Molecular Cell, Vol. 4, 893–902, December, 1999, Copyright 1999 by Cell Press

Presenilin-1 Forms Complexes with theCadherin/Catenin Cell–Cell Adhesion System and IsRecruited to Intercellular and Synaptic Contacts

the endoplasmic reticulum (ER), Golgi, and probablyin vesicles (Cook et al., 1996; Kovacs et al., 1996; DeStrooper et al., 1997; Efthimiopoulos et al., 1998). Mostcellular full-length PS1 (FLPS1) is cleaved within thelarge cytoplasmic loop to yield N-terminal fragments

Anastasios Georgakopoulos,1 Philippe Marambaud,1

Spiros Efthimiopoulos,1 Junichi Shioi,1 Wen Cui,1

Heng-Chun Li,2 Michael Schutte,3 Ronald Gordon,4

Gay R. Holstein,5 Giorgio Martinelli,6 Pankaj Mehta,7

Victor L. Friedrich, Jr.,2 and Nikolaos K. Robakis1,8

1Department of Psychiatry and (PS1/NTF) of approximately 30 kDa and C-terminal frag-ments (PS1/CTF) of approximately 20 kDa. FollowingFishberg Research Center for Neurobiology

2Department of Biochemistry and Molecular Biology cleavage of the full-length protein, PS1 fragments staytogether as a stable 1:1 heterodimer (Thinakaran et al.,3Department of Ophthalmology

4Department of Pathology 1996; Podlisny et al., 1997).Recently it was shown that PS1 binds members of the5Department of Neurology and Cell Biology

6Department of Surgery armadillo family of proteins including d- and b-catenin(Zhou et al., 1997; Yu et al., 1998) and promotes pro-Mount Sinai School of Medicine

New York, New York 10029 cessing and signaling of Notch1 receptor, suggesting aPS1 role in development (De Strooper et al., 1999; Struhl7Institute for Basic Research in Developmental

Disabilities and Greenwald, 1999; Ye et al., 1999). Other studiessuggest that PS1 functions in protein trafficking (Efthimi-1050 Forest Hill Road

Staten Island, New York 10314 opoulos et al., 1998; Naruse et al., 1998; Nishimura etal., 1999), neuroprotection (Giannakopoulos et al., 1997;Shen et al., 1997), chromosome segregation (Li et al.,1997), and processing of selected proteins includingSummaryAPP (De Strooper et al., 1998; Wolfe et al., 1999). Theo-ries proposed to explain the mechanism by which PS1In MDCK cells, presenilin-1 (PS1) accumulates at inter-

cellular contacts where it colocalizes with compo- mutations induce FAD include increased production ofAb, destabilization of b-catenin, inhibition of PS1 prote-nents of the cadherin-based adherens junctions. PS1

fragments form complexes with E-cadherin, b-cate- olysis, and increased apoptosis (for review, see Selkoe,1999).nin, and a-catenin, all components of adherens junc-

tions. In confluent MDCK cells, PS1 forms complexes Classical cadherins, including E- and N-cadherin, area family of cell surface single-pass transmembrane pro-with cell surface E-cadherin; disruption of Ca21-depen-

dent cell–cell contacts reduces surface PS1 and the teins that control critical events in cell–cell adhesion,recognition, and tissue development. These functionslevels of PS1-E-cadherin complexes. PS1 overexpres-

sion in human kidney cells enhances cell–cell adhe- are mediated by the extracellular domain of cadherins,which in the dimeric state promotes Ca21-dependentsion. Together, these data show that PS1 incorporates

into the cadherin/catenin adhesion system and regu- homophilic interactions between same class cadherinson opposing cell surfaces (for review, see Yap et al.,lates cell–cell adhesion. PS1 concentrates at intercel-

lular contacts in epithelial tissue; in brain, it forms 1997). Cadherin-based adherens junctions are special-ized forms of cellular adhesive contacts at which plas-complexes with both E- and N-cadherin and concen-

trates at synaptic adhesions. That PS1 is a constituent malemmal classical cadherins form complexes with cy-tosolic catenins linked to the cortical actin cytoskeleton.of the cadherin/catenin complex makes that complex

a potential target for PS1 FAD mutations. According to this model, the conserved cytoplasmicsequence of cadherin binds to either b- or g-catenin,which in turn binds to a-catenin. The latter proteinIntroductionbinds to a-actinin and g-actin, thus linking the cadherin/catenin system to the cortical actin cytoskeleton (Naga-Presenilin-1 (PS1) mutations are responsible for mostfuchi and Takeichi, 1988; Aberle et al., 1994; Knudsen etcases of early-onset autosomal dominant familial Alzhei-al., 1995; Rimm et al., 1995). Linkage to the cytoskeletonmer’s disease (FAD). PS1 protein is a polytopic trans-increases resistance of the cadherin/catenin complexesmembrane peptide expressed in many tissues includingto extraction with nonionic detergents (Hinck et al., 1994;brain, where it is enriched in neurons (Sherrington et al.,Nathke et al., 1994). In confluent MDCK cells, the cad-1995; Elder et al., 1996). Structural studies suggest thatherin-based intercellular junctions form a continuous beltPS1 crosses the membrane six or eight times with theat the lateral plasma membrane where neighboring cellsN and C termini and the large hydrophilic loop followingcontact each other, whereas they are excluded from thethe sixth transmembrane (TM) sequence, all located inapical plasmalemma (Reinsch and Karsenti, 1994; Yapthe cytoplasm. This topology predicts that only the rela-et al., 1997). Recent reports show that cadherins andtively short segments following TM domains 1, 3, 5, andassociated catenins are found in synaptic junctions ofpossibly 7, are located in the lumen (Doan et al., 1996;the CNS, where they are thought to link pre- and post-Lehmann et al., 1997). PS1 has been mainly localized insynaptic membranes (for review, see Uemura, 1998).

