Neurexin antibodies. For the production of neurexin antibodies, female chickens were immunized with a recombinant neurexin–GST (glutathione S-transferase) fusion protein containing the cytoplasmic tail of neurexin-1. IgY was isolated from egg yolk with the Pierce Eggcellent kit, and antibodies were affinity purified by chromatography on columns carrying the neurexin cytoplasmic tail sequence fused to maltose binding protein. All experiments involving animals were approved by the Institutional Animal Care and Use Committee of the Health Science Division at Columbia University.
Neuronal cultures and transfection.
Cerebellar granule cells were isolated from mice at postnatal day 5 or 6 (P5/P6)33
. For transfection, cells were resuspended at a density of 500,000 per ml in 150 μl of neurobasal medium containing 2 mM Glutamax, 2% B27 supplement (Invitrogen) and 5 ng/ml BDNF (Preprotech). DNA/lipid complexes were formed by incubation of 0.03 μl Lipofectamine 2000 (Invitrogen) with 0.2 μg of DNA in 100 μl OptiMEM (Invitrogen) and added to the cell suspension. After 45 min incubation at 37 °C and 5% CO2
, the cell suspension was plated into one well of an 8-well Lab-Tek Permanox chamber slide coated with polyornithin (Sigma) and Laminin (Invitrogen). After 2–4 h, 200 μl of culture medium containing penicillin and streptomycin (Invitrogen) were added. Cultures were generally maintained in vitro
for 4–10 d before analysis.
Hippocampi were removed from embryonic day 18 (E18) rats and treated with trypsin for 20 min at 37 °C, followed by washing and trituration. Dissociated cells were plated at 100,000 cells/cm2 on poly-lysine-coated glass coverslips (Carolina Biologicals) and cultured in neurobasal medium supplemented with 2 mM Glutamax, and 2% B-27. Cells were transfected with Lipofectamine 2000 (Invitrogen) 12–21 d after plating and analyzed 2–4 d after transfection.
Clustering of neurexin by beads or antibodies. Recombinant GPI-anchored neuroligin was expressed in HEK293 cells and isolated from cell lysates by chromatography on NTA-agarose (Qiagen). The protein bound to the column in phosphate-buffered saline containing 350 mM NaCl, 0.5% Triton X-100 and 6 mM imidazole. Unspecifically bound proteins were removed by extensive washing with PBS containing 1 M NaCl and 0.1% Triton X-100. The detergent was then switched to 25 mM octyl-glucopyranoside (Calbiochem), and bound protein was eluted with an imidazole gradient (10–200 mM). Peak fractions containing purified GPI-neuroligin were used for reconstitution into preformed liposomes composed of egg-Phosphatidylcholine (Avanti Polar lipids) by dialysis of octyl-glucopyranoside against PBS. The reconstituted material revealed a single protein band at the predicted size for GPI-neuroligin by silver staining, which showed immunoreactivity with specific antibodies directed against the hexa-histidine tag (data not shown). Purified GPI-anchored placental alkaline phosphatase was purchased from Sigma.
Coating of cleaned 5-μm silica beads with GPI-neuroligin containing liposomes was done using a method developed by J. T. Groves and colleagues (University of California, Berkeley), and beads were added to dissociated cultures of hippocampal neurons. In control experiments, the lipid bilayers were visualized with fluorescently labeled lipids (1,2-Dipalmitoyl-sn-glycerol-3-phosphoetanolamine-N-(Lissamine Rhodamine B Sulfonyl); Avanti Polar Lipids), and the reconstituted protein was visualized in the bilayer by immunostaining. Cells were fixed and analyzed 24–72 h after addition of beads.
Vesicle turnover on beads was assayed 16 h after addition of beads to hippocampal cultures by incubating cultures for 5 min with a pre-warmed, isotonic depolarization solution containing 10 μg/ml of affinity-purified rabbit anti-synaptotagmin lumenal domain antibodies. After incubation, cultures were briefly washed three times with pre-warmed medium, fixed and processed for immunohistochemistry.
Anti-VSV antibody multimers were generated as previously described for EphB ligands34
by pre-incubation of 50 ng/ml anti-mouse Cy-3 antibody (Jackson Laboratories) with 500 ng/ml mouse anti-VSV antibody (Roche) in neuronal culture medium (neurobasal medium containing 2 mM Glutamax and 2% B-27 supplement). Antibodies were added to dissociated hippocampal cultures and analyzed 18–24 h after addition of antibodies.
Image acquisition and quantitation. Images were acquired with a Leica TCS NT or Zeiss LSM510 confocal microscope. Laserpower and photomultipliers were set such that no detectable bleed-through occurred between the different channels. Eight to ten sections were taken from top to bottom of the specimen and brightest point projections were made. Images were analyzed with OpenLab (Improvision) and Metamorph (Universal Imaging) software (see Supplementary Methods online for details) and processed using Adobe Photoshop software.
