Title: Homo- and heterodimerization of APP family members promotes intercellular adhesion
Abstract: Article29 September 2005free access Homo- and heterodimerization of APP family members promotes intercellular adhesion Peter Soba Corresponding Author Peter Soba ZMBH, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Simone Eggert Simone Eggert ZMBH, University of Heidelberg, Heidelberg, GermanyPresent address: Department of Neurosciences, University of California San Diego, La Jolla, USA Search for more papers by this author Katja Wagner Katja Wagner ZMBH, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Hanswalter Zentgraf Hanswalter Zentgraf Angewandte Tumorvirologie, DKFZ, Heidelberg, Germany Search for more papers by this author Katjuscha Siehl Katjuscha Siehl ZMBH, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Sylvia Kreger Sylvia Kreger ZMBH, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Alexander Löwer Alexander Löwer ZMBH, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Andreas Langer Andreas Langer Department of Pathology, Mental Health Research Institute, University of Melbourne, Parkville, Australia Search for more papers by this author Gunter Merdes Gunter Merdes ZMBH, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Renato Paro Renato Paro ZMBH, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Colin L Masters Colin L Masters Institute for Pharmacia and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Ulrike Müller Ulrike Müller Department of Pathology, Mental Health Research Institute, University of Melbourne, Parkville, Australia Search for more papers by this author Stefan Kins Stefan Kins ZMBH, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Konrad Beyreuther Konrad Beyreuther ZMBH, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Peter Soba Corresponding Author Peter Soba ZMBH, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Simone Eggert Simone Eggert ZMBH, University of Heidelberg, Heidelberg, GermanyPresent address: Department of Neurosciences, University of California San Diego, La Jolla, USA Search for more papers by this author Katja Wagner Katja Wagner ZMBH, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Hanswalter Zentgraf Hanswalter Zentgraf Angewandte Tumorvirologie, DKFZ, Heidelberg, Germany Search for more papers by this author Katjuscha Siehl Katjuscha Siehl ZMBH, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Sylvia Kreger Sylvia Kreger ZMBH, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Alexander Löwer Alexander Löwer ZMBH, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Andreas Langer Andreas Langer Department of Pathology, Mental Health Research Institute, University of Melbourne, Parkville, Australia Search for more papers by this author Gunter Merdes Gunter Merdes ZMBH, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Renato Paro Renato Paro ZMBH, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Colin L Masters Colin L Masters Institute for Pharmacia and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Ulrike Müller Ulrike Müller Department of Pathology, Mental Health Research Institute, University of Melbourne, Parkville, Australia Search for more papers by this author Stefan Kins Stefan Kins ZMBH, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Konrad Beyreuther Konrad Beyreuther ZMBH, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Author Information Peter Soba 1, Simone Eggert1, Katja Wagner1, Hanswalter Zentgraf2, Katjuscha Siehl1, Sylvia Kreger1, Alexander Löwer1, Andreas Langer4, Gunter Merdes1, Renato Paro1, Colin L Masters3, Ulrike Müller4, Stefan Kins1,‡ and Konrad Beyreuther1,‡ 1ZMBH, University of Heidelberg, Heidelberg, Germany 2Angewandte Tumorvirologie, DKFZ, Heidelberg, Germany 3Institute for Pharmacia and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany 4Department of Pathology, Mental Health Research Institute, University of Melbourne, Parkville, Australia ‡These authors contributed equally to this work *Corresponding author. ZMBH, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany. Tel.: +49 6221 546847; Fax: +49 6221 