The Arc coding sequence of rat origin plus a C-terminal histidine tag was inserted into the multiple cloning site of the pGEX plasmid and the sequence was analyzed by DNA sequencing. The plasmid was transformed into BL21 (DE3) CodonPlus competent cells and plated on an agar plate containing 100 μg/ml ampicillin and 34 μg/mL chloramphenicol. A 10 mL LB starter culture was inoculated, grown overnight and diluted in 500 ml LB containing 100 μg/mL ampicillin and 34 μg/ml chloramphenicol in a bacterial shaker at 250 rpm at 37°C until OD600 = 0.6. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.6 mM and incubated for an additional 4 h. The culture was at 4°C for 45 min at 4000 rpm. The supernatant was removed, the pellet was resuspended in lysis buffer (pH 8, 1 M phosphate buffer, 500 mM NaCl, 1 mM PMSF, 1× Roche Protease Inhibitor, 1% Triton-X100, 10 mM imidazole, 0.3% Sarkosyl, 10 mM 2-mercaptoethanol, 5% glycerol), sonicated on ice 3 × 5 s with 1 min interval. The sample was centrifuged at 4°C for 20 min at 14,000 rpm and the supernatant was collected. A Qiagen Ni-NTA column was loaded with 600 μL of equilibration buffer (pH 8, 0.1 M phosphate buffer, 500 mM NaCl, 0.5% Triton-X100, 10 mM imidazole) and spun down at 4°C, 700 g for 4 min. Flow-through was discarded and the column was reloaded with 600 μL of crude GST-Arc lysate and spun down at 4°C, 700 g for 4 min. Flow-through was reloaded 2× on the same column and spun down at 4°C, 700 g for 4 min each time. The column was then washed 3× with 600 μL wash buffer (equilibration buffer with 40 mM imidazole) and spun down at 4°C, 700 g for 4 min. A new collection tube was attached to the column, 200 μL elution buffer (pH 8, 0.1 M phosphate buffer, 50 mM NaCl, 200 mM imidazole, 1.0 mM PMSF, 1× Roche protease inhibitor, 1.0 mM DTT) was added, and Arc-GST was eluted by centrifugation at 4°C, 700 g for 4 min. One additional fraction was eluted in 200 μl elution buffer. Protein concentration was measured with a Nanodrop spectrophotometer.

SH-SY5Y cells (human), HEK293 cells (human) and PC12 cells (rat) were grown in Dulbecco’s modified Eagle medium (DMEM; Sigma) supplemented with 10% fetal bovine serum, penicillin/streptomycin and L-glutamine. Cells were seeded in 97 mm petri dishes or 6-well plates (Nunclon). Cells were lysed in PBS containing 0.1% Triton-X100, 1 mM PMSF and Roche Complete Protease Inhibitor Cocktail. The sample was centrifuged at 4°C for 20 min at 14,000 rpm, the supernatant was collected and protein concentration was determined by bicinchoninic acid assay (BCA assay; Thermo Scientific Pierce).

HEK293 cell lysate containing 200 mg total protein and 5 mM DTT was mixed with 30 μL glutathione sepharose 4B (GE-Healthcare/VWR 17-0756-01) and placed on a rotor for 1 h at 4°C. The supernatant was collected and split into two fractions. One fraction was incubated with glutathione beads loaded with GST or GST-GFP. The other fraction was incubated with beads loaded with GST-Arc, rotating at 4°C overnight. Beads were washed 4× with ice-cold PBS and spun down at 800 rpm. Samples were run on a 10% SDS-PAGE gel, stained for 3 h at room temperature (RT) with 0.25% Coomassie brilliant blue dye R-250 (Bio Rad), and destained overnight at RT in destaining solution.