Here we report that in MDCK cell cultures, PS1 is8 To whom correspondence should be addressed (e-mail: n.robakis@smtplink.mssm.edu). localized at cell–cell adhesion sites, forms complexes

Molecular Cell894

Figure 1. PS1 Immunofluorescence Staining of MDCK Cells

(A) Antibody 222. Affinity-purified polyclonal Ab222 (see Experimental Procedures) against PS1/NTF was used to probe cell extracts from:nontransfected (lane 1) or PS1-transfected (lane 2) HEK293 cells, fibroblast cells derived from wild-type (PS11/1, lane 3) or PS12/2 (lane 4)mice (De Strooper et al., 1998), and MDCK cells (lane 5). Lane 6, MDCK extract stained with Ab222 plus corresponding peptide antigen. FL,full-length PS1; NTF, PS1/NTF.(B) Fibroblast cells from PS11/1 (a) or PS12/2 (b) mice were stained with affinity-purified Ab222. (c and d) Phase contrast images.(C) Confluent MDCK cells stained either with affinity-purified Ab222 (a) or with Ab222 plus corresponding peptide (b). Scale bar, 30 mm.

with components of the cadherin/catenin adhesion sys- along sites of cell–cell apposition and verified the ab-sence of immunoreactivity from both apical and basaltem, and is found at the boundaries between corneal

epithelial cells, suggesting it functions in tissue cell–cell surfaces (Figures 2Ak and 2Bk). This distribution patternsuggested that cell–cell contact might be necessary foradhesion. Overexpression of PS1 in human kidney cells

stimulates cell adhesion. PS1 fragments were found in the surface accumulation of PS1. Indeed, in subcon-fluent cultures, PS1 was strongly concentrated at sur-complexes with brain cadherins, suggesting a PS1 func-

tion in brain cell–cell adhesion. faces directly apposed to other cells but mostly absentfrom surfaces free of intercellular contacts (Figures 2Caand 2Cd).Results

The above data indicate that PS-1 is recruited to sitesof intercellular contact, where MDCK cells form ad-PS1 Concentrates at Cell–Cell Contact Sites,herens junctions (Yap et al., 1997). We then comparedWhere It Colocalizes with E-Cadherinthe distribution of PS-1 with components of the cad-and Cateninsherin-based adherens junctions, including E-cadherin,

We examined the subcellular localization of PS1 inb-catenin, and a-catenin (Ozawa and Kemler, 1992; Ab-

MDCK cells by immunostaining with affinity-purifiederle et al., 1994; Hinck et al., 1994). As expected, both

anti-PS1 antibody 222 (Ab222). This antibody specifi-E-cadherin and b-catenin were concentrated at the lat-

cally recognizes the predicted PS1/NTF in extracts fromeral plasma membrane of confluent cells and were ab-

wild-type (PS11/1) cells but does not bind to extractssent from apical and basal surfaces (Figures 2Af–2Aj

from PS1 knockout (PS12/2) cells (Figure 1A, lanes 3and 2Al, and 2Bf–2Bj and 2Bl). In two-color immuno-

and 4). Ab222 recognizes both FLPS1 and NTF/PS1 influorescence, PS1 colocalized with E-cadherin and

PS1-transfected cells (Figure 1A, lanes 1 and 2) andb-catenin both in confluent and subconfluent cultures;

stains cells derived from PS11/1 mice but not cells de-that is, PS-1, E-cadherin, and b-catenin were concen-

rived from PS12/2 mice (Figure 1B). In confluent cultures,trated together at sites of intercellular contact but were

PS1 showed strong immunofluorescence in circumfer-mostly absent from contact-free surfaces (Figures 2A–

ential rings around each cell at sites of cell–cell contact.2C). PS1 and a-catenin also showed a similar pattern

Weaker PS1 staining was also observed in the perinu-of colocalization (data not shown). For higher resolution,

clear cytoplasm (Figure 1Ca). Pretreatment of Ab222we performed immunogold electron microscopy (EM)

with the corresponding peptide abolished all immuno-of MDCK cells. Morphologically identifiable intercellular

staining (Figure 1Cb), whereas treatment with secondaryjunctions (Boller et al., 1985; Volk and Geiger, 1986) were

antibody only or preimmune serum yielded no stainingclearly immunolabeled with PS1 antibody (Figure 2D).

(data not shown). Identical patterns of immunostainingwere obtained with anti-PS1/NTF antibodies 36097 and52198, and anti-PS1/CTF antibody C-20 (data not PS1 Forms Complexes with Components

of the Adherens Junctions Systemshown; see Experimental Procedures). To further char-acterize PS-1 localization, we examined specimens by In confluent epithelial cells, E-cadherin is found in deter-

gent-insoluble as well as detergent-soluble complexeslaser scanning confocal microscopy. Serial optical sec-tions parallel to the substrate showed PS1 distributed with other adherens junctions components, including

b- and a-catenin (Ozawa and Kemler, 1992; Hinck et al.,extensively along the lateral surfaces of cells but absentfrom basal and apical surfaces (Figures 2Aa–2Ae and 1994). To test for a possible interaction between PS1

and these junctional components, digitonin extract from2Ba–2Be). X–Z scans perpendicular to the substrateshowed surface immunoreactivity distributed vertically confluent MDCK cells was treated with Ab222, and the

