Volume 4, Issue 6, Pages 893-902 (December 1999) Presenilin-1 Forms Complexes with the Cadherin/Catenin Cell–Cell Adhesion System and Is Recruited to Intercellular and Synaptic Contacts  Anastasios Georgakopoulos, Philippe Marambaud, Spiros Efthimiopoulos, Junichi Shioi, Wen Cui, Heng-Chun Li, Michael Schütte, Ronald Gordon, Gay R Holstein, Giorgio Martinelli, Pankaj Mehta, Victor L Friedrich, Nikolaos K Robakis  Molecular Cell  Volume 4, Issue 6, Pages 893-902 (December 1999) DOI: 10.1016/S1097-2765(00)80219-1

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 (PS1+/+, lane 3) or PS1−/− (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 PS1+/+ (a) or PS1−/− (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 μm. Molecular Cell 1999 4, 893-902DOI: (10.1016/S1097-2765(00)80219-1)

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-cadherin monoclonal (Af–Aj and Al) or Ab222 (Ba–Be and Bk) and anti-β-catenin monoclonal antibody (Bf–Bj and Bl). Sets (a)–(e) and (f)–(j) represent serial 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 β-catenin (B). Scale bars, 20 μm. Vertical spacing (Aa–Ae, Af–Aj, Ba–Be, and Bf–Bj), 1.2 μm. (C) Subconfluent MDCK cells double immunostained for PS-1 (a) and E-cadherin (b) or PS1 (d) and β-catenin (e). (c and f) Phase contrast images. Arrows, free surfaces without PS1. Scale bar, 20 μm. (D) Transmission electron micrograph of confluent MDCK cells fixed (see Experimental Procedures) and treated with affinity-purified Ab222 followed by anti-rabbit IgG coupled to 10 nm collodial gold. Gold particles representing PS1 localization are seen at an intercellular junction. No labeling was obtained with preimmune serum (not shown). Scale bar, 100 nm. Molecular Cell 1999 4, 893-902DOI: (10.1016/S1097-2765(00)80219-1)

Figure 3 PS1 Forms Complexes with Components of the Adherens Junctions (A) PS1 fragments coimmunoprecipitate with E-cadherin, β-catenin, and α-catenin. Extract from confluent MDCK cells in 1% digitonin were treated with the antibodies indicated at the top of the figure, and the resulting IPs were probed on WBs with antibodies against the proteins indicated on the right of the figure. In the lane marked R222+Pep, extracts were immunoprecipitated with Ab222 pretreated with the corresponding peptide. For reference, 10 μg of MDCK cell lysate was probed with the indicated antibodies. The time of film exposure in a, b, and c was five times less than in d, e, and f. TR, transferrin receptor. (B) Antibody 33B10. Mouse monoclonal antibody 33B10 recognizing PS1/CTF was used to probe extract from: nontransfected (lane 1) or PS1-transfected (lane 2) HEK293 cells, fibroblast cells from wild-type (PS1+/+, lane 3) or PS1−/− (lane 4) mice, or MDCK cells (lane 5). Lane 6, MDCK extract stained with 33B10 plus corresponding antigen. FL, full-length PS1; CTF, PS1/CTF. (C) PS1 fragments, E-cadherin, and β-catenin form a single complex. A sample of MDCK cell extract in 1% digitonin was treated with anti-E-cadherin antibodies, and the IP was placed 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-β-catenin antibodies, and the IP was placed on lane 3. An identical aliquot was treated first with anti-β-catenin antibodies, and the IP was placed on lane 4. The supernatant was treated again with anti-β-catenin antibodies, and the IP was placed on lane 5 and then with anti-E-cadherin antibodies, and the IP was placed on lane 6. All samples were then tested for PS1/NTF and PS1/CTF on WBs. (D) PS1 fragments, E-cadherin, and β-catenin comigrate as a high–molecular weight complex. MDCK cell extract in 1% digitonin was separated in a linear 10%–40% glycerol velocity centrifugation gradient, and collected fractions were analyzed on WBs with antibodies indicated at the left 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 (R222+Pep), and the resulting IPs 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 either with anti-E-cadherin antibody DECMA-1 or with biotin. DECMA-1-labeled cells were solubilized in 1% digitonin, and immunocomplexes were dissolved in Laemmli buffer and placed on lane 1. An aliquot of the biotin-labeled cells was solubilized in 1% digitonin, and the labeled surface protein was precipitated with streptavidin, dissolved in Laemmli buffer, and placed in lane 4. A similar aliquot from the biotin-labeled cells was dissolved in 1% SDS and placed in lane 3. Lane 2, total cell extract. Molecular Cell 1999 4, 893-902DOI: (10.1016/S1097-2765(00)80219-1)

Figure 4 PS1 Localization at Intercellular Contacts Depends on Extracellular Ca2+ and Is Resistant to Detergent Extraction (A) Effects of Ca2+-induced cell–cell adhesion on PS1 localization. Confluent MDCK cells grown in the presence of 1.8 mM CaCl2 were stained with Ab222 (a) or anti-E-cadherin antibody (d). Cultures were then switched to Ca2+-free medium for 20 hr and examined again for PS1 (b) or E-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) or E-cadherin (f) staining. (B) Extract from confluent MDCK cultures grown as above were immunoprecipitated with the antibodies shown at the top of the figure, and the IPs were examined on WBs using antibodies indicated at the right of the figure. Lysates were used to probe for total cellular E-cadherin or PS1/NTF. The blot probed for E-cadherin was overexposed to visualize the weak signal in lane 33B10(−). Scale bar, 50 μm. (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 and d) 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 μm. Molecular Cell 1999 4, 893-902DOI: (10.1016/S1097-2765(00)80219-1)

Figure 5 PS1 Concentrates at Intercellular and Synaptic Contact Sites In Vivo (A) PS1 is localized at intercellular junctions of corneal epithelium. (a) Rat cornea was stained with Ab222. The double arrow (20 μm) spans the epithelium; s, stroma. Corneal epithelial cells display strong PS-1 immunoreactivity in areas of reciprocal contact. Arrowheads indicate examples of multiple cell contact zones. (b) A section of the same specimen stained with preimmune serum (symbols are as in [a]). A 15 μm frozen section is shown. (B) Brain PS1 IPs contain E-cadherin, N-cadherin, and β-catenin. Total mouse brain extract was immunoprecipitated with Ab222, and the isolated IPs were probed on WBs with the indicated antibodies. (C) PS1 immunostaining of synaptic junctions in mouse cerebral cortex. Both immunoperoxidase (a) and immunogold (b) show prominent staining of postsynaptic densities (arrows). Controls, including (separately) antibody pretreated with cognate peptide, an irrelevant primary antibody, and secondary antibody, only showed negligible immunoreactivity. Scale bar, 100 nm. Molecular Cell 1999 4, 893-902DOI: (10.1016/S1097-2765(00)80219-1)

Figure 6 PS1 Transfectant Cells Show Increased Cell–Cell Aggregation 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 ± SEM. Aggregation was significantly increased by WTPS1 (groups 1–3: p < 0.003, 0.001, 0.05) but was not changed by mutant PS1. Molecular Cell 1999 4, 893-902DOI: (10.1016/S1097-2765(00)80219-1)