Unidentified gel bands were cut into 1.0 mm cubes. Pieces were washed in 100 μL wash solution at RT for 20 min with slow agitation. The supernatant was discarded and the wash was repeated. Gel pieces were then dried in a vacuumed Rotavapor until dry and 50 μL of 10 mM DTT was added and incubated at 56°C for 45 min. Samples were cooled, DTT was removed, and 50 μL of 55 mM iodoacetamide was added and incubated in the dark at RT for 30 min. Iodoacetamide was removed and washed twice, as earlier described. Gel pieces were dried in a vacuumed Rotavapor. To digest proteins, 30 μL of 3 ng/μL trypsin from porsine were added to each sample and rehydrated on ice for 30 min. Samples were incubated for 16 h at 37°C with slow agitation and then cooled and centrifuged at 1300 rpm for 2 min. The supernatant was saved as the first extraction. To extract peptides, 50 μL of 1% trifluoracetic acid was added and incubated at RT for 20 min with gentle agitation. The supernatant was added to the first extraction. Fifty microliter of 60% ACN/0.1% TFA was added to the gel samples and incubated for 20 min with gentle agitation. The supernatant was pooled with the first two extractions. Extractions were then dried in the vacuumed Rotavapor until 10–15 μL sample remained.

Samples were then concentrated by STop And Go Extraction (STAGE) tips. To make a STAGE tip, a Hamilton needle (22 gauge) with a liquid chromatography tip was pressed against an Empore 3M Extraction Disk to wedge a small piece in the opening. The needle tip was placed into a 20 μL gel-loader tip. Using 0.37 mm diameter capillary tubing, the piece of Empore disk was pushed and packed into the gel-loader tip, forming the column to be used for peptide concentration. The gel-loader tip was cut 2–5 mm below this column. The column was washed by slowly pushing 10 μL MeOH through the tip with a syringe. The column was “wetted” with 10 μL 60% CAN/0.1% TFA, knocked down by flicking the gel-loader tip, and 70% was pushed out. The column was “conditioned” with 10 μL 0.1% TFA, knocked down and 70% was pushed out. Ten microliter of the sample was then added to the column, knocked down and 70% was pushed out. The sample was washed with 0.1% TFA and then dried by pushing air through the column. The sample was eluted directly on a MALDI plate with 1.0 μL alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix containing 50% CHCA-1 (60% CAN and 0.2% TFA) and 50% CHCA-2 (ACN:MeOH:H₂O; 60:30:10). A peptide calibration standard was also plated adjacent to eluted samples. Samples were analyzed by MALDI-ToF-MS UltraFlex instrumentation and Mascot was used to identify peptides.

Arc cDNA residues 1–396 of rat origin were amplified with HindIII and BamHI sites and ligated into the pEYFP-C1 vector (Clontech). This full-length EYFP-tagged Arc was used to amplify various regions of the Arc sequence (see “Results” Section). Suitable primers were used to yield a PCR product with HindIII and BamHI sites, which were also ligated into pEYFP-C1. The calnexin signal sequence required for ER localization was inserted upstream of the mCherry fluorophore and the C-tail was ligated into the multiple cloning site of mCherry-C1. Arc cDNA of human origin was inserted into the multiple cloning site of EGFP-C1. Constructs were checked by DNA sequencing by the dideoxy chain termination method in an automated DNA sequencer (ABI Prism 310).

HEK293 cells at ~80% confluency in 100 mm Petri dishes were transfected with 8 μg Arc-EYFP plasmid deletion mutants, using 30 μL Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. After 24 h, cells were washed, lysed in lysis buffer, scraped off and centrifuged. To IP calnexin, Protein G Sepharose 4 Fast Flow beads (GE Healthcare) were washed in PBS and then incubated with 2.5 μg calnexin C-20 antibody (Table 1) for 1 h at RT. Beads were washed 3× in lysis buffer and incubated with 500 μg total protein overnight at 4°C. Bound fractions were washed 3× in lysis buffer and analyzed by western blot with GFP antibody (Table 1). A western blot of the input fractions ensured that equal amounts of each yellow fluorescent protein (YFP-Arc) construct were used in each IP.

