithing t roun 1.75-fol stge (t not shown), initing wil-type musle tivity 23. These results suggest tht AJM-1 n the HMR HMP omplex lolize n funtion mutully inepenently. Furthermore, AJM-1 is not require for the irumferentil lignment of mirofilments n for the positioning n funtion of oy wll musle in the emryo. AJM-1 is require for juntionl integrity ut not epithelil polrity. To ress the funtionl role of AJM-1 in pil juntions speifilly, we nlyse jm-1 emryos t the ultrstruturl level y TEM. Apil juntions of C. elegns epitheli pper s ontinuous lose pposition of neighouring ells. They ontin eletronense mteril t the ytoplsmi fe of the memrnes n n eletron-ense re in the prellulr spe (Fig. 2g n Fig. 3g i). Thin setions of hypoerml ells of jm-1 emryos isply pil juntions tht re properly positione (t not shown). However, the lose pposition of these juntions is isrupte intermittently y ring-shpe seprtions long the whole length of the juntionl elt. These seprtions rete prellulr gps tht re typilly etween 50 n 200 nm in imeter, wheres properly sele omins re 15 nm wie with prellulr gps of 0 2 nm (Fig. 3g i). Anlysis of seril thin setions revele tht the pil sl extent of the seprtions is 100 nm n tht they omprise only the eletron-ense pil omin. Close memrne pposition is oserve oth pil to n sl to the seprtions (t not shown). We onlue tht AJM-1 is require for mintining the integrity of the eletron-ense omin of pil juntions. To ress further whether AJM-1 is require for epithelil polrity, we nlyse the loliztion of pil n sl mrkers in jm-1 emryos. As pil mrkers we use ntioies ginst SMA- 1 (βh-spetrin), whih lolizes pilly in hypoerml ells (J. Austin, personl ommunition), n PAR-3 n PAR-6, whih lolize pilly of AJM-1 in the phrynx n intestine (t not shown). Antioies ginst UNC-70 (βg-spetrin) were use s solterl mrker in the hypoermis 24, n s sl mrkers we use the monolonl ntioies MH46 n MH5, whih reognize omponents of hemiesmosomes involve in oy wll musle tthment 25. In eh se, protein loliztion ws unltere in jm-1 emryos in omprison with the wil type (t not shown). This suggests tht AJM-1 is not require for epithelil polrity. AJM-1 n DLG-1 physilly intert n funtion in ommon pthwy. To ientify proteins tht re require for AJM-1 loliztion n funtion, we performe yest two-hyri sreen with fusion protein etween AJM-1 n the DNA-ining omin of LexA s it. The plexa AJM-1 onstrut ws me with prtil jm-1 DNA enoing mino is of the protein, whih inlue the preite oile-oil omin. Of the 200,000 trnsformnts sreene, two inepenently isolte DNAs were foun orresponing to the lg-1 gene. lg-1 enoes the C. elegns homologue of Drosophil Diss lrge (Fig. 4) 26,27. Both lg-1 DNAs isolte in the two-hyri ssy enoe mino is of the preite DLG-1 protein. To onfirm this intertion in n inepenent ssy, we performe n in vitro pull-own experiment with fusion of glutthione S-trnsferse (GST) n DLG-1 (mino is ). Full-length 35 S-lelle AJM-1 (mino is 1 868) ws proue y trnsltion in vitro n inute with es rrying GST DLG-1 or GST. Riolelle AJM-1 oun to GST DLG-1 ut not to GST lone (Fig. 4). DLG-1 is memer of the fmily of memrne-ssoite gunylte kinses, ontining three PDZ omins, n SH3 omin n gunylte kinse omin. Amino is of the protein ontin the first two PDZ omins (mino is n ) n itionl resiues mino-terminl n roxy-terminl to those omins (Fig. 4). PDZ omins hve een previously implite in protein protein intertion in Drosophil Diss lrge n other MAGUK proteins 7,28. PDZ omins of Diss lrge-like MAGUKs fvour ining prtners with C-terminl E(S/T)X(V/I) omin NATURE CELL BIOLOGY VOL 3 NOVEMBER

4 GAL4 DLG-1 LexA AJM-1 GST DLG-1 GST Input (20%) GAL4 IRP [ 35 S]AJM-1 GAL4 C. elegns DLG-1 NH 2 PDZ1 PDZ2 PDZ3 SH3 GuK COOH Anti-DLG lg-1(rnai) lg-1(rnai) jm-1(ok160) Anti-DLG Anti-DLG Figure 4 AJM-1 n DLG-1 physilly intert n funtion in ommon pthwy., AJM-1 DLG-1 intertion in yest. An unise yest two-hyri sreen ws onute with AJM-1 (mino is ) fuse to the LexA DNA-ining omin s it. Two inepenently isolte DNAs orrespon to lg-1. Speifiity of the AJM-1 DLG-1 intertion ws onfirme in irete intertion tests with yest ontining the plexa AJM-1 plsmi n either the ientifie DNAs or ontrol plsmis. β-gltosise itivity ws etete in yest expressing the Gl4 tivtion omin DLG-1 fusion protein (Gl4 DLG-1) n LexA AJM-1 (top strek). Control ssys using Gl4 IRP (iron response protein) or Gl4 resulte in omplete lk