545891; E-mail: [email protected] The EMBO Journal (2005)24:3624-3634https://doi.org/10.1038/sj.emboj.7600824 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The amyloid precursor protein (APP) plays a central role in Alzheimer's disease, but its physiological function and that of its mammalian paralogs, the amyloid precursor-like proteins 1 and 2 (APLPs), is still poorly understood. APP has been proposed to form dimers, a process that could promote cell adhesion via trans-dimerization. We investigated the dimerization and cell adhesion properties of APP/APLPs and provide evidence that all three paralogs are capable of forming homo- and heterocomplexes. Moreover, we show that trans-interaction of APP family proteins promotes cell–cell adhesion in a homo- and heterotypic fashion and that endogenous APLP2 is required for cell–cell adhesion in mouse embryonic fibroblasts. We further demonstrate interaction of all the three APP family members in mouse brain, genetic interdependence, and molecular interaction of APP and APLPs in synaptically enriched membrane compartments. Together, our results provide evidence that homo- and heterocomplexes of APP/APLPs promote trans-cellular adhesion in vivo. Introduction Research on the amyloid precursor protein (APP) has been focused on generation of the amyloid-β (Aβ) peptide, which is proteolytically derived from its precursor APP by consecutive cleavages by BACE-1 and γ-secretase (Aguzzi and Haass, 2003). Despite the wealth of studies regarding the physiological role of APP, there is little consensus about its function in vivo. Among other proposed functions, APP was found to promote neurite outgrowth, cell adhesion, and cell proliferation (Annaert and De Strooper, 2002). However, genetic studies have shown that APP knockout mice are viable, exhibiting only a mild phenotype including reduced forelimb grip strength and locomotor activity (Zheng et al, 1995; Li et al, 1996). Two paralogs of APP are known in mammals, termed amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2). Mice with single knockouts of APLP1, APLP2, or double knockouts of APP/APLP1 are viable, while combinations of APLP2 and APP or APLP1 knockouts are lethal shortly after birth (von Koch et al, 1997; Heber et al, 2000), suggesting functional redundancy. Interestingly, triple knockout mice lacking all the three APP family members exhibit cortical dysplasia with ectopic neurons resembling a human type II lissencephaly phenotype (Herms et al, 2004). The observed phenomenon is in accordance with a redundant function of APP family proteins in neuronal cell adhesion. Redundancy of APP/APLPs is also reflected in a similar protein domain structure, where all the three family members share high homology of intra- and extracellular regions (Coulson et al, 2000). Moreover, all APP family members are processed in a similar manner by the same protease activities (Eggert et al, 2004), and it has also been shown that they interact with the Notch pathway during Drosophila development (Merdes et al, 2004). Despite the high degree of conservation between APP, APLP1, and APLP2, several differences can be noted. The expression of APLP1 is limited to the nervous system (Lorent et al, 1995), whereas APP and APLP2 are ubiquitously expressed (Tanzi et al, 1988; Slunt et al, 1994). Additionally, biochemical analyses suggested that APLP1 accumulates at the postsynapse (Kim et al, 1995), while APP (Ferreira et al, 1993) and APLP2 (Lyckman et al, 1998) are transported to presynaptic sites. On the basis of its structural features, APP has been proposed to have a receptor-like function and it binds to different extracellular matrix proteins, such as heparin and collagen (Small et al, 1992; Beher et al, 1996). In this context, it is of interest that APP forms dimers (Scheuermann et al, 2001), which is reminiscent of classical receptor dimerization described for the EGF receptor (Schlessinger, 2002). However, it is unclear whether APP dimerization could also occur in an intercellular manner, as described for different cell adhesion molecules (CAMs), such as cadherins and nectins (Takai