PS1 Is a Component of Adherens Junctions895

Figure 2. Confocal and Electron Microscopy of MDCK Cultures

Laser scanning confocal micrographs of confluent MDCK cells double stained with anti-PS1 Ab222 (Aa–Ae and Ak) and anti-E-cadherinmonoclonal (Af–Aj and Al) or Ab222 (Ba–Be and Bk) and anti-b-catenin monoclonal antibody (Bf–Bj and Bl). Sets (a)–(e) and (f)–(j) representserial scans parallel to and at different distances from the substrate. (k)–(m) represent X–Z scans oriented perpendicular to substrate. (m)Superimposed images of (k) and (l); yellow represents PS-1 colocalization with E-cadherin (A) or b-catenin (B). Scale bars, 20 mm. Verticalspacing (Aa–Ae, Af–Aj, Ba–Be, and Bf–Bj), 1.2 mm. (C) Subconfluent MDCK cells double immunostained for PS-1 (a) and E-cadherin (b) orPS1 (d) and b-catenin (e). (c and f) Phase contrast images. Arrows, free surfaces without PS1. Scale bar, 20 mm. (D) Transmission electronmicrograph of confluent MDCK cells fixed (see Experimental Procedures) and treated with affinity-purified Ab222 followed by anti-rabbit IgGcoupled to 10 nm collodial gold. Gold particles representing PS1 localization are seen at an intercellular junction. No labeling was obtainedwith preimmune serum (not shown). Scale bar, 100 nm.

resulting immunoprecipitates (IPs) were analyzed on digitonin was immunoprecipitated quantitatively withanti-E-cadherin antibodies, and the supernatant wasWestern blots (WBs). Figure 3A (lane R222) shows that

in addition to PS1/NTF, these IPs contained E-cadherin, precipitated with anti-b-catenin antibodies. A similar ali-quot was treated first with anti-b-catenin antibodies andb-catenin, a-catenin, and PS1/CTF. IPs prepared with

monoclonal antibody 33B10, specific for PS1/CTF (see then with anti-E-cadherin antibodies. All IPs were thenprobed in WBs for PS1 fragments. Figure 3C (lanes 4–6)Experimental Procedures and Figure 3B for character-

ization of 33B10), also contained E-cadherin, b-catenin, shows that b-catenin antibodies immunoprecipitated allE-cadherin-PS1 complexes. In contrast, E-cadherin an-a-catenin, and PS1/NTF (Figure 3A, lane 33B10). In re-

verse experiments, both PS1/NTF and PS1/CTF were tibodies immunoprecipitated most, but not all, b-catenin-bound PS1 fragments (Figure 3C, lanes 1–3). These datafound in IPs prepared either with anti-E-cadherin or anti-

b-catenin antibodies. Other transmembrane proteins, show that all E-cadherin-PS1 complexes also containb-catenin, but there is also a pool of E-cadherin-inde-including desmoglein of the desmosomal adhesion

complex and transferrin receptor (TR), were not found pendent b-catenin-PS1 complexes. Densitometry of thesignals in our gels showed that about one-third of thein our IPs (Figure 3A). These results indicate that PS1

fragments form complexes with components of the PS1-b-catenin complexes contain no E-cadherin.The size of the PS1-cadherin-catenin complexes wascadherin/catenin adhesion system, including E-cad-

herin, b-catenin, and a-catenin. We observed that levels determined in a linear velocity glycerol gradient (Pindet al., 1994). Both PS1 fragments migrated together andof PS1 were much higher in E-cadherin and b-catenin

IPs than in a-catenin IPs (data not shown). Since both peaked at about 400 kDa. E-cadherin, b-catenin, andlow amounts of a-catenin were also present in the PS1PS1 and a-catenin bind b-catenin (Hinck et al., 1994;

Yu et al., 1998), the low yield of the PS1-a-catenin com- peak (Figure 3D). Immunoprecipitation of this fractionwith Ab222 coimmunoprecipitated PS1/CTF, E-cad-plexes (see also Figure 3A; d, e, and f), suggests that

PS1 and a-catenin may be held together indirectly, by herin, b-catenin, and a-catenin (Figure 3E). The last threeproteins, which collectively have a molecular mass ofbinding to b-catenin, rather than by binding directly to

each other. about 310 kDa, are components of the cadherin/catenincell adhesion apparatus and form 1:1:1 complexesTo examine whether all three proteins are present in

a single complex, an aliquot of MDCK cell extract in (Ozawa and Kemler, 1992; Aberle et al., 1994). Since the

Molecular Cell896

Figure 3. PS1 Forms Complexes with Com-ponents of the Adherens Junctions

(A) PS1 fragments coimmunoprecipitate withE-cadherin, b-catenin, and a-catenin. Extractfrom confluent MDCK cells in 1% digitoninwere treated with the antibodies indicated atthe top of the figure, and the resulting IPswere probed on WBs with antibodies againstthe proteins indicated on the right of the fig-ure. In the lane marked R2221Pep, extractswere immunoprecipitated with Ab222 pre-treated with the corresponding peptide. Forreference, 10 mg of MDCK cell lysate wasprobed with the indicated antibodies. Thetime of film exposure in a, b, and c was fivetimes less than in d, e, and f. TR, transferrinreceptor.(B) Antibody 33B10. Mouse monoclonal anti-body 33B10 recognizing PS1/CTF was usedto probe extract from: nontransfected (lane1) or PS1-transfected (lane 2) HEK293 cells,fibroblast cells from wild-type (PS11/1, lane3) or PS12/2 (lane 4) mice, or MDCK cells (lane5). Lane 6, MDCK extract stained with 33B10plus corresponding antigen. FL, full-lengthPS1; CTF, PS1/CTF.(C) PS1 fragments, E-cadherin, and b-cateninform a single complex. A sample of MDCKcell extract in 1% digitonin was treated withanti-E-cadherin antibodies, and the IP wasplaced on lane 1. The resulting supernatant