of β-gltosise tivity (mile n ottom streks, respetively)., AJM-1 DLG-1 intertion in vitro. Sephrose es rrying GST DLG-1 fusion protein (mino is of DLG-1) or GST were inute with riolelle AJM-1 (mino is 1 868). Boun protein ws elute n nlyse y SDS PAGE. Bining ws oserve etween AJM-1 n GST DLG-1 (left lne) ut not etween AJM-1 n GST lone (mile lne). The right lne shows lelle AJM-1 protein equivlent to 20% of the mteril nlyse in the experimentl lnes., DLG-1 omin struture. The lg-1 enoes 967-mino-i protein, ontining three PDZ omins, n SH3 omin n GuK omin. The r inites the region enoe y oth DNAs ientifie in the yest two-hyri sreen (mino is )., DLG-1 loliztion n lg-1(rnai) phenotype. A wil-type emryo ws immunostine with n nti-dlg ntioies rise ginst Drosophil Diss lrge 31. The upper left pnel shows stining in hypoerml ells. The upper mile pnel shows nti-dlg in the sme fol plne. Both ptterns overlp ompletely in merge imge (upper right pnel). Anlysis of lg-1(rnai) emryo with Nomrski vieo-mirosopy revels phenotype similr to tht of jm-1 emryos. The emryo is rreste t roun the 2-fol stge of elongtion n forms lrge vuole in the posterior oy vity (rrow, ottom left pnel). A lg-1(rnai) emryo stine with nti-dlg ntioies shows omplete sene of stining (ottom mile pnel), wheres n jm-1(ok160) emryo shows proper nti-dlg-1 stining (ottom right pnel). Sle r, 10 µm. (single-letter mino i revitions) 29. However, this motif is not foun in AJM-1. Furthermore, the AJM-1 protein use in the two-hyri sreen (ut not in the GST pull-own ssy) lke the C-terminl 59 mino is. This suggests tht the intertion with DLG-1 oes not involve the C terminus of AJM-1. Similrly, the oile-oil protein ingulin hs een shown to intert with the MAGUK proteins ZO-1, ZO-2 n ZO-3 through its N-terminl omin 30. We propose tht AJM-1 either ontins novel internl PDZ-intertion omin, or lterntively tht the intertion oes not involve the PDZ omins. Aitionl struture funtion nlyses will ress these possiilities. To nlyse the expression pttern of DLG-1 in C. elegns emryos, we stine emryos with polylonl ntioy rise ginst the highly onserve mino is of Drosophil Diss lrge 31. Consistent with nother report tht ws pulishe while this work ws eing reviewe ws our oservtion, y lser snning onfol mirosopy nlysis, of omplete o-loliztion with AJM-1 to the pil juntions in the hypoermis (Fig. 4), phrynx n the intestine (t not shown) 27. We onlue tht AJM-1 n DLG-1 physilly intert t pil juntions. We resse the funtion of DLG-1 protein y using RNAmeite interferene (RNAi) 32. At the level of Nomrksi mirosopy, lg-1(rnai) emryos isply n rrest phenotype tht is morphologilly similr to, ut slightly more severe thn, tht of jm-1 emryos. Elongtion is rreste immeitely efore or t the 2-fol stge t 165 ± 6.7 min (men ± SEM, n = 6) fter the initition of elongtion (Fig. 4 n t not shown). Therefore, the elongtion of lg-1(rnai) emryos is signifintly slower thn tht of oth wil-type n jm-1 mutnts. Like jm-1 mutnts, lg- 1(RNAi) emryos typilly isply lrge vuole in the posterior 986 NATURE CELL BIOLOGY VOL 3 NOVEMBER

5 let-413(rnai) Wil type lg-1(rnai) lg-1; let-143(rnai) e f g nti-dlg h i j let-413 h k l m lg-1 Figure 5 LET-413 n DLG-1 oopertively ontrol AJM-1 loliztion. Comprison of the n ptterns in wil-type, lg-1(rnai), let- 413(RNAi) n lg-1;let-413(rnai) emryos with onfol mirosopy., Wil type. () n () show ontinuous juntionl loliztion n the merge imge revels tht oth ptterns re losely ssoite (). g, let- 413(RNAi). The pttern revels long strethes of norml AJM-1 loliztion tht re seprte intermittently y gps ompletely lking AJM-1 (); mgnifition of the oxe re is shown in ( ). Juntionl is oserve ut is reue ompre with the wil type (e). Ajent loliztion of the signl n is mintine, s oserve in the merge imge (f). Anti-DLG stining of the sme emryo revels pttern tht is lrgely ientil to the pttern (g). h j, lg-1(rnai). The pttern is hrterize y ggregtes seprte y lrge regions in whih stining is gretly reue (h); mgnifition of the oxe re is shown in (h ). The pttern is unltere ompre with the wil type (i), n the remining signl is losely ssoite with (j). k m, let-413; lg-1(rnai). Juntionl stining is ompletely olishe (k), wheres the pttern is similr to tht in let- 413(RNAi) emryos (l). In the merge imge no signifint overlp of n is oserve (m). Sle rs, 10 µm (, g) n 2 µm (h ). region (Fig. 4). We oserve no nti-dlg-1 