et al, 2003). This possibility is supported by the recent crystal structure of the APP extracellular carbohydrate domain (E2), which crystallized in the form of a trans-dimer (Wang and Ha, 2004). These intrinsic properties of APP would suggest a role in cell–cell adhesion via trans-dimerization. In order to address this hypothesis, we investigated homo- and heterointeraction of APP family proteins and analyzed their ability to promote intercellular adhesion. Here we report for the first time that homo- and heterocomplexes of APP family proteins promote cell adhesion via trans-cellular interaction. We further demonstrate the existence of endogenous heterocomplexes of APP family proteins in mouse brain and synaptic compartments, suggesting a role for APP family proteins in cell–cell interaction. Results Homo- and heterointeraction of APP, APLP1, and APLP2 To address the question whether APP and its paralogs can interact with each other in a cellular system, we performed coimmunoprecipitation analyses of myc- and HA-tagged APP family proteins transiently expressed in COS7 cells. We tested if HA-tagged APP can be coimmunoprecipitated with the corresponding myc-tagged APP construct by using specific anti-myc and anti-HA antibodies (Figure 1A). As a control, the analysis was also carried out with cells expressing myc-tagged APP and vector only. Additionally, in order to exclude artificial postlysis aggregation, extracts from transfected cells expressing myc- or HA-tagged APP only were mixed after lysis and examined by immunoprecipitation as well (Figure 1A, lanes 'mix'). Figure 1.Homo- and heterointeraction of APP family proteins. (A) Homointeraction of myc- and HA-tagged APP, APLP1, and APLP2. Pairs of myc- and HA-tagged APP (myc-APP and HA-APP), APLP1 (myc-A1 and HA-A1), or APLP2 (myc-A2 and HA-A2) were expressed in COS7 cells. In all, 1/25 of each lysate was used as an input control (DL). HA-tagged APP/APLPs were immunoprecipitated and immunoblotted for myc- and HA-tagged constructs. As controls, cells transfected with the corresponding myc-tagged APP family member or postlysis mixtures (lanes 'mix') of separately transfected myc- and HA-tagged APP/APLPs were immunoprecipitated. (B) Heterointeraction of myc- and HA-tagged APP, APLP1, and APLP2. Heterotypic pairs of myc- and HA-tagged APP, APLP1, or APLP2 were transfected into COS7 cells as indicated. In all, 1/25 of the each lysate was loaded as input control (DL). Myc-tagged APP/APLPs were immunoprecipitated and immunoblotted for myc- and HA-tagged APP/APLPs. Controls were performed as above. Download figure Download PowerPoint Under these conditions, myc-tagged APP was specifically interacting with HA-tagged APP in cotransfected cells, but not in the control or after postlysis mixing (Figure 1A). With the same experimental setup, APLP1 and APLP2 were also found to specifically interact in a homophilic fashion to a similar extent as observed for APP (Figure 1A). Interestingly, we observed interaction of both mature and immature forms of APP/APLPs. To prove the specificity of our approach, we performed the same experiment in reverse by immunoprecipitating the myc-tagged APP/APLP constructs. Again, we detected the coimmunoprecipitated corresponding HA-tagged constructs at comparable levels as in the experiments above (Supplementary Figure 1). These results show that APP/APLPs specifically interact in a homophilic fashion. We further asked whether APP/APLPs are capable of forming heterocomplexes as well. For this purpose, we expressed all possible combinations of myc- and HA-tagged APP/APLPs, and immunoprecipitated the according myc-tagged constructs as described above (Figure 1B). We found that all APP family proteins were efficiently coimmunoprecipitated in double-transfected cells, while no interaction was detected after postlysis mixing of single transfected cells. Comparable amounts of APP/APLP1, APP/APLP2, and APLP1/APLP2 heterocomplexes were recovered under these conditions independent of the type of tag used for the according APP/APLP construct (see Supplementary Figure 1). Together, these results demonstrate