Construction and expression of GST fusion proteins in E. coli
Glutathione S-transferase (GST) fusion constructs were generated by cloning various parts of Cupidinα/Homer2a cDNA  into the GST fusion vector pGEX-KG (see, Fig. 1). Escherichia coli JM109 expressing GST-fusion proteins were lysed in lysis buffer (50 mM Tris-HCl pH 7.4, 25% sucrose, 1% Triton X-100, 5 mM MgCl2). GST fusion protein lysates (10 mg) were coupled to glutathione-Sepharose 4B (Amersham Pharmacia Biotech, Piscataway, NJ) by rotating for 1 h at 4°C. After washing three times with 1% Triton X-100/phosphate-buffered saline, GST fusion protein-coupled Sepharose was mixed with 1 mg of protein lysates prepared from mouse cerebella, which were pre-cleared with glutathione-Sepharose for 1 h at 4°C. After rotating for 1 h at 4°C, the GST fusion protein complex was washed five times with cell lysis buffer and subjected to immunoblotting.
Ligand overlay assay with Cdc42
Bacterially expressed GST-Cdc42 protein was purified using glutathione-Sepharose column chromatography according to a previously described procedure . One μg samples of non-degraded GST-fusion proteins were separated by 10% SDS-PAGE and blotted onto nitrocellulose membranes (Hybond-ECL; Amersham Pharmacia Biotech, Piscataway, NJ). A ligand overlay assay was carried out as described previously . Briefly, after the GST-fusion proteins on the blots were re-natured, the protein blots were probed by incubating with each GST-Rho family fusion protein loaded with [35S]-GTPγS at an equal specific activity. After washing three times, the ligand-bound blots were air-dried and the radioactivities were analyzed using a BAS2000 Bioimaging analyzer (Fujix, Japan). The relative radioactivities were respectively measured from consistently sized areas using IPLab software (Scanalytics, Fairfax, VA), and normalized as described in the figure legends (Fig. 1).
Western blot analysis
After boiling proteins in sample buffer (0.4 M Tris-HCl pH 6.8, 8% sodium dodecyl sulfate, 40% (v/v) glycerol, 0.04% bromophenol blue) for 5 min, equal portions of protein solution were separated by SDS-PAGE and electro-transferred onto nitrocellulose membrane filters (GE Healthcare). Blots were reacted with diluted primary antibodies: anti-Cupidin antibody (1:5000) , anti-pan Homer antibody (1:1000) , anti-Drebrin antibody (1:400) (D029-3, MBL), anti-Flag monoclonal antibody (1:1000) (F3165, Sigma), anti-GFP antibody (1:400) (11814460001, Roche), anti-Myc monoclonal antibody (1:1000) (sc-40, Santa Cruz). Immunoreactivity was detected with ECL (GE Healthcare).
Bacterially expressed GST-CPD, GST-CPD N, GST-CPD C, GST-CPDΔ191–230, GST-CPDΔ191–283, and GST-CPDΔ231–283 proteins were digested with thrombin to remove the GST moiety, and dialyzed against a crosslinking buffer (10 mM HEPES-NaOH, pH 7.5, 2 mM EDTA, 1 mM MgCl2, 0.05% Tween 20, 5 mM DTT, and 1 mM GDP). Each GST- protein (25 μg/ml) was incubated with 10 mM dimethyl pimelimidate (DMP) (Pierce, Rockford, IL) for 1 hr at room temperature. Equal amounts of DMP-treated protein mixtures were analyzed by Western blotting using anti-CPD antibody.
For examination of the effects of Cdc42 binding on Cupidin multimerization, COS7 cells were triple-transfected with Flag-tagged CPD, GFP-tagged CPD and either myc-tagged Cdc42V12 or myc-tagged Cdc42N17. Similarly, COS7 cells were triple-transfected with Flag-tagged CPDΔ191–230, GFP-tagged CPDΔ191–230, and either myc-tagged Cdc42V12 or myc-tagged Cdc42N17. To prepare protein extracts from these transfected cells, cells were lysed and homogenized in 1% Triton X-100 buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 1 mM Na3VO4, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). After centrifuging at 14,000 × g for 10 min, protein solutions (containing approximately 1 mg proteins) were mixed with anti-Flag antibody, and incubated for 1 h on ice. Protein-antibody complex was precipitated with protein G-Sepharose (GE Healthcare) followed by repeated centrifugation at 2000 × g for 5 min at 4°C. The precipitated proteins were subjected to Western blot analysis using anti-CPD antibody. Signal intensities in areas of consistent size were measured using IPLab software, and the efficiency of multimerization was calculated as described in Fig. 2.