stining in lg- 1(RNAi) emryos, onfirming tht the ntioy reognizes DLG-1. jm-1 mutnt emryos isply DLG-1 expression pttern tht is ientil to the wil type (Fig. 4). Next, we eliminte lg-1 funtion in jm-1 emryos. The phenotype oserve ws ientil to tht of lg-1(rnai) emryos with respet to the stge of emryoni rrest, the morphology of the emryos n the rte of elongtion (t not shown). Tken together, these results inite tht DLG- 1 is require for proper emryoni elongtion; DLG-1 proly ts upstrem of AJM-1 n eomes lolize to pil juntions inepenently of AJM-1. LET-413 n DLG-1 oopertively regulte AJM-1 loliztion. LET- 413 is LAP1 protein, ontining leuine-rih repets n one PDZ omin. LET-413 is lolize to the solterl omins of epithelil ell memrnes, n in let-413 mutnt emryos pil juntions isply severe struturl efets. In ition, the loliztion of pil proteins, inluing AJM-1, is isrupte 9. AJM-1 misloliztion in lg-1(rnai) emryos hs lso een esrie 27. To explin in more etil how AJM-1 loliztion is regulte y LET-413 n DLG-1, we refully ompre AJM-1 loliztion in emryos lking either LET-413 or DLG-1, or oth. Our results onfirm tht let-413(rnai), let-413(s128) n sdf35 emryos (the efiieny eleting the let-413 gene) onsistently rupture fter the 1.5-fol stge of elongtion (t not shown) 9.We nlyse AJM-1 loliztion in these three kgrouns n otine ientil results for eh. We oserve n AJM-1 pttern showing regions of norml loliztion seprte y intermittent gps (Fig. 5, ). In let-413(rnai) emryos, juntionl loliztion of is oserve onsistently, lthough t lower levels thn in wil-type emryos (Fig. 5, e). Similr results were otine from immunostining of let-413(rnai) emryos n let- 413(s128) mutnts with nti-hmp-1 (t not shown). The remining AJM-1 protein is losely ssoite with, suggesting tht it is lolize to pil juntions s in the wil type (Fig. 5, f). We next ompre DLG-1 n AJM-1 loliztion in let-413(rnai) emryos. Invrily, the ptterns of oth proteins re ientil n ompletely overlp in merge imges (Fig. 5, g n t not shown). lg-1(rnai) emryos onsistently show n AJM-1 misloliztion pttern tht is istint from tht in let- 413(RNAi) emryos: AJM-1 lolizes to pil juntions, ut unlike the even, uninterrupte AJM-1 pttern of wil-type emryos, we oserve regions of AJM-1 ggregtion tht lternte with lrge regions in whih AJM-1 expression is gretly reue; this pttern is reminisent of es on string (Fig. 5, h, h ). The HMP- 1 GFP pttern in these emryos is unltere, n AJM-1 remins losely jent to t pil juntions (Fig. 5i, j). In emryos eprive of oth LET-413 n DLG-1 y RNAi, the juntionl loliztion of AJM-1 is lmost ompletely olishe. HMP- 1 GFP shows reue expression ut remins juntionl (Fig. 5k m). Tken together, these results suggest tht LET-413 n NATURE CELL BIOLOGY VOL 3 NOVEMBER

6 let-413(rnai) DLG-1 GFP Wil type Wil type lg-1 0 min let-413 lg-1; let-413 e f 75 min g h e X LET-413 i j k l DLG-1 AJM-1 Integrity of pil juntions Fig. 6 LET-413 is require for rpi pil onfinement of DLG-1. Dynmi nlyses of DLG-1-GFP expression in eveloping let-413(rnai) n wil-type emryo were performe with time-lpse multiphoton lser snning mirosopy. DLG-1-GFP istriution long iniviul pil juntions ws orrelte with piosl istriution of the protein over time. During erly epithelil morphogenesis (orsl interltion), DLG-1-GFP shows puntte pttern in ll orsl epithelil ells of the let-413(rnai) emryo; for further nlysis, one ell orer is mrke with n rrowhe (, n enlrge in e). A Z-projetion of the mrke ell orer revels tht DLG-1-GFP is lolize pilly s well s lterlly (f). The equivlently stge wil-type emryo shows ompletely ontinuous DLG-1-GFP pttern (, g), n DLG-1-GFP is onfine to nrrow pil omin within the juntion (h). At 75 min. lter, fter the ompletion of orsl interltion, further DLG-1-GFP hs umulte in the pil juntions of the let-413(rnai) emryo (, i); the DLG-1-GFP pttern of the mrke juntion hs eome more similr to tht of the wil-type juntion (, k). Comprison of the piosl istriution of DLG-GFP t this stge revels tht the protein hs eome onfine pilly within the let-413(rnai) juntion (j), similr to the wil-type juntion (l). Sle rs represent 10 mm () n 2 mm (l). Figure 7 Moel for the ontrol of AJM-1 loliztion y LET-413 n DLG-1., In the wil type, AJM-1 (re) is istriute in ontinuous pttern t pil juntions in the presene of LET-413 (yellow), whih lolizes solterlly, n DLG-1 (lue), whih ins AJM-1 t pil juntions., In