was treated again with anti-E-cadherin antibodies, and the IP was placed on lane 2 and then with anti-b-catenin antibodies, and the IP wasplaced on lane 3. An identical aliquot was treated first with anti-b-catenin antibodies, and the IP was placed on lane 4. The supernatant wastreated again with anti-b-catenin antibodies, and the IP was placed on lane 5 and then with anti-E-cadherin antibodies, and the IP was placedon lane 6. All samples were then tested for PS1/NTF and PS1/CTF on WBs.(D) PS1 fragments, E-cadherin, and b-catenin comigrate as a high–molecular weight complex. MDCK cell extract in 1% digitonin was separatedin a linear 10%–40% glycerol velocity centrifugation gradient, and collected fractions were analyzed on WBs with antibodies indicated at theleft side of the figure. Fraction 12 corresponds to the top of the gradient. Arrows at the bottom indicate mobility of protein markers.(E) Peak PS1 fraction 5 was immunoprecipitated either with Ab222 (R222) or Ab222 plus corresponding peptide (R2221Pep), and the resultingIPs were analyzed on WBs with antibodies indicated at the left of the figure.(F) PS1 associates with cell surface E-cadherin. Polarized MDCK cells on polycarbonate filters were labeled in the basolateral surface eitherwith anti-E-cadherin antibody DECMA-1 or with biotin. DECMA-1-labeled cells were solubilized in 1% digitonin, and immunocomplexes weredissolved in Laemmli buffer and placed on lane 1. An aliquot of the biotin-labeled cells was solubilized in 1% digitonin, and the labeled surfaceprotein was precipitated with streptavidin, dissolved in Laemmli buffer, and placed in lane 4. A similar aliquot from the biotin-labeled cellswas dissolved in 1% SDS and placed in lane 3. Lane 2, total cell extract.

molecular mass of PS1 is about 50 kDa, the 400 kDa 4). In the SDS isolates, however, only E-cadherin wasdetected (Figure 3F, lane 3), suggesting that PS1 frag-complex may incorporate additional proteins and/or

multiple copies of some components (see also Hinck et ments were not labeled directly but were precipitatedas part of a complex with cell surface protein. Our at-al., 1994).tempts to label surface PS1 using antibody 519 (DeStrooper et al., 1997) directed against a lumenal PS1PS1 Forms Complexes with Cell Surface E-Cadherin

Live confluent MDCK cells were treated with anti-E- sequence were also unsuccessful (data not shown).cadherin antibodies at 48C, and following homogeniza-tion in digitonin, immunolabeled surface E-cadherin wasprecipitated with a mixture of proteins A and G. The IPs Cell Surface PS1 Depends on Ca21-Induced Cell–Cell

Contacts and Is Resistant to Detergent Extractioncontained E-cadherin, PS1/NTF, PS1/CTF, b-catenin,and a-catenin but not the ER protein calnexin (Figure In MDCK cell cultures, Ca21-dependent intercellular

binding triggers accumulation of adherens junction3F, lane 1). Neither uncleaved E-cadherin, which is foundonly intracellularly (Shore and Nelson, 1991), nor TR components, including E-cadherin, at cell–cell contact

sites. Removal of extracellular Ca21 induces disruptionwas found in these IPs (data not shown). These resultssuggest that PS1 fragments form complexes with cell of intercellular junctions and movement of E-cadherin

from the cell surface to the cytoplasm (Contreras et al.,surface E-cadherin. We then attempted to directly labelsurface PS1 using biotin. Surface-labeled cells were ho- 1992; Rajasekaran et al., 1996). To determine whether

the presence of PS1 at the cell surface also dependsmogenized in either digitonin or SDS, and biotinylatedprotein was collected with streptavidin. The digitonin on Ca21, confluent MDCK cultures grown in the presence

of Ca21 were switched to Ca21-free media. After 20 hr,isolates contained E-cadherin, PS1/NTF, PSN/CTF, b-cat-enin, and a-catenin but no calnexin (Figure 3F, lane cells lost intercellular contacts and became rounded. At

PS1 Is a Component of Adherens Junctions897

Figure 4. PS1 Localization at Intercellular Contacts Depends on Extracellular Ca21 and Is Resistant to Detergent Extraction

(A) Effects of Ca21-induced cell–cell adhesion on PS1 localization. Confluent MDCK cells grown in the presence of 1.8 mM CaCl2 were stainedwith Ab222 (a) or anti-E-cadherin antibody (d). Cultures were then switched to Ca21-free medium for 20 hr and examined again for PS1 (b) orE-cadherin (e) staining. Following that, medium CaCl2 was adjusted back to 1.8 mM, and cells were examined again after 5 hr for PS1 (c) orE-cadherin (f) staining.(B) Extract from confluent MDCK cultures grown as above were immunoprecipitated with the antibodies shown at the top of the figure, andthe IPs were examined on WBs using antibodies indicated at the right of the figure. Lysates were used to probe for total cellular E-cadherinor PS1/NTF. The blot probed for E-cadherin was overexposed to visualize the weak signal in lane 33B10(2). Scale bar, 50 mm.(C) Triton extraction of PS1. MDCK cells were extracted with CSK buffer plus 0.5% Triton X-100. Nonextracted (a and c) or extracted (b andd) cultures were treated either with anti-PS1 Ab222 (a and b) or anti-E-cadherin antibody (c and d) and examined for immunofluorescence.Scale bar, 30 mm.