For examination of the Cupidin-Drebrin interaction, mouse cerebella (ICR, Nihon SLC, Hamamatsu, Japan) were lysed and homogenized in 1% Triton X-100 buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 1 mM Na3VO4, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). After centrifuging at 14,000 × g for 10 min, protein solutions (containing approximately 1 mg proteins) were mixed with primary antibody (non-immune serum, anti-CPD C antibody, or anti-pan Homer antibody), and incubated for 1 h on ice. Protein-antibody complex was precipitated with protein G-Sepharose (GE Healthcare) followed by repeated centrifugation at 2000 × g for 5 min at 4°C. The precipitated proteins were subjected to Western blot analysis using anti-Drebrin antibody.
Cell morphology of transfected HeLa cells
HeLa cells were transfected with CPD alone, CPDΔ191–230 alone, Cdc42V12 alone, Cdc42V12 and CPD, or Cdc42V12 and CPDΔ191–230 using the calcium phosphate precipitation method described previously . At 24 hours after transfection, cells were fixed with 4% formalin in PBS and stained with Alexa Fluor568-conjugated phalloidin (1:1000) (A12380, Invitrogen). Fluorescence was observed with a microscope (Eclipse E800; Nikon, Tokyo, Japan) equipped with a CCD camera (SPOT; Diagnostics Instruments Inc., Sterling Heights, MI). The phalloidin images were captured following confirmation of completing single/double transfection by detection of distinct fluoroprobes, as described by Shiraishi et., al . The number of spikes protruding from the cellular edge were counted in 10 cells respectively, and represented as the means ± SE per 10 μm of cell edge; data were compared by a two-tailed unpaired Student t tests using Excel software (Microsoft Corporation, Tokyo, Japan).
Preparation of primary hippocampal cell cultures
Hippocampal primary cell cultures were prepared from embryonic day 17 Wistar rats (Nippon SLC, Shizuoka, Japan) as described previously . Briefly, hippocampi were dissected after rats had been anesthetized with diethyl ether; excised hippocampi were treated with 45 U of papain (Worthington, PAPL, Lakewood, NJ), 0.01% DNase I (Boehringer-Mannheim, Indianapolis, IN), 0.02% DL-cysteine, 0.02% bovine serum albumin, and 0.5% glucose in PBS for 20 min at 37°C. After adding 20% bovine serum, cells were dissociated by repeatedly passing them through a 1-mL plastic pipette tip. Dispersed cells were plated at a density of 1.1 × 104 cells/cm2 onto poly-L-lysine-coated glass coverslips (Matsunami, Tokyo, Japan) in neurobasal medium (GIBCO BRL, Life Technologies, Rockville, MD) containing 2% B27 supplement (Invitrogen), 500 mM L-glutamine, 0.1 mg/mL streptomycin (Meiji, Tokyo, Japan), and 100 U/mL penicillin (Banyu, Tokyo, Japan). Cultures were maintained in a humidified atmosphere of 5% CO2 in air at 37°C.
Construction of and infection with recombinant adenovirus vectors
The EGFP-coding region (referred as to GFP in this study) derived from pEGFP-C1 (Clontech, Cambridge, UK) was fused in frame to the N-terminus of the full-length or mutated constructs of Cupidin to generate GFP-CPD. The GFP fragment was also fused to Cupidin with a deletion of amino acid residues 191–230 (CPDΔ191–230) to generate GFP-CPDΔ191–230. Replication-deficient adenovirus vectors carrying these GFP-fused constructs were generated by the COS-TPC method, as described previously . Briefly, the DNA fragment of GFP-CPD or GFP-CPDΔ191–230 was inserted into the SwaI site of the pAxCAwt cosmid cassette (Takara, Tokyo, Japan). The resultant cosmid DNA was co-transfected with the complex of the EcoT22I-digested Ad5-dlx DNA and the terminal protein into HEK293 cells, and recombinant adenoviruses were thus obtained by homologous recombination between them. The viruses were propagated in HEK293 cells, and were concentrated and purified by double CsCl step gradient centrifugation. The titers of viruses were measured by the 50% tissue culture infectious dose (TCID50) method. Hippocampal cultures at 19 days in vitro (DIV) were infected with the viruses at a multiplicity of infection (m.o.i.) of 100–200, and were analyzed at post-infection 2 days, corresponding to 21 DIV.