the sene of DLG-1, pil AJM-1 is erese, n in ition the protein is unevenly istriute within the pil omin., In the sene of LET-413, rpi pil onfinement of DLG-1 n AJM-1 oes not our, resulting in erese in the pil loliztion of oth proteins n misistriution to the lterl omin., In the sene of oth LET- 413 n DLG-1, loliztion of AJM-1 to pil juntions is lmost ompletely lost. The protein is rnomly istriute long the piosl xis or lost from ell orers. e, A propose pthwy of AJM-1 loliztion. LET-413 is require for the loliztion of DLG-1, whih iretly ontrols AJM-1 loliztion. In ition, n unknown ftor is require to ount for the signifint mount of pil DLG-1 in the sene of LET-413 (protein X; ). LET-413 lso ffets AJM-1 loliztion vi DLG-1-inepenent pthwy; loss of oth the DLG-1-epenent n DLG-1-inepenent pthwys les to mrke misloliztion of AJM-1 in the sene of oth LET-413 n DLG-1 () ompre with the loss of DLG-1 lone (). DLG-1 oopertively regulte AJM-1 loliztion to pil juntions, wheres norml juntionl loliztion of HMP-1 is prtly epenent on LET-413 ut not DLG-1. To ress how LET-413 funtions in lolizing DLG-1 n AJM-1 to pil juntions, we nlyse the ynmis of the piosl istriution of DLG-1 n AJM-1 in iniviul hypoerml juntions of wil-type n let-413(rnai) emryos over time. Time-lpse multi-photon nlysis of DLG-1 GFP fusion protein revele tht uring n erly phse of hypoerml morphogenesis (orsl interltion) in let-413(rnai) emryos, DLG-1 GFP shows puntte loliztion to pil hypoerml juntions n is prtly lolize to the lterl memrne omin (Fig. 6, e, f). In ontrst, omprle wil-type emryos isply ontinuous pttern of DLG-1 GFP tht is ompletely pil (Fig. 6, g, h). As hypoerml morphogenesis proees, DLG-1 GFP umultes t nrrow pil region of the hypoerml juntions of let- 413(RNAi) emryos (Fig. 6,i, j), forming pttern omprle with tht of wil-type emryos (Fig. 6, k, l). Similr results were otine for AJM-1 GFP (t not shown). We onlue tht LET-413 is require for rpi onfinement of DLG-1 n AJM-1 to nrrow pil region in C. elegns epitheli. Apil onfinement of oth proteins ours in the sene of LET-413, ut more slowly. Disussion Our results inite tht AJM-1 is novel oile-oil protein tht lolizes to istint pil omin of epithelil juntions of C. elegns n is require for mintining the integrity of this omin. Juntionl loliztion of AJM-1 is regulte y oth the Diss lrge homologue DLG-1 n the LAP1 protein LET-413. AJM-1 seems to intert iretly with DLG-1 t pil juntions, wheres LET-413 meites the rpi pil loliztion of oth DLG-1 n AJM-1. Anlysis of AJM-1 loliztion y immunofluoreene n immunogol TEM revels tht AJM-1 oupies istint pil juntionl omin in C. elegns epitheli tht is sl to the HMR HMP (herin tenin) omplex. AJM-1 is require uring emryogenesis, euse jm-1 mutnt emryos re onsistently rreste t the 2 3-fol stge of elongtion. This phenotype lerly iffers from tht resulting from ompromise HMR HMP omplex. Mutnts lking ny omponent of the omplex show inomplete enlosure of the emryo y the hypoermis n susequent rupture uring elongtion, s well s mislignment of mirofilments. This efet proly results from filure to form herens juntions etween leing ventrl hypoerml ells uring enlosure, or from filure to nhor properly the mirofilments tht trnsmit ontrtile fores to the juntions uring elongtion 14,19.In jm-1 emryos, no epithelil ruptures, oy shpe efets or improper mirofilment lignment re oserve. Consistent with this ws the oservtion tht AJM-1 lolizes normlly in the sene of the HMR HMP omplex 14,19 ; here we report norml loliztion of HMP-1 (α-tenin) in the 988 NATURE CELL BIOLOGY VOL 3 NOVEMBER

7 sene of AJM-1. AJM-1 therefore seems to perform funtion tht is inepenent of the HMR HMP omplex, whih is onsistent with loliztion of AJM-1 to istint pil omin. Ultrstruturl nlysis of the hypoermis of jm-1 emryos revels pil juntions tht re properly positione n hve, in prt, norml morphology. However, wheres wil-type juntions pper s ontinuous lose ppositions etween neighouring epithelil ells, numerous smll seprtions of the juntions re oserve in jm-1 mutnts. Aprt from the pil juntion to whih AJM-1 lolizes, we oserve no morphologilly istint strutures in the ell orers etween epithelil ells of C. elegns tht oul e nites for mintining the prellulr sel. This pil juntion might therefore serve s permeility rrier. Consistent with this hypothesis is the oservtion tht this omin is sl to the herin tenin