that APP family proteins specifically form homo- and heterocomplexes, suggesting a strong tendency for dimerization/multimerization of APP/APLPs in a cellular context. Interaction of APP/APLPs depends on the E1 domain To characterize the site of interaction, we generated deletion constructs of APP/APLPs lacking the N-terminal growth factor-like and copper-binding domains (ΔE1), the carbohydrate domain (ΔE2), the entire ectodomain (ΔEC), or the intracellular domain (ΔCT) (Figure 2A). Both HA- and myc-tagged deletion constructs of all the three homologs were made and analyzed in coimmunoprecipitation experiments. Figure 2.Mapping of APP/APLP homo- and heterointeraction. (A) Schematic drawing of APP/APLP constructs used. HA- and myc-tagged APP/APLP constructs lacking either the E1 domain (ΔE1), the E2 domain (ΔE2), the entire ectodomain (ΔEC), or the cytoplasmic domain (ΔCT) were analyzed. (B) Homointeraction of full-length HA-tagged APP, APLP1, and APLP2 with different deletion constructs in COS7 cells. HA-tagged APP (HA-APP FL) was coexpressed with vector only, myc-tagged APPΔE1 (ΔE1), APPΔE2 (ΔE2), APPΔEC (ΔEC), or APPΔCT (ΔCT). In all, 1/25 of each lysate was used for the DL. HA-tagged APP was immunoprecipitated from cell extracts and immunoblotted for myc- and HA-tagged constructs. The identical setup was used for APLP1 (HA-A1) and APLP2 (HA-A2) homointeraction with the corresponding deletion constructs. Lower levels of APLP2ΔCT interaction are due to lower expression levels of this construct. (C) Heterointeraction of APP, APLP1, and APLP2 with different deletion constructs in COS7 cells. As for the mapping of homointeractions, a HA-tagged APP family member was coexpressed with vector only or different APP/APLP deletion constructs and immunoprecipitated (as indicated). In all, 1/25 of each lysate was used as an input control (DL). Download figure Download PowerPoint To assess homotypic interaction, HA-tagged full-length APP was coexpressed with the different myc-tagged APP deletion constructs and immunoprecipitated as described above. We found comparable amounts of coimmunoprecipitated myc-tagged full-length APP, APPΔE2, and APPΔCT (Figure 2B). Intriguingly, APP constructs lacking the E1 domain or the entire ectodomain (ΔEC) displayed poor or no detectable interaction, respectively. We also did not observe postlysis aggregation of the different deletion constructs (data not shown). Similarly, little or no interaction of full-length APLP2 with APLP2ΔE1 or APPΔEC constructs was observed, respectively (Figure 2B). For APLP1, consistently lower, but detectable, amounts of myc-tagged APLP1ΔE1 or APLP1ΔEC were coimmunoprecipitated, while interaction levels of APLP1ΔE2 and APLP1ΔCT remained unchanged (Figure 2B). These data suggest the E1 domain as the major region required for homodimerization of all three homologs. Additionally, the transmembrane (TM)/juxtamembrane region, especially of APLP1, might be involved in mediating homotypic binding. Analogously, we performed a systematic mapping of heterotypic interaction of APP family proteins. APP/APLPs lacking the E2 domain or the intracellular domain were still interacting to a similar extent as the full-length proteins. In contrast, like already seen for homointeraction, all mutant APP/APLP1 lacking the E1 domain or the entire ectodomain were weakly, or not, coimmunoprecipitated, respectively (Figure 2C). Taken together, these results show that the E1 domain is the major interface for homo- as well as heterotypic interaction of APP/APLPs. Dimerization of APP, APLP1, and APLP2 at the cell surface We further asked whether interaction of APP/APLPs also occurs at the cell surface, which would be prerequisite for dimerization-induced cell–cell adhesion. Therefore, we used a crosslinking approach utilizing the membrane impermeable crosslinker DTSSP to covalently link cell surface APP/APLPs in intact cells. COS7 cells expressing APP, APLP1, or APLP2 were labeled with [35S]methionine and incubated with DTSSP at 4°C to prevent endocytosis. Afterwards, cells were lysed and the according APP family members were immunoprecipitated