Analysis of spine morphology
Hippocampal cultures were fixed with 4% formaldehyde for 10 min and directly incubated with Oregon Green phalloidin (Molecular Probes, 1:200) overnight at 4°C. DiI (Molecular Probes) emulsion, mixed with cod liver oil at 1 μg/μl, was put onto the somata of neurons, which were identified by phalloidin staining, as a droplet of 20–30 μm in diameter, using a manually handled injector (Narishige, Tokyo, Japan). After incubation overnight at 4°C, the excess un-penetrated DiI emulsions were removed by suction, and DiI images were captured by confocal microscopy (MRC1024; BioRad, Hercules, CA) with 100×, 1.4 NA lens. Digital images were processed using Adobe Photoshop 6.0 software (Adobe Systems, San Jose, CA). Numbers of either GFP-CPD or GFP-CPD puncta were manually counted on the secondary dendrites of 10 neurons and the results presented as the means ± SE; data were compared by a two-tailed unpaired Student t test using Excel software. Spine morphology was categorized into five types as described in the legend for Fig. 4. Over 1,000 protrusions on the secondary dendrites of 20–30 neurons were analyzed for evaluation of spine morphology.
All immunocytochemical procedures were performed as described previously . Briefly, primary-cultured neurons (21 DIV) overexpressing GFP-constructs by adenovirus-mediated infection were fixed with 4% paraformaldehyde for 30 min at 37°C, washed three times with PBS, and then permeabilized with 0.2% Triton X-100 in PBS for 10 min. After preincubation with 5% BSA in PBS for 1 h, cells were incubated with primary antibody (anti-synaptophysin or anti-Drebrin antibody) for 1 h at 37°C. After washing three times with PBS, the cells were incubated with Alexa Fluor 568-conjugated anti-mouse IgG (Invitrogen). Fluorescence and phase-contrast images of immunostained cells were captured by confocal microscopy (MRC1024; BioRad, Hercules, CA) to acquire a single focal plane with a 100×, 1.4 NA lens. Digital images were processed using Adobe Photoshop 6.0 software (Adobe Systems, San Jose, CA). The number of punctate immunopositive signals larger than 1 pixel (0.16 × 0.16 μm2/pixel with a 255-gradient signal intensity; signals lower than 165 on the scale were cut off to eliminate noise) was counted by measuring the area with a signal above 165 on the scale, using IPLab software. Scores from the secondary dendrites of 10 neurons were normalized to each control (= 1.0). Results presented as mean ± SE were compared by two-tailed unpaired Student t tests using Excel software.
Glass coverslips with infected cells (as indicated by GFP fluorescence) at 21 DIV were transferred to an experimental chamber and superfused with modified Krebs-Ringer solution (in mM): NaCl 150, KCl 4, CaCl2 2, glucose 5, pyruvate 2, HEPES 5 (pH 7.4 with NaOH). Tetrodotoxin (1 μM) and picrotoxin (50 μM) were added to block action potentials and inhibitory synaptic transmission, respectively. The experimental chamber, consisting of an acrylic frame with a glass bottom, was mounted on the stage of an inverted microscope equipped with interference-contrast optics (Axiovert 100S, ZEISS, Germany). Patch pipettes were pulled from glass capillaries (Clark Electromedical Instruments, Pangbourne, U.K.) with a horizontal puller (P-97 Flaming/Brown Micropipette Puller, Sutter Instrument Company, U.S.A.). The pipettes had direct current resistance of 3–6 MΩ (tip diameter ~1–2 μm) when filled with solution (in mM): K-gluconate 25, KOH 80, CsCl 60, methane sulfonic acid 60, MgCl2 4, CaCl2 0.8, EGTA 2, Na2-ATP 4, Na2-GTP 0.2, glutathione 1, glucose 5 and HEPES 30 (pH 7.2 with CsOH, ≅ 330 mosm/l). The pipettes were connected to a patch-clamp amplifier and filtered with a 1-kHz Bessell low-pass filter (AXOPATCH 200B, Axon Instruments, U.S.A.). Data acquisition was done with Clampex software (Axon Instruments, U.S.A.). Miniature EPSCs sampled at 50 kHz were detected and fitted to a template function using custom software  written in IDL (Research System Inc., Boulder, CO). Peak amplitudes and interval were calculated for about 200 mEPSCs from each cell. Detection threshold was set to 5 pA amplitude. The data from 12 cells for each construct were compared using the Kolmogorov-Smirnov nonparametric test. Significance was set at p < 0.01. Recordings were performed at room temperature (22–25°C).