omplex n ontins Diss lrge, whih is nlogous to the Drosophil septte juntion, propose permeility rrier 10,11. We speulte tht the juntionl seprtions oserve in jm-1 emryos llow the exessive pssge of flui ross the hypoermis through the prellulr spe, using emryoni rrest owing to osmoti imlne etween the pseuooelom n the exterior of the emryo. The lrge vuole tht onsistently forms in the posterior region of elongting jm-1 emryos might result from the umultion of flui in the pseuooelom. However, our ttempts to nlyse epithelil permeility of sizespeifi yes in jm-1 emryos hve so fr een inonlusive (M. K., P. A. S. n J. D. H., unpulishe oservtions). The lose pposition of epithelil ells pil n sl to the juntionl seprtions in jm-1 mutnts might still provie suffiient physil rrier to restrit ye movement in suh experiments. jm-1 is preite to enoe novel ytoplsmi protein. Severl splie vrints of the jm-1 trnsript were ientifie. However, AJM-1 funtion requires only two essentil omins: short N-terminl omin without ovious seonry struture, followe y puttive oile-oil omin. The roles of lterntive N n C termini re not known. Our GFP-tgging experiments hve emonstrte tht the oile-oil omin is suffiient for lolizing AJM-1 to juntions. The funtion of the ritil N-terminl omin is urrently unler. Severl oile-oil proteins with struturl similrity to the oile-oil omin of AJM-1 hve een shown to in n rosslink ytoskeletl omponents. These inlue the mmmlin proteins trihohylin, lesmon n pletin In ition, reent stuies hve implite oile-oil motifs in memrne-nhore sffoling of struturl n funtionl proteins t the ytoplsmi fe of verterte tight juntions. The oile-oil-ontining protein ingulin n the intrellulr oile-oil motif of oluin intert physilly with other tightjuntion omponents, inluing the MAGUK proteins ZO-1, ZO-2 n ZO-3, n intert iretly with the tin ytoskeleton 30,36,37. Similrly, AJM-1 might ontriute to the ssemly n integrity of pil juntions etween epithelil ells of C. elegns y lustering ytoplsmi n/or trnsmemrne omponents t juntions. Testing this moel will require the ientifition of other iret ining prtners of AJM-1. A potentil requirement for AJM-1 in orgnizing tin filments t pil juntions ws not onfirme y the use of phlloiin stining; however, more in-epth nlysis t the ultrstruturl level might e neessry to ress this possiility etter. Our results suggest tht AJM-1 loliztion to pil juntions is ontrolle oopertively y the MAGUK protein DLG-1 n the LAP-1 protein LET-413. We hve shown tht DLG-1 might e iretly require for proper AJM-1 loliztion t pil juntions (Fig. 7); DLG-1 n AJM-1 intert physilly in yest s well s in vitro, n oth proteins lolize together t pil juntions of C. elegns. Furthermore, loss of DLG-1 uses similr evelopmentl rrest to tht use y the loss of AJM-1, n loss of oth DLG-1 n AJM-1 uses phenotype inistinguishle from tht proue y the loss of DLG-1 only. This suggests tht oth proteins t in ommon pthwy. In the sene of DLG-1, the juntionl AJM-1 level is erese n misistriute long the juntionl ring (Fig. 7), wheres the formtion n pil loliztion of the herin tenin omplex s well s generl piosl polrity re unffete. Thus, one wy in whih DLG-1 seems to ontrol the integrity of pil juntions is y meiting the proper istriution of AJM-1 long the juntions. LET-413 h een shown to lolize to the sl n solterl memrnes of epithelil ells n to e require for the proper loliztion of pil proteins 9. We hve shown tht DLG-1 n AJM-1 re foun in the juntionl elt in the sene of LET-413. However, the normlly ontinuous istriutions of DLG-1 n AJM-1 re eqully isrupte y gps, n oth proteins prtly mislolize to the lterl memrne omin (Fig. 7). Moreover, our results inite tht LET-413 meites rpi umultion of DLG-1 n AJM-1 t pil juntions. An pil umultion of oth proteins ours in the sene of LET-413 ut is signifintly elye. We propose two moels for how LET-413 might funtion in this proess. Apil loliztion of DLG-1 n AJM-1 might involve n initil, LET-413-inepenent loliztion to the lterl memrne omin, given the lterl loliztion of the proteins oserve in the sene of LET-413. This is followe y LET-413- epenent trnslotion to the pil omin. Eqully plusily, DLG-1 n AJM-1 might e trgete iretly to the pil memrne omin vi urrently unientifie mehnism, n LET- 413 might exlue these proteins from the lterl memrne omin. Clerly, these two mehnisms re not mutully exlusive. A more temporlly