Samples were separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane (Hybond-C; Amersham). Membranes were blocked for 1 h at RT in Tris-buffered saline/0.1% Tween-20 (TBST) and 3% non-fat dry milk. Primary antibodies were diluted in blocking buffer containing TBST and 5% BSA and applied on membranes overnight at 4°C with constant shaking. Following three washes with TBST, blots were incubated for 1 h at RT in horseradish peroxidase-conjugated secondary antibody diluted in TBST. Blots were then visualized using enhanced chemiluminescence (ECL Western Blotting Substrate; Pierce).

Hippocampi were dissected from E18 rat embryos (Wistar RjHan:WI, Janvier labs, France), trypsinized, and plated at 30,000 cells/cm² on Nr.1 glass coverslips coated with poly-D-lysine (Sigma P6407). Cells were cultured in minimal essential medium (Gibco 21430), supplemented with B27 (Gibco 17504), antibiotics (Sigma-Aldrich P4083), 2 mM glutamine, 0.6% glucose and 1 mM pyruvate, and conditioned on astrocyte cultures. Where indicated, cells were treated with 100 ng/mL human BDNF diluted in culture medium (Alomone labs; B-250) for 4 h prior to fixation.

Cells were washed twice with PBS, fixed for 10 min with 4% paraformaldehyde/4% sucrose/PBS, washed with PBS, permeabilized 10 min with 0.2% Triton-X-100/PBS, washed with 50 mM NH₄Cl/PBS, washed with PBS, and blocked for 1 h with 1% Roche Blocking Reagent (Roche 11096176001). Primary antibodies (Table 1) were diluted in blocking buffer. Coverslips with neurons were then inverted onto a drop of antibody solution on parafilm in a humidified chamber at 4°C overnight. Coverslips were washed with PBS, then incubated on a drop of secondary antibody (Table 1) in blocking buffer for 1 h at RT, washed again with PBS, dipped in water and mounted with ProLongGold Antifade Reagent containing DAPI (Invitrogen).

Adult wild type male Sprague-Dawley rats (180–250 g; NTac:SD; Taconic, Denmark) were anesthetized with urethane (i.p. 1.5 g/kg). Rats were placed in a stereotaxic frame and body temperature was maintained at 37°C throughout the experiment. A bipolar stimulation electrode (NE-200; 0.5 mm tip separation; Rhodes Medical Instruments, Wood hills, CA, USA) was positioned ipsilaterally into the perforant path (7.9 mm posterior to Bregma, 4.2 mm lateral to midline and 2.5 mm ventral from the brain surface). Evoked potential was measured by positioning an insulated tungsten recording electrode (0.075 mm; A-M Systems) in the dentate gyrus (DG; 3.9 mm caudal to Bregma, 2.3 mm lateral to the midline and 2.5–3.3 mm ventral from the brain surface). The recording electrode was lowered into the brain in 0.1 mm increments while monitoring the laminar profile of the response waveform evoked by a 300–400 μA test pulse stimulus. Following 20 min of baseline recording, HFS was applied that consisted of 400 Hz, 8-pulse stimulus trains repeated four times with 10 s between each train. HFS was applied three times with 5 min between each session. Total HFS duration was 10.5 min and the total pulse number was 96 (pulse-width was 0.15 ms). The stimulus intensity used for HFS was twice of that used for test pulses. Evoked responses were recorded for 120 min after HFS. Changes in the fEPSP slope were expressed in percent of baseline (20 min preceding HFS). After recordings were completed, the electrodes were removed, the animal was transcardially perfused with 4% paraformaledhyde (PFA). The brain was dissected and immersed in 4% PFA over night at 4°C, then in 30% sucrose for 2 days at 4°C. Twenty micrometer coronal sections were cut using Tissue-Tek (Sakura), mounted on Superfrost GOLD slides (Braunschweig, Germany), and stored at 4°C.

