Isolation of cDNAs for Necl proteins
Total RNA was isolated from purified rat Schwann cells or rat DRG neurons with the RNAqueous-4PCR kit (Ambion); mRNA from mouse brain was purified using the mRNA isolation kit (Roche). First-strand templates were prepared by RT-PCR with an oligo-dT primer (Promega). cDNAs for Necl-1–4 were amplified using primers based on rodent sequences and cloned into the pcDNA3.1 directional TOPO cloning vector (Invitrogen).
To clone the rat Necl-1, sense (ATGGGGGCCCCTTCCGCCCT) and antisense (CTAGATGAAATATTCCTTCTTGTCATCCCCGCC) primers were used to PCR amplify a full-length cDNA from rat DRG neuron first strand (GenBank/EMBL/DDBJ accession no. DQ272743
). To clone Necl-2, we designed primers based on the mouse cDNA for TSLC1 (Fukami et al., 2002
). We amplified a full-length cDNA from adult mouse brain with sense (ATGGCGAGTGCTGTGCTGCC) and antisense (CTAGAAGTACTCTTTCTTTTCTTCGGAGTT) primers. This mouse isoform of Necl-2 corresponds to isoform 4 of SynCAM 1 (Biederer et al., 2002
) that lacks the putative mucin-like domain of the predicted rat sequence. The mouse Necl-2 cDNA is available from GenBank/EMBL/DDBJ (accession no. DQ279856
). Necl-3 was amplified from rat Schwann cell first strand using sense (ATGATTTGGAAACGCAG) and antisense (TTAAATGAAATACTCTTTTTTCTC) primers, and the sequence is available from GenBank/EMBL/DDBJ (accession no. DQ272744
). A full-length Necl-4 cDNA was amplified from rat Schwann cell first strand using sense (ATGGGCCGGGCCCGGCGCTTCC) and antisense (AATGAAGAATTCTTCTTTCCGTTTGTGTCCATCGCCGC) primers, and the sequence is available from GenBank/EMBL/DDBJ (accession no. DQ272745
Generation of permanently transfected CHO cell lines expressing Necl proteins
CHO cells were transfected with full-length constructs for each Necl protein and with the empty pcDNA3.1 vector using the LipofectAMINE 2000 reagent (Invitrogen); permanently transfected cells were selected by maintenance in G418-containing medium (α-MEM, 10% FBS, and 750 μg/ml G418). To facilitate analysis, the sequence encoding the influenza HA epitope was added by PCR to the N terminus of each Necl protein immediately after the signal peptide as predicted by the SignalP program. CHO cells were stained for the HA epitope and selected by FACS (MoFlo; DakoCytomation).
Generation of Necl-Fc fusion proteins
Constructs encoding the extracellular domain of each Necl protein fused to the hinge region of the human IgG-Fc were subcloned into the pcDNA 3.1 TOPO vector (Invitrogen) and transiently transfected into HEK 293FT cells (Invitrogen). Transfected cells were maintained in media containing DME, 1% Ultra Low IgG FBS (Invitrogen), 1 mM nonessential amino acids (Invitrogen), and 2 mM l-glutamine. Conditioned media was collected after 3 d and alkalinized by 1 M Hepes buffer (Invitrogen) at 10% vol, and the salt concentration was increased by adding 10× Dulbecco's PBS (dPBS) at 25% vol. The conditioned media was then filtered, and Necl-Fc proteins were collected batchwise with protein A–agarose beads (Roche). The beads were washed with 6× dPBS plus 0.2% Triton X-100 (Sigma-Aldrich) followed by 6× dPBS and 1× dPBS washes. The proteins were eluted off the beads with 200 mM glycine buffer at pH 2.8, immediately neutralized with 1 M Hepes, pH 8.5, at 10% vol, and dialyzed overnight against dPBS (1:500 vol/vol) using Slide-A-Lyser dialysis cassettes (Pierce Chemical Co.). Protein concentrations were assessed with the BCA Protein Assay (Pierce Chemical Co.). Biochemical analysis confirmed that each construct encoded proteins of the predicted size of ~65 kD after deglycosylation.