refine ynmi nlysis of DLG-1 n AJM-1 eposition might help to istinguish etween these possiilities. Signifintly, we oserve tht the AJM-1 pttern is more severely ffete in emryos lking the funtions of oth LET-413 n DLG-1 thn in emryos lking the funtion of either protein lone (Fig. 7). This result rgues ginst stritly liner pthwy in whih LET-413 ffets AJM-1 exlusively y ontrolling DLG-1 loliztion. We propose tht, in ition, LET-413 ontrols AJM- 1 loliztion vi prllel pthwy tht is inepenent of DLG-1 (Fig. 7e). Our nlysis llows us to infer itionl spets of juntionl regultion in C. elegns. Unientifie ftors proly ontriute to the loliztion of DLG-1 to pil juntions, euse there is signifint mount of pil DLG-1 n AJM-1 in the sene of LET-413. Tht juntionl loliztion of HMP-1 (α-tenin) is erese in the hypoermis is onsistent with stuy showing erese in juntionl HMP-1 in the gut epithelium of let-413 mutnt emryos 27, n extens these finings to the hypoermis. We i not oserve the essentilly ytoplsmi loliztion of HMP-1 in the hypoermis tht h previously een reporte for 50% of let-413 mutnt emryos in n itionl stuy 9.Tken together, these results rgue tht itionl pil ues exist tht funtion in prllel with LET-413 (Fig. 7e). A more omplete unerstning of pil trgeting of proteins wits the ientifition of itionl ftors tht omplement the roles of LET-413. Our moel for the regultion of juntionl integrity in C. elegns y LET-413, DLG-1 n AJM-1 iffers signifintly from the roles propose for the LAP4 protein Srile n Diss lrge in Drosophil, whih re require for eh other s loliztion n the ontrol of generl epithelil polrity 8. The severe polrity efets use y oth lg n sr mutnts hve me it iffiult to ientify iret trgets of Srile n Diss lrge tht re require for speifi spets of juntionl integrity in Drosophil. The Protein 4.1 fmily memer Corle hs een implite s possile trget of Diss lrge. Corle lolizes to septte juntions n is require for mintining the tightness of the prellulr spe 11. Although the protein is mislolize in lg mutnts, no physil intertion of Diss lrge n Corle hs een oserve; euse Corle loliztion proly requires proper piosl polrity of the juntions, regultion of Corle y Diss lrge oul e iniret 6,38. Our emonstrtion tht AJM-1 is iret ining prtner of DLG-1 tht oes not ffet piosl polrity lrifies the role of proteins of the NATURE CELL BIOLOGY VOL 3 NOVEMBER

8 Diss lrge fmily in regulting the struturl integrity of epithelil juntions. A etter unerstning of the nture n funtion of the pil juntionl omin to whih DLG-1 n AJM-1 lolize in C. elegns will require the ientifition of other ining prtners for these proteins. This in turn shoul she itionl light on the funtion n regultion of epithelil juntions in generl. Methos Strins. The Bristol strin N2 ws use s the wil type n hnle s esrie 39. JJ1136 (un119(e2498::t1)iii; zvex24(un119(+) hmp-1::gfp) ws gift from Jmes Priess. The strins BC2991 (py-18(e364)/et1 III; py-11(e224) let-413(s128) un-42(e270)/et1 V) n BC2511 (py-18(e364)/et1 III; un-60(e677) py-11(e224) sdf35/et1 V) were reeive from the Cenorhitis Genetis Center. jm-1 loning. To ientify the originl jm-1 DNA esignte mh-1, iretionl DNA expression lirry of mixe-stge C. elegns ws onstrute in lgt11 (Promeg Corp., Mison, Wisonsin) with the EoRI NotI restrition sites in orne with the mnufturer s protool. The originl titre of this lirry ws plque-forming units ml 1, with 97% of the phge ontining inserts rnging from 0.5 to 2.0 k; the verge size of insert ws 1.2 k. Clones otine y sreening the lgt11 DNA lirry y DNA hyriiztion were igeste with SfiI n NotI, gel-purifie n ligte into pgem- 11Zf igeste with the sme enzymes. RT PCR ws performe using Supersript II regents (Gio BRL, Rokville, Mryln). Poly(A) + RNA from mixe-stge N2 nimls ws use s templte for firststrn synthesis. SL-1 primer n poly(t) primer were use for first-strn synthesis for the 5 en n 3 en, respetively. Exons 2 n 4 were shown to e lterntive SL-1-trns-splie trnsription strt sites. The thir 5 strt site ws ientifie y sequening of the EST yk2852; it extens into exon 1, whih is ontine in H08J11 (ll Kohr DNAs were gifts from Y. Kohr). One 3 en ws ientifie y sequening of the ESTs yk2811 n yk2852; oth exten into puttive 3 untrnslte region (UTR) fter exon 9. The seon 3 en ws ientifie y sequening of RT PCR lones n the ESTs yk344g1, yk1613 n yk152f7; ll lones exten into puttive 3 UTR fter