Neurexin binding assays and cell aggregation assays. The soluble neurexin-1–alkaline phosphatase fusion protein was expressed in HEK293 cells and collected in the culture medium. For binding assays, HEK293 cells in 35 mm dishes were transfected with various neuroligin expression constructs in triplicates. We removed the culture medium 36 h after transfection and added the fusion protein in Hank’s buffered saline containing 2 mM CaCl2,1 mM MgCl2 and 5% fetal bovine serum. After incubation for 90 min at 4 °C, the unbound protein was removed quantitatively, and the bound protein was collected in a solution containing 1% Triton X-100 and 10 mM Tris-HCl, pH 8.0. Alkaline phosphatase activity was measured kinetically with 3 mg/ml para-nitro-phenyl-phosphate (Sigma) as substrate in a solution containing 100 mM Tris-HCl (pH 9.5), 50 mM MgCl2 and 100 mM NaCl.
For aggregation assays, 80% confluent PC12 cells were transfected separately with epitope-tagged neurexin-1 and neuroligin-1 or neuroligin mutants using Lipofectamine 2000 (Invitrogen). The cells were resuspended 36–48 h after transfection, carefully triturated, and mixed at a final density of 5 × 106 cells per ml. Cells were incubated in 1.5-ml microcentrifuge tubes at 37 °C with rotation in a total volume of 0.3 ml HBSS containing 10 mM CaCl2 and 20 mM MgCl2. At 0, 20, 40, 60 and 80 min, an aliquot was removed and aggregation determined by calculating N0 (number of particles at time 0) divided by Nt (number of particles at time t).
Analysis of neuroligin complexes. Recombinant soluble neuroligin was expressed in HEK293 as secreted protein with a C-terminal hexa-histidine tag. The protein was purified on NTA-agarose and eluted with a 10–200 mM imidazole gradient.
For chemical cross-linking, 1 μg of purified recombinant neuroligin-1 was incubated in phosphate-buffered saline for 30 min at ambient temperature with 20 μM BS3
(Pierce). The cross-linker was quenched by addition of Tris-HCl (pH 8.0) to a final concentration of 200 mM and cross-linking products were analyzed by western blot.
For native gel electrophoresis, HEK293 cells on 35-mm culture dishes were transfected with wild-type or mutant neuroligin expression vectors. Thirty-six hours after transfection, cells were washed with ice-cold phosphate-buffered saline and 150 μl of ice-cold lysis buffer was added: 20 mM Tris-HCl (pH 6.8), 50 mM NaCl, 5 mM MgCl2,2 mM CaCl2,2 mM DTT and 1% (w/v) Triton X-100 containing protease inhibitors “complete EDTA-free” (Roche). After 5 min incubation on ice, cell lysates were homogenized by repeated pipetting and centrifuged for 5 min at 14,000 r.p.m. The supernatants, which contained more than 95% of total neuroligin protein, were analyzed by electrophoresis (8 h, 60 V at 4 °C) in 8% poly-acrylamide gels; electrophoresis buffer contained 375 mM Tris-HCl (pH 8.8), 0.5% Triton X-100 with 250 mM Tris-base and 190 mM glycine. Neuroligin complexes were detected by western blot.
For analysis of oligomers by co-immunoprecipitation, myc-tagged wild-type neuroligin-1 was co-transfected into HEK293 cells with HA-tagged wild-type or mutant neuroligin-1. Cells were lysed in 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM CaCl2,1 mM MgCl2, 0.5% Triton X-100 and 1 mM DTT containing “complete EDTA-free” protease inhibitor cocktail (Roche) at 4 °C. Insoluble material was pelleted by 5 min centrifugation at 13,200g, and supernatants were incubated with 9E10 anti-myc antibodies for 1 h at 4 °C. Lysates were then re-centrifuged for 5 min and immune complexes in the supernatants were recovered on protein G sepharose (Pharmacia). Immunoprecipitates were washed twice with lysis buffer containing 0.1% Triton X-100 and analyzed by western blotting with anti-HA antibodies (Roche). Expression levels of both transfected proteins in the cell lysates were verified by western blotting with anti-HA and anti-myc antibodies.
Sequence alignments between neuroligin-1 and mouse AChE were carried out using clustalX35
. Homology modeling between aligned sequences was performed with MODELLER36
. Figures showing the mouse AChE and modeled neuroligin-1 structures were generated with RIBBONS37
A more detailed account of the methods and the DNA constructs and antibodies used in this study can be found in Supplementary Methods online.