Antigen was retrieved by microwaving the mounted sections for 10 min at 600 W in citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0). After cooling for 20 min, sections were washed with PBS, permeabilized for 1.5 h with 0.5% Triton-X-100/PBS, washed with PBS and blocked with 5% horse serum/5% bovine serum albumin/PBS for 1 h at RT. Primary antibodies (Table 1) were diluted in blocking buffer, incubated overnight at 4°C, washed with PBS, then incubated with secondary antibodies (Table 1) for 1 h at RT, washed for 30 min, mounted, and coverslipped with ProLongGold Antifade Reagent containing DAPI (Invitrogen).

PLA was performed using the Duolink PLA Kit¹ with red (DUO92008) or orange (DUO92007) detection reagents, anti-mouse minus probe (DUO92004), and anti-rabbit plus probe (DUO92002). Manufacturer’s instructions were followed for cultured neurons except that Roche blocking solution was used. For F-actin staining, phalloidin-FITC (Sigma; 0.5 μg/mL) was added in the penultimate wash step (wash buffer B) for 10 min. On brain sections, antigen retrieval was performed as above, all incubation times and wash steps were doubled, and the blocking buffer consisted of PBS containing 5% horse serum and 5% bovine serum albumin.

Images were taken on a Leica SP5 Laser Scanning confocal microscope. Immunofluorescence of cultured neurons was imaged with a 63× objective, a 561 nm laser for Alexa Fluor 568, a 633 laser for Alexa Fluor 647, and a 402 laser for DAPI. Two optical sections were imaged—one at the dendritic level and the other at the equatorial plane of the nucleus. Dendritic PLA and phalloidin-FITC staining were imaged using a 100× objective. For PLAs of cultured neurons, 24 z-stacks of 30 optical sections were taken from each coverslip using a 40× objective, 402 nm excitation for DAPI, and 598 nm excitation for the red PLA signal or 561 nm for the orange PLA. For PLA on brain sections, tile scans were taken at 40× of 5× 4 z-stacks of 14 optical sections, covering the DG.

Confocal stacks were analyzed with Imaris (Bitplane, RRID:SCR_007370) using the 3D “spot” function for PLA spots and “surface” function for nuclei. Optimal parameters for these functions were determined using the negative control (no antibodies) and positive control (Arc/Arc) from each experiment and kept constant for all images belonging to this experiment. In cell culture, PLA was quantified as the number of spots divided by the number of cells. In brain sections, the volumes of the molecular and granule cell layers (GCLs) were measured by creating a “surface object”, defined by the lateral boundaries of the layers in XY direction, and the tissue surface in Z direction. PLA was quantified as spot density (PLA/mm³) inside this defined volume. All analyses were performed by investigators blind to the experimental conditions.

SH-SY5Y human neuroblastoma cells were plated on 1 cm coverslips coated with collagen at a concentration of 0.3 μg/mL and were maintained in complete DMEM. Cells were transiently transfected at 70% confluence using Lipofectamine 2000 (Invitrogen) and 2 μg of Arc-GFP, calnexin C-terminus-mCherry, or both. Transferrin uptake assays were performed 24 h after transfection. Cells were serum-starved for 6 h prior to experiments (DMEM + 0.5% FBS). Alexa Fluor 647-conjugated Transferrin (Life Technologies T-23366) was added to each sample to a final concentration of 15 μg/mL for 30 min on ice. The samples were then returned to 37°C for 15 min to restore endocytosis. Cells were immediately placed on ice, washed twice with cold PBS, and fixed using 1% PFA on ice for 30 min. Coverslips were mounted using Prolong Gold anti-fade reagent with DAPI.

All cells were imaged on a Leica SP2 confocal microscope using a 60× objective. Identical settings were used for each sample. Images were analyzed in ImageJ (RRID:SCR_003070). Endocytosis was quantified by integrating the background-subtracted Alexa Fluor 647 signal within the boundaries of each cell. Transfected cells within each sample, determined by the presence of GFP (Arc), mCherry (calnexin), or both, were compared to internal controls (untransfected cells from the same slide, in the same field of view), and conditions were compared to each other using ANOVA after first normalizing to internal controls for each replicate. All analyses were performed by investigators blind to the experimental conditions.