exon 20. Northern lot nlysis ws performe essentilly s esrie previously 40. Poly(A) + RNA of mixe-stge N2 worms ws use n northern lots were proe with proes speifi to exon 1, exons 5 8, the preite 3 UTR of exon 9, n exons Northern sizes were in greement with oneptul DNAs erive from RT PCR lones n sequene ESTs (t not shown). jm-1 mutnt isoltion. jm-1(j18) ws isolte in linke lethl sreen. un-27 n py-6 mp within 0.5 mp unit from the jm-1 gene lous on hromosome X; un-27(e155)/py-6(e14) L4 nimls were mutgenize with ethyl methyl sulphonte, n 5000 F 1 progeny were single n sreene for the sene of the py-6 phenotype in the F 2 genertion. Initil positives were re-sreene for e emryos n the remining positives were teste for the sene of AJM-1 expression y stining with. jm-1(ok160) ws isolte in PCR-se eletion sreen performe y the C. elegns Knokout Consortium in Oklhom s esrie previously 41. An externl primer pir (5 -ACTATTTCGCACCTTGGCTC-3 n 5 -ATAGTGATGACGGTGGG-3 ) spnning 3.5 k n neste pir (5 -TACCGACCGAGAAGT- GATGAC-3 n 5 -ACAGGAGTCATGCGGATTTC-3 ) spnning 3.1 k were use. The eletion rekpoints of jm-1(ok160) on osmi C25A11 re 6232 n Eletron mirosopy. For immuno-tem of lrve with, post-emeing immuno-tem proeures were onute on nimls fixe in uffere lehyes n emee in LR Gol resin 42,43. ntioy (1/500 to 1/2000 ilution) ws lolize on thin setions on nikel mesh gris with gol-lelle seonry ntioies (AuroProe; Amershm Life Siene). For nlysis of hypoerml juntions in wil-type n jm-1 mutnts, emryos were mounte on thin gr p n immoilize with low-melting-point geltin gr uner overslip. A smll piee of gr ontining the emryos ws ut out of the mount n frozen t high pressure in Blzer HPM high-pressure freezer. After freeze sustitution in 2% OsO4, 1% urnyl ette, smples were emee in Epon n nm setions were post-stine with urnyl ette n le itrte. Setions were viewe on Philips CM 120 eletron mirosope t kv elerting voltge. For mutnt nlysis, strin homozygous for jm-1(ok160) n resue y n extrhromosoml rry ontining jm-1::gfp ws use. Mutnts were ientifie y onfol mirosopy to sore the loss of jm-1::gfp n stge with Nomrski optis. RNAi. Templtes for RNA synthesis for RNAi experiments were generte y PCR-mplifying DNAs from pbluesript vetor (Strtgene, L Joll, Cliforni). Primers flnking T3 n T7 sites were use (5 - TTGTAAAACGACGGCCAG-3 n 5 -CATGATTACGCCAAGCTC-3 ) n trnsription in vitro ws performe with Amion Megsript regents (Amion, Austin, Texs). yk2811 ws use for jm- 1(RNAi); yk25e5 ws use For lg-1(rnai) n yk5247 ws use for let-413(rnai). In eh se, oule-strne RNA t onentrtion of 1 4 mg ml 1 ws injete into the oy vity of ult worms 32. jm-1::gfp n lg-1::gfp expression nlysis. The jm-1::gfp lone (pjs191) ws onstrute y loning right mutnt GFP (S65A, V68L, S72A) in frme into exon 8 of genomi lone ontining exons 2 9. Trnsgeni lines were generte with prf4 (rol(su1006)) 44 s o-injetion mrker, n the rry ws integrte into hromosome IV of wil-type worms to rete the integrte rry jis1 (strin SU93). jis1 ws rosse into the jm- 1(ok160) mutnt kgroun n shown to resue mutnts to viility. jis1 ws use to etermine the AJM-1 expression pttern. To ress the requirement of mino is for AJM-1 funtion, n oligonuleotie enoing stop oons in ll reing frmes ws lone into BglII site in exon 3 of pjs191, essentilly eliminting exon 2 s trnsription strt n llowing expression only from exon 4. To generte strin of nemtoes expressing lg-1::gfp using its own promoter (strin OR113), we rete the free rry oex33(lg-1::gfp; rol(su1006)). The plsmi CR16, GFP trnsltionl fusion t the C terminus of genomi lg-1, ws me y loning 5-k XhoI/ApI frgment from the osmi C25F6 into the GFP vetor PD95.75 (Any Fire). Live imging of AJM-1 GFP n DLG-1 GFP ws performe vi utomte time-lpse multiphoton lser snning mirosopy, essentilly s esrie previously 13. Three-imensionl stks ontining slies spe 1 µm prt were tken every 5 min over severl hours. Z-projetions of the stks were one with NIH Imge softwre. Antioies n immunostining. Antioy stining of emryos ws performe with the freeze-rking metho essentilly s esrie previously 18. The following primry ntioies were use t the ilutions inite: t 1:500 n MH46 t 1:100 (gifts from R. Wterston), R224 t 1:100 ( gift from A. Kgw), nti-hmp-1 t 1:100 ( gift from J. Priess) nti-dlg t 1:3000 (gift from V. Bunik), nti-par-3 t 1:40 n nti- PAR-6 t 1:20 (gifts from K. Kemphues), nti-sma-1 