Antibodies and immunofluorescence
Guinea pig polyclonal antibodies were generated against Necl-1, -3, and -4 by immunization with the corresponding Necl-Fc fusion protein. Antibodies to the human Fc moiety were removed by passing antiserum over an agarose–human IgG (Jackson ImmunoResearch Laboratories) column. Rabbit polyclonal antibodies to Necl-2 have previously been described (Masuda et al., 2002
; Surace et al., 2004
). A rabbit polyclonal antibody was raised to the C-terminal sequence (AEGGQSGGDDKKEYFI) of Necl-1 coupled to keyhole limpet hemocyanin and injected into rabbits, and the resulting antibodies were affinity purified against the immunizing peptide coupled to Sepharose beads. Other antibodies included mouse monoclonal antibodies to MBP (SMI-94) and neurofilament (SMI-31 and SMI-32; all obtained from Sternberger Monoclonals) and the HA epitope (HA.11; Covance). Polyclonal antibodies specific to Krox-20 and Oct-6 (provided by D. Meijer, Erasmus University, Rotterdam, Netherlands), Caspr (gifts from E. Peles [Weizmann Institute, Rehovot, Israel] and M. Bhat [University of North Carolina, Chapel Hill, NC]), MAG (Pedraza et al., 1990
), and neurofilament (Covance) were also used. Secondary antibodies included donkey anti–mouse (FITC, rhodamine X, Cy5, and amino-methylcoumarin conjugated), donkey anti–guinea pig (FITC and rhodamine X conjugated), donkey anti–rabbit (FITC and rhodamine X conjugated), donkey anti–chicken (Cy5 and amino-methylcoumarin conjugated), and goat anti–human-Fc (FITC and rhodamine X conjugated; Jackson ImmunoResearch Laboratories). For FACS analysis, we also used phycoerythrin (PE)-conjugated donkey anti–mouse (eBioscience).
Immunofluorescent preparations were examined by epifluorescence on a confocal microscope (LSM 510; Carl Zeiss MicroImaging, Inc.). Confocal images were acquired with Neofluor 40× NA 1.3 oil or Apochromat 63× NA 1.4 oil objectives (Carl Zeiss MicroImaging, Inc.) on an 8-bit photomultiplier tube (Carl Zeiss MicroImaging, Inc.) using LSM software (Carl Zeiss MicroImaging, Inc.). In some cases, brightness and contrast were adjusted with Photoshop 7.0 (Adobe).
Neuron and Schwann cell cultures
Establishment of primary rat Schwann cell and DRG neuron cultures has been described previously (Einheber et al., 1993
). In brief, neurons were isolated from embryonic day 16 DRGs by trypsinization and plated on a collagen substrate (Biomedical Technologies Inc.) in standard neuronal medium (neurobasal medium, 2% B27 supplement, 2 mM l
-glutamine, 0.4% glucose, and 50 ng/ml 2.5S NGF). Nonneuronal cells were removed by feeding the cultures every 2 d alternately with standard neuronal medium supplemented or not supplemented with 5-fluorodeoxyuridine and uridine (both at 10 μM) over a week. Schwann cells prepared from postnatal day 2 sciatic nerves (Brockes et al., 1979
) were expanded in D media (DME, 10% FBS, and 2 mM l
-glutamine) supplemented with 4 μM forskolin and 5 ng/ml of the EGF domain of rhNRG-1-β1 (over a period of 2–3 wk; R&D Systems). Schwann cells were then maintained in D media for 3 d before use. Myelinating Schwann cell–neuron cocultures were established by seeding purified DRG neuron cultures with 200,000 Schwann cells in C media (MEM, 10% FBS, 2 mM l
-glutamine, 0.4% glucose, and 50 ng/ml 2.5S NGF). After 3 d, cocultures were supplemented with 50 μg/ml ascorbic acid to initiate basal lamina formation and myelination and were maintained for an additional 10–21 d.