with specific antibodies. As a control, equally treated cells without addition of DTSSP were analyzed in parallel. For all the three APP family members, specific bands corresponding to the size of homodimers (200–220 kDa) could be detected in DTSSP-treated cells, but not in control cells (Figure 3). To assess the nature of the crosslinked protein species, the crosslinking products were excised from the gel and the contained proteins were extracted. The samples were then denatured under reducing conditions in order to dissociate the crosslinker. After SDS–PAGE and autoradiography, we found that the recovered major bands were corresponding to the size of the APP/APLPs monomers (100–120 kDa) (Figure 3, lanes 'el.d.', eluted dimers), indicating that all APP family proteins form homodimers at the cell surface. Figure 3.Dimerization of APP family proteins at the cell surface. [35S-Met]-labeled COS7 cells expressing APP, APLP1, or APLP2 were incubated either with or without the membrane-impermeable crosslinker DTSSP as indicated. APP/APLPs were immunoprecipitated with anti-APP (22734), anti-APLP1 (57), or anti-APLP2 (D2-II) antibodies, respectively. The samples were denatured without β-mercaptoethanol (β-ME) and analyzed on 3–8% Tris-acetate gels by autoradiography. Long and short exposures are shown as indicated to visualize crosslinked dimeric (d) or the monomeric (m) forms of APP/APLPs, respectively. Longer exposures show the crosslinked dimers compared to control cells without crosslinker. The crosslinking products (d) were subsequently extracted from the gel, denatured under reducing conditions (el.d.: eluted dimers), and analyzed on 3–8% Tris-acetate gels. The asterisks indicate unspecific signals present in all samples and controls. Download figure Download PowerPoint Homophilic intercellular interaction of APP, APLP1, and APLP2 Our experiments so far have shown that mature, but also immature, APP/APLP forms are interacting in COS7 cells, suggesting that the large intracellular pool of APP family proteins forms lateral cis-dimers. However, to exclusively address trans-interactions at the cell surface, we asked if APP and its homologs are able to promote cell adhesion. Drosophila Schneider (S2) cells are a powerful tool to investigate trans-cellular interaction of cell adhesion proteins (Islam et al, 2004) and receptor–ligand binding (Klueg and Muskavitch, 1999). Expression of two interacting proteins in two separated pools of S2 cells causes coclustering after mixing of both cell pools, thus giving a direct readout for interaction of the two proteins. We examined the properties of APP and its mammalian paralogs APLP1 and APLP2 to induce homotypic cell clustering. For this purpose, we transfected S2 cells with APP, APLP1, APLP2, or different extracellular deletion constructs thereof (ΔE1, ΔE2, or ΔEC; Figure 4A). Transfected single-cell suspensions were aggregated for 2 h and analyzed by immunocytochemistry. The transfection efficiency was within a comparable range of 20–25%, with high cell surface expression of all constructs (Figure 4A). Expression of APP/APLP full-length or ΔE2 constructs caused homotypic cell clustering, whereas no specific aggregation was observed for cells expressing constructs lacking the E1 domain or the entire ectodomain (Figure 4A). Recombinant expression of the full-length Notch receptor or GFP only did not induce homotypic cell clustering (data not shown), confirming the specificity of our assay. Quantification revealed that 6% of all APP- or APPΔE2-expressing cells were clustered, showing statistically significant interaction compared to APPΔE1- or APPΔEC-expressing cells (Figure 4A; P<0.05). Strikingly, 32 and 36% of APLP1- and APLP2-expressing cells were clustered, respectively (Figure 4A). The interaction of APLP1- or APLP2-transfected cells was again strictly dependent on the presence of the E1 domain, but not of the E2 domain, as APLP1ΔE1- and APLP2ΔE1-expressing cells were not clustered, respectively (Figure 4A; P<0.001). Consistent with these data, confocal microscopy studies of APLP1-expressing aggregated cells revealed that