the same time, PS1 and E-cadherin staining disap- cell–cell boundaries of epithelial cells (Figure 5Aa). Atthis resolution, most labeling appeared at the cell sur-peared from the cell surface and became mostly cyto-face with little staining in the cytoplasm. Treatment withplasmic (Figures 4Ab and 4Ae). Removal of extracellularpreimmune serum showed no specific labeling (Fig-Ca21 also caused a significant reduction in PS1-E-cad-ure 5Ab).herin complexes, although the total cellular levels of these

Recently E- and N-cadherin were found in the mam-proteins did not change appreciably (Figure 4B). Addi-malian CNS, and components of the cadherin/catenintion of Ca21 restored cell–cell contacts and induced thecomplex were localized at the neuronal synapse (Uem-reappearance of both PS1 and E-cadherin staining atura, 1998). PS1 IPs from mouse brain homogenates con-cell adhesion sites (Figures 4Ac and 4Af). In confluenttained E-cadherin, N-cadherin, and b-catenin, but notMDCK cells, a fraction of the surface cadherin/catenindesmoglein or TR (Figure 5B). None of the above pro-complex is linked to the actin cytoskeleton, renderingteins were detected when homogenates were treatedits component proteins, including E-cadherin, resistantwith antibodies preabsorbed with the correspondingto extraction with Triton X-100. In contrast, most or allpeptide (data not shown). Since PS1 is localized in neu-of the cytoplasmic E-cadherin is soluble in this detergentronal processes (Elder et al., 1996), our data suggest that(Nathke et al., 1994). Extraction of confluent MDCK cellsPS1 fragments may form complexes with the synapticwith CSK buffer (Refolo et al., 1991) containing 0.5%cadherin/catenin adhesion system (Uemura, 1998). In-Triton X-100 resulted in a dramatic reduction of cyto-deed, EM immunoperoxidase and immunogold stainingplasmic PS1 staining, while significant staining of thisshow PS1 antigens concentrated at brain synaptic adhe-protein persisted at established cell–cell contact sitessions (Figure 5C).

(Figure 4C). In agreement with these observations, WBsshowed that more than half of total PS1 remained in the PS1 Stimulates Cell–Cell AdhesionTriton-insoluble fraction (data not shown). These data To further explore the role of PS1 in cell–cell adhesion,suggest that, like other components of the cadherin/ we examined the effects of PS1 overexpression on cellcatenin complex, PS1 at intercellular sites is regulated aggregation using stable PS1 transfectants of humanby Ca21-dependent mechanisms and may be linked to kidney cell line HEK293. These cells express surfacethe cortical cytoskeleton. E-cadherin and contain PS1-E-cadherin (S. E., unpub-

lished observations) and PS1-b-catenin (Yu et al., 1998)PS1 Is Found In Vivo at Intercellular Contacts complexes. Three independent groups were selected,and Synaptic Adhesions each comprising a vector-, a WTPS1-, and PS1 mutantPS1 mRNA has been detected in the cornea and lens D257A–transfected clone. The PS1-transfected clonesepithelia (Frederikse and Zigler, 1998). To investigate expressed comparable levels of exogenous PS1 (datawhether PS1 also localizes in cell–cell contacts in vivo, not shown). In all groups, WTPS1-transfected cellsfrozen sections of whole rat eye were stained with Ab222 showed a 25%–30% increase in aggregation rate com-and examined for PS1 immunofluorescence. In the cor- pared to vector-transfected cells, indicating that overex-

pression of PS1 stimulated cell–cell adhesion (Figureneal epithelium, abundant labeling was observed at the

Molecular Cell898

Figure 6. PS1 Transfectant Cells Show Increased Cell–Cell Aggre-gation

The aggregation rate was assayed using three independent groups(1–3) of vector-, WTPS1-, or mutant PS1-transfected HEK293 clones.Each bar represents the average of six independent experiments 6

SEM. Aggregation was significantly increased by WTPS1 (groups1–3: p , 0.003, 0.001, 0.05) but was not changed by mutant PS1.

at cell–cell contact sites along the length of the lateralplasma membrane in close association with E-cadherinand b-catenin, both of which are components of thecadherin-based adherens junctions (Yap et al., 1997).In nonconfluent cultures, PS1 concentrates only in areasof cell–cell contact and is scant or absent from cellsurface areas free of such contact, suggesting that PS1is recruited specifically at cell–cell adhesion sites. Im-munogold EM localized PS1 at intercellular junctionsalong the lateral plasma membrane, providing additionalsupport for the association of PS1 with adherens junc-tions.

That PS1 concentrates at cell–cell contact sites inclose association with E-cadherin and b-catenin sug-

Figure 5. PS1 Concentrates at Intercellular and Synaptic Contact gests that PS1 may be a part of the cadherin/cateninSites In Vivo adhesion system. In support of this suggestion, we(A) PS1 is localized at intercellular junctions of corneal epithelium. found PS1 fragments in complexes with E-cadherin,(a) Rat cornea was stained with Ab222. The double arrow (20 mm)

b-catenin, and a-catenin, all of which are important com-spans the epithelium; s, stroma. Corneal epithelial cells displayponents of this system. Furthermore, both PS1 frag-strong PS-1 immunoreactivity in areas of reciprocal contact. Arrow-ments form large molecular complexes together withheads indicate examples of multiple cell contact zones. (b) A section

of the same specimen stained with preimmune serum (symbols are E-cadherin, b-catenin, and a-catenin. Thus, detectionas in [a]). A 15 mm frozen section is shown. of complexes containing PS1 and the basic structural(B) Brain PS1 IPs contain E-cadherin, N-cadherin, and b-catenin. components of the cadherin/catenin adhesion systemTotal mouse brain extract was immunoprecipitated with Ab222,