t 1:125 ( gift from J. Austin) n nti-unc-70 t 1:500 ( gift from V. Bennett). Phlloiin stining ws performe s previously esrie 18. Imges were pture on Bio-R MRC1024 onfol mirosope. Yest two-hyri nlysis. The yest two hyri-sreen ws performe with yest strin L40ur s previously esrie 45.In rief, the plexa AJM-1 plsmi ws onstrute with prtil jm-1 DNA enoing mino is L40ur yest ells ontining this plsmi were trnsforme with rnom-prime DNA lirry, LmACT-RB-2. The lirry trnsformtion ws plte on SD-Trp-Leu-His pltes ontining 5 mm 3-minotrizole. Colonies were pike fter 6 ys n teste for reporter (lz) expression. The DNA-ontining plsmis of those testing positive were reovere n sequene. The speifiity of the intertions ws resse in irete ssys y introuing plexa AJM-1 into L40 together with reovere DNA plsmis, n for negtive ontrols, with the empty pgl4 (Gl4 tivtion omin) plsmi lone or pgl4 IRP (Iron Response Protein). In eh se, lz expression ws ssye in triplite using X-Gl olony filter ssy s esrie previously 45. In irete two-hyri tests, pbtmkndb n pact2 were use s vetors for the LexA n Gl4 tivtion omin (Gl4) plsmis, respetively (gifts from M. Wikens). GST pull-own ssy. The GST DLG-1 fusion ws generte y loning lg-1 DNA (enoing mino is ) into pgex-5x-1 n introue into the E. oli strin BL21(DE3) (Strtgene, L Joll, CA). GST ws generte using empty pgex-5x-1. Bteri were grown in TB plus 2% Gluose to log phse t 37 C, inue with 0.1 mm isopropylthiogltosie for 2 h t 25 C n hrveste. Cells were lyse with B- PER regent (Piere, Rokfor, Illinois), with Complete protese inhiitor oktil (Boehringer- Mnnheim, Ininpolis, Inin), in orne with the mnufturers iretions. Reominnt protein ws oun to glutthione Sephrose es (Amershm Phrmi Bioteh, Pistwy, New Jersey) n repetely wshe with uffer A (20 mm Tris-HCl ph 7.9, 0.2 mm EDTA, 0.1 M NCl, 1 mm ithiothreitol, 0.2% Noniet P40, n Complete protese inhiitor oktil). Riolelle AJM-1 protein (mino is 1 868) ws generte y in vitro trnsltion with the TNT Couple Retiuloyte Lyste System (Promeg). The Kohr lone yk2852 ws use s templte. For the protein ining ssys in vitro, lelle AJM-1 ws e to equivlent quntities of es rrying GST DLG-1 or GST lone. The quntities of ouple protein were similr for eh protein, s juge y stining of the proteins fter SDS PAGE. Riolelle protein ws inute with es in uffer A t 4 C, with roking. Bes were pellete, wshe four times in uffer A n elute y oiling in SDS PAGE smple uffer. Elute lelle protein ws nlyse y SDS PAGE. An equivlent of 20% of the lelle AJM-1 use in the ining ssys ws run s omprison. RECEIVED 26 JANUARY 2001; REVISED 10 JULY 2001; ACCEPTED 9 AUGUST 2001; PUBLISHED 8 OCTOBER Guminer, B. M. Cell hesion: the moleulr sis of tissue rhiteture n morphogenesis. Cell 84, (1996). 2. Tepss, U. Geneti nlysis of herin funtion in niml morphogenesis. Curr. Opin. Cell Biol. 11, (1999). 3. Willott, E. et l. 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J. Cell Biol. 140, (1998). 39. Brenner, S. The genetis of Cenorhitis elegns. Genetis 77, (1974). 40. Smrook, J., Fritsh, E. F. & Mnitis, T. Moleulr Cloning, A Lortory Mnul (Col Spring Hror Lortory, Col Spring Hror, New York, 1989). 41. Dernurg, A. F. et l. Meioti reomintion in C. elegns initites y onserve mehnism n is ispensle for homologous hromosome synpsis. Cell 94, (1998). 42. Selkirk, M. E., Gregory, W. F., Yznkhsh, M., Jenkins, R. E. & Mizels, R. M. Cutiulr lolistion n turnover of the mjor surfe glyoprotein (gp29) of ult Brugi mlyi. Mol. Biohem. Prsitol. 42, (1990). 43. Hll, D. H. Eletron mirosopy n three-imensionl imge reonstrution. Methos Cell Biol. 48, (1995). 44. Mello, C. & Fire, A. DNA trnsformtion. Methos Cell Biol. 48, (1995). 45. Brtel, P. L. & Fiels, S. The Yest Two-hyri System (Oxfor Univ. Press, New York, 1997). ACKNOWLEDGEMENTS We thnk memers of the Hrin lortory for helpful isussion n ritil reing of the mnusript, J. Pwley for help with onfol mirosopy, n the Wikens lortory, in prtiulr D. Bernstein, for tehnil ssistne with two-hyri experiments n the GST pull-own ssy. This work ws supporte y NSF grnt DBI n NIH grnt GM58038 wre to J.D.H. The Bio- R MRC 1024 onfol mirosope is supporte y NSF grnt Corresponene n requests for mterils shoul e resse to J.D.H. The GenBnk ession numers for the C25A11.4 (jm-1) sequene n the lg-1 sequene re U39650 n AJ295228, respetively. NATURE CELL BIOLOGY VOL 3 NOVEMBER