To identify novel Arc binding partners, we performed GST-affinity purification using recombinantly-expressed full-length rat Arc as bait (Arc-GST) and HEK293 cell lysate (Figure 1). The prominent band at 81 kDa on the Coomassie Brilliant Blue-stained SDS-PAGE gel was GST-Arc, as confirmed by MALDI-TOF mass spectrometry. The unidentified 90 kDa band seen in the experimental lane, but not in the control (GST), was excised from the gel and also analyzed by mass spectrometry (Figure 1A). The band was identified as a transmembrane protein of the endoplasmic reticulum, calnexin (Peptide Identification Score: 142; Peptides identified: APVPTGEVYFADSFDR and KIPNPDFFEDLEPFR). This finding was confirmed by probing GST-Arc-bound protein on a western blot with anti-calnexin antibody (Figure 1B, upper blot). GST-Arc also bound calnexin from non-stimulated SH-SY5Y neuroblastoma cells, a neuronal cell line in which Arc protein is endogenously expressed (Figure 1B, middle blot). Since both HEK293 and SH-SY5Y cells are of human origin, while our GST-Arc construct contained the rat sequence, we also performed the GST-pull-down with rat PC12 cells, a neuron-like cell line from the adrenal gland. Western blot analyses confirmed the presence of calnexin in each GST-Arc lane (Figure 1B).

We then sought to determine the specific region of Arc protein that binds to calnexin. We constructed plasmids encoding the full-length rat Arc mRNA sequence, N-terminally fused to YFP-Arc (Figure 2C). Three deletion mutants of this construct, covering amino acids 1–100, 101–240 and 241–396, respectively, were ectopically expressed in HEK293 cells and subjected to IP with a calnexin-specific antibody. We found that YFP-Arc (101–240) co-immunoprecipitated with calnexin, while YFP-Arc (1–100) and YPF-Arc (241–396) failed to do so (Figure 2A, left panel). To further narrow down the binding region, we made sequential N-terminal deletions of YFP-Arc, expressing amino acids 125–396, 150–396, 175–396, 200–396 and 225–396, respectively. Of these fragments, only one failed to co-IP with calnexin, namely the shortest fragment YFP-Arc (225–396). Since YFP-Arc (200–396) is only 25 amino acids longer, but does co-IP with calnexin, Arc residues 200–225 are essential for the interaction with calnexin (Figure 2B, left panel).

We then examined whether the interaction between Arc and calnexin occurred in neurons. To induce Arc expression in primary hippocampal neurons, we stimulated the cells with brain-derived neurotrophic factor (BDNF) for 4 h. As expected, Arc was detected in the nucleus, neuronal soma and dendrites by immunofluorescence (Figure 3A). Since calnexin is a transmembrane protein, with the C-terminus located in the cytosol and the N-terminus located in the lumen of the endoplasmic reticulum, we used antibodies directed against either the C- or N-terminus. Both antibodies resulted in very similar staining patterns—a reticular cytoplasmic staining, including the nuclear membrane, and a weaker punctate dendritic pattern (Figure 3A). Partial colocalization was observed between Arc and both the calnexin C- and N-termini in dendrites (Figure 3A, arrowheads). This colocalization was rather sparse and merely indicates that a small fraction of both Arc and calnexin localize at or near the same sub-cellular site. To validate the interaction in neurons, we performed PLAs, which detect protein proximity below 40 nm. PLAs detect the proximity of two primary antibodies by labeling the interaction site with a fluorophore, which is then detected by fluorescence microscopy and quantified (Fredriksson et al., 2002).