indicates that PS1 is a part of this system. In supportand the isolated IPs were probed on WBs with the indicated anti-of this conclusion, we found PS1 fragments in stablebodies.complexes with cell surface E-cadherin.(C) PS1 immunostaining of synaptic junctions in mouse cerebral

cortex. Both immunoperoxidase (a) and immunogold (b) show prom- Accumulation of components of the cadherin-basedinent staining of postsynaptic densities (arrows). Controls, including adhesion system, including E-cadherin, on the surface(separately) antibody pretreated with cognate peptide, an irrelevant of MDCK cells depends on the presence of Ca21-primary antibody, and secondary antibody, only showed negligible

induced cell–cell contacts (Contreras et al., 1992; Rajase-immunoreactivity. Scale bar, 100 nm.karan et al., 1996). Our experiments show that removalof Ca21 from the growth medium results in reduced cellsurface PS1 and increased cytoplasmic PS1 staining.6). Cells overexpressing PS1 mutant D257A showed no

increased aggregation (Figure 6). Thus, similar to other components of the cadherin/catenin adhesion system, PS1 is recruited to Ca21-inducedcell–cell contact sites. Interestingly, removal of extracel-Discussionlular Ca21 resulted in a specific decrease of the cellularPS1-E-cadherin complexes. Under the same conditions,Here we show that PS1 is a component of the cadherin-

based cell–cell adhesion system, is recruited to inter- we also observed a reduction of the PS1-b-catenin com-plexes (P. M. et al., unpublished data). These data sug-cellular junctions, and functions to modulate cell–cell

adhesion. In confluent MDCK cultures, PS1 is localized gest the presence of signal transduction mechanisms

PS1 Is a Component of Adherens Junctions899

that regulate the formation or stability of PS1–cadherin/ 1997; Discussion in Li and Greenwald, 1998]), to label cellcatenin complexes in response to changes in extracellu- surface PS1, cannot exclude the possibility that PS1 is inlar Ca21 or Ca21-induced cell–cell adhesion. Cadherin/ fact incorporated into the plasma membrane.catenin complexes are present in the Triton X-100 solu- The localization of PS1 at cell–cell contacts in vitro andble and insoluble fractions of confluent MDCK cell ex- in vivo and its association with the cadherin adhesiontract (Hinck et al., 1994). At cell–cell contacts, a signifi- system suggest that PS1 functions in cell–cell adhesion.cant fraction of the components of these complexes, This suggestion is supported by our data that PS1-over-including E-cadherin, is insoluble in Triton X-100, sug- expressing cells displayed a statistically significant in-gesting a linkage to the cortical actin cytoskeleton (Na- crease in cell–cell aggregation compared to vector-gafuchi and Takeichi, 1988; Nathke et al., 1994). Our transfected controls and to cells overexpressing PS1data shows that in confluent MDCK cells, a significant mutant D257A. PS1 residue Asp257 is considered func-fraction of PS1 at cell–cell contact sites is resistant to tionally important as it is conserved in many speciesTriton X-100 extraction, suggesting that, similar to other and the D257A mutation inhibits PS1 endoproteolysiscomponents of the cadherin/catenin adhesion system, (Wolfe et al., 1999). Accordingly, transfectant PS1D257APS1 at these sites is linked to the cortical cytoskeleton. was not cleaved in HEK293 cells (data not shown). The

In summary, this report presents multiple lines of evi- inability of this mutant to increase cell aggregation sug-dence indicating that PS1 is an integral component of gests that a functional PS1 is necessary for stimulationthe cadherin-based adherens junctions: Confocal mi- of cell adhesion. Since PS1 functions in the traffickingcroscopy showed PS1 at cell–cell contacts on the lateral

and/or processing of several proteins (see Introduction),plasma membrane in close proximity with E-cadherin

the PS1 effects on cell aggregation may reflect a PS1and b-catenin. Immunogold EM localized PS1 at inter-participation in the transport of components of the cad-cellular junctions. PS1 forms complexes with severalherin/catenin complex to the cell surface, a PS1 role incomponents of the cadherin/catenin adhesion system,the processing and maturation of E-cadherin (Shore andincluding cell surface E-cadherin. Similar to E-cadherin,Nelson, 1991), or a novel PS1 function in cell adhesion.cell surface localization of PS1 depends on the presence

HEK293 cells contain no detectable Notch1 receptorof Ca21-induced cell–cell contacts. Similar to other com-(Ray et al., 1999) and, in our cultures, PS1 overexpres-ponents of the cadherin-based adhesion system, a frac-sion had no effect on Notch1 (data not shown), sug-tion of PS1 at cell–cell contacts is linked to the cytoskel-gesting that PS1 effects on cell–cell adhesion are proba-eton, and PS1 is found in intercellular contacts ofbly not mediated by Notch1 signaling. Recent reportsepithelial tissue in vivo.suggest that PS1 mediates processing of Notch1, a sur-It is not clear whether PS1 binds directly to E-cad-face receptor involved in cell–cell interactions duringherin; their interaction might be mediated by other com-development (Artavanis-Tsakonas et al., 1999; Deponents of the cadherin/catenin system. b-catenin is aStrooper et al., 1999; Ye et al., 1999). This suggestion,potential mediator of the PS1-E-cadherin complex be-however, is inconsistent with the intracellular localiza-cause it binds both PS1 and E-cadherin. However, undertion of PS1 (Hardy and Israel, 1999). Our data that PS1certain experimental conditions we observed PS1-E-is recruited to intercellular contact sites suggests thatcadherin complexes in the absence of b-catenin (P. M.