APLP1 strongly accumulated at sites of cell–cell contact transfected cells (Figure 4A), but not at contact sites of nontransfected cells (Figure 4A). Figure 4.Homo- and heterotypic intercellular interactions of APP, APLP1, and APLP2. (A) Quantification and immunostainings of homotypic S2 cell clusters (indicated by arrows) expressing APP/APLPs or the corresponding deletion constructs. The percentage of clustered transfected cells from at least three independent experiments is shown for the different constructs as indicated (n⩾3, ±s.d., t-test, scale bar=20 μm). Lower panel: confocal analysis of APLP1 expressing S2 cells stained with an anti-myc antibody (APLP1) and overlaid with the transmission image (scale bar=3 μm). (B) Quantification and immunostainings of heterotypic cell contacts (indicated by arrows) of mixed pools of S2 cells expressing APP/APLPs or the corresponding extracellular deletion constructs as indicated. Direct heterotypic cell contacts were quantified from at least three independent experiments, and is given as the percentage of total transfected cells (n⩾3, ±s.d., t-test, scale bar=20 μm). Lower panel: confocal analysis of immunostained mixed pools of S2 cells expressing APLP1 (anti-myc) and APP (40090, scale bar=3 μm). Download figure Download PowerPoint These data demonstrate that APP family proteins present at the cell surface can stabilize cell–cell interactions by homophilic trans-interaction via the E1 domain. Heterotypic intercellular interaction of APP, APLP1, and APLP2 We further investigated heterotypic interactions between APP family proteins as our coimmunoprecipitation experiments already suggested a high degree of heterointeraction. Again, S2 cells were transiently transfected with APP, APLP1, APLP2, or the corresponding extracellular deletion constructs. Pairs of differentially transfected single-cell suspension pools were mixed, aggregated, and analyzed by immunocytochemistry. Intriguingly, 28% of mixed cell populations expressing APP and APLP1 displayed heterotypic cell contacts, suggesting a high degree of hetero-trans-interaction (Figure 4B). Similar results were obtained for mixed pools of APP and APLP2, or APLP1- and APLP2-expressing S2 cells, where heterocluster formation was counted for 8 or 12% of transfected cells, respectively (Figure 4B). Additionally, no interaction of GFP-expressing cells with APP/APLP-transfected cells was observed (Supplementary Figure 3), strongly suggesting that the observed cell contact formation requires direct interaction of APP family proteins. This is further supported by the finding that cells expressing APP/APLPs lacking the ΔE1 domain were not incorporated into clusters of APP-, APLP1-, or APLP2-expressing cells, respectively (Figure 4B). Consistently, confocal microscopy studies revealed that heterotypic aggregates of APP- and APLP1-expressing cells displayed accumulated APLP1 immunoreactivity at cell–cell contact sites, not only adjacent to APLP1-expressing cells as observed for homotypic clusters, but also at contact sites of APP-expressing cells (Figure 4B). APP, in turn, was also localized at cell contact sites and cellular protrusions framing adjacent APLP1-expressing cells (Figure 4B). Together, these results demonstrate that APP, APLP1, and APLP2 have homo- and hetero-trans-interaction properties resulting in cell–cell adhesion, which specifically depend on E1 domain association. APP/APLPs promote intercellular adhesion of mouse embryonic fibroblasts (MEFs) Having shown that overexpressed APP family proteins are capable of promoting cell adhesion via trans-cellular interaction, we asked if a lack of APP/APLPs would lead to loss of cell adhesion. To investigate this hypothesis, we used MEFs derived from APP/APLP knockout animals. We tested the cell adhesion properties of MEFs derived from wild-type (WT), APP or APLP2 knockout (APP−/−, APLP2−/−), and APP/APLP2 double knockout (Dko) mice in a modified cell aggregation assay under calcium- and magnesium-free conditions to avoid cadherin-mediated adhesion (Miura et al, 1992). MEFs were initially treated with 1 mM EDTA and single-cell suspensions were aggregated for 60 min at 80 r.p.m. As cell clusters are formed during aggregation, the number of particles (single cells or cell cluster are equal to one particle) decreases over time. Thus, stronger cell adhesion causes an increase in cell clustering, resulting in a decreased number of particles. We detected solid and comparable cellular adhesion of WT and APP−/− MEFs shown by a decrease in particle number of approx. 