We used well-characterized Arc-directed antibodies for the positive control, namely: (1) polyclonal rabbit anti-Arc, directed against full-length Arc (Synaptic Systems); and (2) monoclonal mouse anti-Arc (C-7, Santa Cruz). The probability of observing proximity between these antibodies is high, given that they bind to the same protein, thus forming intramolecular PLA. Expectedly, PLA signal for Arc/Arc was low in naïve neuronal cultures, in agreement with low basal Arc expression. After induction of Arc expression with BDNF for 4 h, Arc/Arc PLA was significantly upregulated (Figure 3B). Arc/Arc PLA punctae were frequently found at phalloidin-labeled dendritic punctae, most likely dendritic spines or synapses (Figure 3C). They were also located in the dendritic shaft and neuronal soma, but rarely in the nucleus. Arc/Arc PLA signal thus confidently reflects Arc expression and is suitable as a positive control.

To probe the interaction between Arc and calnexin, we used the same antibodies as for immunofluorescence (rabbit anti-calnexin C-terminus and mouse anti-Arc). This was important, since Arc is a cytoplasmic protein (lacking a signal peptide), and thus should only be able to interact with the calnexin C-terminus. PLA between calnexin and Arc was low in naïve neurons, and clearly positive in BDNF-treated neurons (Figure 3B). Thus, PLA confirmed the proximity between Arc and calnexin in cultured neurons. The Arc/calnexin C-terminus PLA was readily observed in neuronal soma and dendritic shaft, but was not in detected spines (Figure 3C).

We performed PLA with different combinations of antibodies to minimize the possibility of false-positive artifacts. PLA with rabbit anti-Arc and mouse anti-calnexin C-terminus gave the same result as the reverse above, which was significantly higher than the negative control in BDNF-treated neurons (Figure 3D). When using the rabbit anti-calnexin N-terminal antibody, PLA with Arc was not significantly different from the negative controls, although it seemed slightly elevated (Figure 3D). These results reflect the further distance between the lumenal N-terminus of calnexin and cytoplasmic Arc. Inconsistencies during sample preparation (most importantly, membrane permeabilization by Triton-X-100) could enhance or reduce this proximity.

PLAs were also performed in coronal brain sections following HFS of the medial perforant path input to the DG of adult anesthetized rats. HFS induces robust upregulation of Arc in the granule cells of the DG, which is necessary for LTP maintenance (Steward et al., 1998; Messaoudi et al., 2007). Two hours after unilateral application of HFS, brains were transcardially fixed, embedded, cryosectioned and subjected to dual immunofluorescence or PLA. As expected, immunofluorescence showed that Arc was robustly upregulated in the DG granule cells of the ipsilateral hemisphere (Figure 4A), present both in nuclei and neuronal soma of the GCL (Figure 4B). Within the GCL, calnexin was seen in the perinuclear region of granule cell soma, but was excluded from the nuclei, as expected (Figure 4B). No obvious difference in calnexin expression was seen between the ipsilateral and contralateral brain hemispheres (data not shown). PLAs were then performed on neighboring sections of the same brains. PLA between Arc/Arc followed the same pattern as Arc expression—low in the contralateral hemisphere and high in the ipsilateral GCL (Figure 4C). PLA between calnexin and Arc is also significantly increased in the ipsilateral hemisphere, particularly in the GCL, but not the molecular layer (ML; Figures 4C,D). These assays demonstrate that the calnexin C-terminus and Arc interact in the DG GCL following HFS in vivo.

Lastly, we began to investigate the role of the Arc-calnexin interaction. Arc regulates endocytosis of AMPA receptors, where co-overexpression of Arc with endophilin increases AMPAR endocytosis (Chowdhury et al., 2006). Calnexin also regulates endocytosis, but overexpression of full-length calnexin or calnexin C-terminus impairs clathrin-mediated endocytosis (Li et al., 2011). We therefore examined whether the interaction affects receptor endocytosis by carrying out receptor uptake assays in neuroblastoma cells. The amount of endocytosed transferrin was quantified via confocal microscopy in three independent experiments (n = 63, 51 and 39 cells, respectively). No significant difference in endocytosis was detected between the Arc, calnexin, and Arc/calnexin groups (Figure 5; p = 0.246).