et al., unpublished data), suggesting a direct PS1-E- PS1 processes Notch1 at the plasma membrane at sitescadherin interaction. Thus, PS1 at intercellular contacts of cell–cell contact and could therefore constitute partmay bind both E-cadherin and b-catenin. The latter pro- of the Notch signaling system.tein may then mediate the linkage of cell surface PS1 Recently, the cadherin/catenin system was localizedto a-catenin and ultimately to the cortical actin cy- in brain synaptic junctional complexes, suggesting thattoskeleton. it functions in the CNS to link pre- and postsynaptic

The presence of PS1 at cell–cell adhesion sites and membranes (Uemura, 1998). Our data that brain PS1its transmembrane configuration (see Introduction) sug- is concentrated at synaptic adhesion sites and formsgest that this protein is an integral part of the lateral complexes with components of the cadherin/cateninplasma membrane. However, our attempts to label the system (see Results) indicate that this protein is an inte-extracellular sequence of PS1 in intact cells using either gral component of the synaptic cadherin/catenin com-biotin or anti-PS1 antibodies failed even though both plex. This association makes the brain cadherin/cateninmethods did successfully label cell surface E-cadherin

system a potential target for PS1 FAD mutations as they(see Results). These results most likely reflect limitations

could affect any of the steps required for the interac-of the experimental methods. In contrast to E-cadherin,tion of PS1 with other components of this system.which has a relatively large extracellular sequence ex-Such perturbations of protein–protein interactions withintending outward from the plasma membrane (Yap et al.,multimeric complexes is a mechanism for dominant1997), all extracellular domains of PS1 are very short and“gain-of-aberrant-function or loss-of-function” effectsconnect intramembrane segments (Doan et al., 1996;of disease-associated mutations (Kaushal and Khorana,Lehmann et al., 1997). This configuration suggests that1994; Nishimura et al., 1999). Cadherin-based adhesionthe extracellular loops of PS1 may lie very close to theis influenced by a number of extracellular signals, includ-extracellular surface of the lipid bilayer and might for thating growth factors, Ca21, and peptide hormones, andreason be inaccessible to labeling reagents. In addition,these signals may trigger a range of intracellular signalPS1 at intercellular junctions may be shielded by othertransduction pathways (Yap et al., 1997). The incorpora-proteins in the junctional complex. Thus, our failure, andtion of a mutated PS1 into the cadherin adhesion com-those of others (Cook et al., 1996; Doan et al., 1996; De

Strooper et al., 1997 [see however, Dewji and Singer, plex may interfere with any of these signaling pathways.

Molecular Cell900

Experimental Procedures in 25 mM HEPES (pH 7.2), 150 mM NaCl, and 0.4% digitonin. Apoferi-tin, b-amylase, and bovine albumin were used as molecular massmarkers. Samples were centrifuged at 35,000 rpm for 22 hr in a SW41PS1 Antibodies

Rabbit polyclonal Ab222 (Figure 1A) was raised against human PS1 rotor and then fractionated into 1.0 ml aliquots. Thirty microliters ofeach fraction was analyzed on WBs.amino acids 2–12 and affinity purified using cognate peptide cou-

pled to CNB4-activated Sepharose 4B (Pharmacia). Mouse mono-clonal antibody 33B10 was raised against a glutathione S-trans- Ca21 Switch and Triton X-100 Extractionferase (GST) fusion protein containing human PS1 amino acids Confluent MDCK cultures grown on collagen-coated coverslips in259–410, synthesized in E. coli. This antibody recognizes an epitope Ca21-free DMEM plus 10% dialyzed FBS and 1.8 mM CaCl2 werewithin PS1 sequence 331–350 (J. S. et al., unpublished observations) washed in Ca21-free PBS and then incubated in Ca21-free DMEMand is specific for PS1/CTF (Figure 3B). Both antibodies recognized plus 10% dialyzed FBS for 20 hr. After that, CaCl2 was adjustedthe corresponding PS1 antigens in cells from PS11/1 mice but not back to 1.8 mM, and cells were incubated for an additional 5 hr. Atfrom PS12/2 mice (see Results for further characterization of PS1 the appropriate times, cells were fixed, permeabilized, treated withantibodies). Antisera 36097 and 52198 against PS1 sequence 1–25 antibodies, and examined for immunofluorescence. For Triton X-100and 1–133, respectively, were a gift from Dr. G. Schellenberg, and extraction, MDCK cells on collagen-coated coverslips were washedantiserum C-20 against PS1 sequence 449–468 was from Santa with PBS containing 1 mM Ca21 and 0.5 mM Mg21, extracted withCruz Biotechnology, CA. Antibodies 222 and 33B10 were used for CSK buffer (50 mM NaCl, 300 mM sucrose, 10 mM PIPES [pH 6.8],most of our work because they were available in sufficient quantities. 3 mM MgCl2, 0.5% Triton X-100) for 5 min at 48C, washed in PBS,All other antibodies were obtained from Transduction Laboratories fixed with 2.5% paraformaldehyde, and incubated with the corre-except anti-E-cadherin DECMA-1 (Sigma) and anti-transferrin re- sponding primary and secondary antibodies as described above.ceptor (Zymed).