40% (Figure 5A). Strikingly, cellular interaction of APLP2−/− and Dko MEFs was dramatically impaired, leading to significantly less cell aggregation (Figure 5A; P<0.01). However, after supplementation of calcium, APLP2−/− and Dko MEFs still exhibited similar aggregation properties as WT and APP−/− cells, indicating that the reduced amount of cellular interaction is not due to a lack of cadherin-mediated adhesion (Figure 5B). Since APP−/− MEFs were still showing strong cellular aggregation and MEFs do not express APLP1 endogenously (Supplementary Figure 4), we reasoned that APLP2 might be the essential family member for mediating cell–cell adhesion. To prove this hypothesis, we retransfected Dko cells with APLP2 (DkoAPLP2re). In addition, as we have shown strong trans-interaction of APLP1 in S2 cells, we also analyzed Dko cells stably expressing APLP1 (dkoAPLP1re) and APP−/− MEFs stably expressing APP (APP−/−APPre). The expression of APP family proteins in the different MEF lines was verified by Western blotting (Supplementary Figure 4). APP−/− and APP−/−APPre MEFs both showed strong cellular aggregation, with no significant difference (Figure 5A). Intriguingly, we observed strong cellular aggregation for DkoAPLP1re and DkoAPLP2re cell lines, showing that the cell adhesion properties observed in WT cells could be restored by APLP1 or APLP2 (Figure 5A; P<0.01). Figure 5.APP family proteins are required for cell adhesion. (A) Quantitative cellular aggregation of WT, APP knockout (APP−/−), APLP2 knockout (APLP2−/−), or APP/APLP2 double-knockout MEF cells (Dko). MEF cells were aggregated in suspension under calcium- and magnesium-free conditions for 1 h at 80 r.p.m. The number of particles (single cells and cell clusters) was counted at the indicated time points (Nt). The relative decrease in particle counts compared to t=0 (Nt/N0) is shown over time as a measure of aggregation of the different MEF cells (n⩾3, ±s.d., t-test). APP−/− MEFs were compared with APP retransfected cells (APP−/−APPre), and APLP1 (DkoAPLP1re) or APLP2 (DkoAPLP2re) rescued Dko cells were compared with parental Dko cells. (B) Typical micrographs of the different MEF lines after 60 min of aggregation. APLP2−/− and Dko cells were additionally aggregated in the presence of 1 mM Ca2+ to induce cadherin-mediated adhesion (scale bar=100 μm). (C) Equal amounts of differentially labeled Dko, DkoAPLP1re, and DkoAPLP12re cells (as indicated) were coaggregated for 60 min and analyzed by fluorescence microscopy (scale bar=100 μm). Download figure Download PowerPoint We further assessed the specificity of APLP-induced cell adhesion by coaggregation experiments with the parental Dko cell line. For this purpose, calcein-red-labeled Dko cells were coaggregated with calcein-labeled dkoAPLP1re or dkoAPLP2re cells. We observed predominant homotypic aggregation of dkoAPLP1re and dkoAPLP2re MEFs, with only weak random coclustering of dko cells (Figure 5C). Coaggregation of labeled dkoAPLP1re and dkoAPLP2re cells, however, resulted in mixed cell aggregation, largely consisting of homotypic cell clusters (Figure 5C). Taken together, our results clearly show that APLP2 is both required and sufficient for calcium-independent cell adhesion of MEFs. Moreover, APLP1 and APLP2 mediate MEF cell adhesion in a predominantly homotypic fashion, suggesting direct trans-cellular interaction. Heterointeraction of APP/APLPs in vivo We further aimed to investigate whether heterophilic interactions between APP family members exist in vivo. Therefore, we performed coimmunoprecipitations from WT mouse brain extracts and, to verify specificity, from APP−/−, APLP1−/−, and APLP2−/− mouse brain extracts (Figure