Immunoprecipitation of Cell Surface E-Cadherinand BiotinylationCell Cultures, Immunofluorescence, and Confocal MicroscopyPolarized MDCK cells on polycarbonate filters were cooled on iceUnless otherwise stated, cell cultures were grown in DMEM pluswater and washed with DMEM/BSA buffer (DMEM, 0.6% BSA, 2010% fetal bovine serum (FBS), penicillin, and streptomycin in 5%mM HEPES [pH 7.3]), and the basolateral surface was exposed forCO2 and 378C. For immunofluorescence, cells were plated on 22 31 hr to 60 mg/ml anti-E-cadherin rat monoclonal antibody DECMA-122 mm collagen-coated glass coverslips, fixed in 2.5% paraformal-(Vestweber and Kemler, 1985). Cells were then washed with ice-dehyde in phosphate-buffered saline (PBS), and permeabilized incold PBS-Ca-Mg and solubilized in 1% digitonin, and cell surface5% acetic acid and 95% ethanol for 10 min at 2208C. Followingcadherin with associated proteins was precipitated with a mixture ofwashing, cells were treated with 10% goat serum in TBS (25 mMprotein A and protein G beads. Cell surface biotinylation of polarizedTris–HCl [pH 7.4], 150 mM NaCl) plus SuperBlock (Pierce) for 1 hr,MDCK cells was performed as described (Graeve et al., 1989). Fol-incubated overnight with primary antibody, washed in TBS, and thenlowing labeling with NHS-SS-biotin, cells were washed and split inincubated with fluorescein-conjugated anti-rabbit and/or Texas red–two aliquots, one treated with TNE plus 1% digitonin, and the otherconjugated anti-mouse secondary antibodies (Amersham). Cellswith TNE plus 1% SDS. Samples were centrifuged at 17,000 3 g,were washed in TBS, mounted with Fluoromount-G medium (South-and the supernatants were incubated with streptavidin beads forern Biotechnology Associates, Inc.), and photographed on a Zeiss30 min. Streptavidin beads were recovered by centrifugation, dis-Axiophot microscope or on a Leica TCS-4D confocal laser scanningsolved in Laemmli buffer, and analyzed on WB.microscope.

Cell Aggregation AssaysImmunoelectron Microscopy Cell aggregation assays were performed as described (NagafuchiSpecimens were fixed with buffered aldehydes by immersion (con- and Takeichi, 1988). In brief, human kidney HEK293 cells stablyfluent MDCK cells) or by vascular perfusion under anesthesia. For transfected with WTPS1, mutant PS1, or vector, were suspendedimmunogold staining, specimens were treated sequentially with os- in Ca21-free HCMF buffer (10 mM HEPES [pH 7.4], 137 mM NaCl, 5mium tetroxide, uranyl acetate, and tannic acid and embedded in mM KCl, 5.5 mM glucose, 0.4 mM Na2HPO4), disaggregated, andepoxy resin. Ultrathin sections mounted on nickel grids were treated then adjusted to 1.5–2 3 106 single cells/ml in 1% BSA/HCMF. Cellwith primary antibody followed by 10 nm of gold-labeled secondary suspensions were adjusted to 2 mM in Ca21, and 250 ml aliquotsantibody and stained with uranyl acetate and lead citrate. For immu- were added to the wells of 24-well plates precoated with 1% BSAnoperoxidase staining, sucrose-infiltrated vibratome sections were and incubated for 45 min at 378C with 80 rpm agitation. The numberfrozen and thawed for enhanced permeability and treated with pri- of particles was then determined using a hemocytometer. Aggrega-mary antibody followed by visualization with biotinylated secondary tion was calculated as the ratio (N0–N45)/N0, where N0 and N45 areantibody, peroxidase-labeled avidin–biotin complex, and diamino- the total number of particles at time zero and at 45 min of incubation,benzidine, then treated with osmium tetroxide followed by metha- respectively, and values are expressed as percent of control. Thenolic uranyl acetate, and embedded in epoxy resin. Controls in- significance of differences was determined by two-way analysis ofcluded (separately) PS1 antibody pretreated with cognate peptide, variance.an irrelevant primary antibody, and omission of primary antibody.

Acknowledgments

Immunoprecipitation, Immunoblotting, and GlycerolWe thank Dr. P. Safting for fibroblasts and tissue from PS12/2 andVelocity GradientsPS11/1 mice; and Dr. D. Selkoe for PS1D257A cDNA. This work wasConfluent MDCK cells in 150 mm dishes were washed in PBS, centri-supported by grants AG-08200 and AG-05138, by the Alzheimer’sfuged, and suspended in cold TNE buffer (25 mM Tris–HCl [pH 7.6],Association, and by the A. P. Slaner family.150 mM NaCl, 13 Complete Protease Inhibitor Cocktail (Boehringer

Mannhein), 1 mM EDTA) plus 1% digitonin. Following centrifugationReceived April 28, 1999; revised October 4, 1999.at 17,000 3 g, supernatants (4 mg/ml protein) were treated with 20

ml of either protein G- or protein A–agarose beads per ml lysate forReferences2 hr. Beads were removed, and supernatants were incubated with

primary antibody overnight at 48C and then treated with proteinAberle, H., Butz, S., Stappert, J., Weissig, H., Kemler, R., andG–agarose (monoclonal antibodies) or protein A–agarose (poly-Hoschuetzky, H. (1994). Assembly of the cadherin-catenin complexclonal antibodies) for 2 hr. IPs were washed in TBS plus 0.2% NP-in vitro with recombinant proteins. J. Cell Sci. 107, 3655–3663.40, dissolved in 1% SDS, and analyzed on WBs. Glycerol gradient

centrifugation was performed as described (Pind et al., 1994). In Artavanis-Tsakonas, S., Rand, M.D., and Lake, R.J. (1999). Notchsignaling: cell fate control and signal integration in development.brief, 15 mg of MDCK cell extract in 0.5 ml of TNE plus 1% digitonin

was applied to a 11.5 ml of 10%–40% (w/v) linear glycerol gradient Science 284, 770–776.

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