DME was obtained from BioWhittaker Bioproducts. α-MEM, MEM, neurobasal media, B27 supplement, l-glutamine, trypsin, and G418 were purchased from Life Technologies. Glucose, forskolin, 5-fluorodeoxyuridine, uridine, and ascorbic acid were purchased from Sigma-Aldrich. FBS was obtained from Gemini, and NGF was purchased from Harlan Bioproducts.
Preparation of teased sciatic nerves
Sciatic nerves were removed from 10–12-mo-old C57BL mice, fixed with 4% PFA (Electron Microscopy Sciences) for 2 h, and stored in dPBS (Invitrogen) at 4°C until teased. Teased sciatic nerve fibers were mounted on glass slides, dried overnight at room temperature, and stored at −80°C until use for immunofluorescence staining.
Necl binding and FACS analysis
To analyze the binding of Necl proteins to Schwann cells, DRG neurons, and CHO-Necl clones, cells were rinsed with L15 medium (Invitrogen) and incubated for 45 min at RT with conditioned media from transiently transfected HEK 293FT cells secreting specific Necl-Fcs. Cultures were washed once with L15 and incubated with a FITC- or rhodamine X–conjugated goat anti–human Fc for 45 min at room temperature. After washing with dPBS, cultures were fixed with 4% PFA. Schwann cells and CHO-Necl clones were mounted in the presence of Hoechst dye (Invitrogen) nuclear stain. DRG neurons were further stained for neurofilaments before mounting.
To further characterize homophilic versus heterophilic Necl binding, we performed a FACS analysis with Necl-Fc constructs. CHO cells expressing Necl proteins were briefly trypsinized for 1 min with 0.125% trypsin and 0.5 mM EDTA at room temperature, collected in ice-cold HBSS medium (Invitrogen) containing 10% FBS to terminate trypsinization, and washed twice in HBSS medium. Cells were diluted in HBSS to a final concentration of 750,000 cells in 200 μl and incubated with either one of the Necl-Fc proteins or whole human IgG at a final concentration of 200 nM; cells were incubated on ice for 30 min. Anti-HA antibody at 1:500 was also added to detect the CHO-Necl cells. After incubation, cells were washed once with ice-cold HBSS and incubated with PE-conjugated donkey anti–mouse (to detect expressing cells) and FITC-conjugated anti–human Fc (to detect Necl-Fc binding; both at 1:100) for 30 min on ice. After two washes in ice-cold HBSS and one wash in Ca2+/Mg2+-free ice-cold dPBS, fluorescence measurements of individual cells were performed using FACS (FACScan; Becton Dickinson). Log fluorescence was collected for FITC (channel FL1-H) and PE (channel FL2-H) and displayed as double-parameter (PE/FITC) graphs. For each CHO-Necl clone, the FL2-H channel was calibrated by labeling cells for the HA tag only (no FITC), whereas the FITC channel (no PE) was calibrated by detecting Necl-Fc, giving the strongest binding as determined initially by binding to cells in culture. Additional controls included omitting anti-HA antibodies and Necl-Fc proteins, with only PE- and FITC-conjugated secondary antibodies added. Analysis was first gated on single cells on the basis of forward and side light scatter based on data acquisition from 10,000 cells. Each CHO-Necl clone was incubated with human IgG as a control. The FITC fluorescence value below which 99% of the events were found was noted and used as the boundary between no binding versus binding (Fig. S4 B, left and right red boxes). Binding of Necl-Fc was determined by counting the number of cells in the binding gate and dividing by the total number of cells. FACS analysis was performed with the FlowJo software package (Tree Star, Inc.).
Cell adhesion assay
Necl-Fc proteins serially diluted in HBSS were spotted (2 μl/spot) onto 100-mm nontissue culture polystyrene dishes (Fisherbrand; Fisher Scientific) to minimize nonspecific cell attachment to the plastic. Duplicate spots were made for each dilution of Necl-Fc per experiment. Plates were incubated in a 37°C tissue culture incubator for 1 h, washed twice with HBSS, blocked with 1% BSA in HBSS for 1 h at 37°C, washed twice with HBSS, and washed once with D media. Schwann cells were briefly trypsinized, resuspended in D media at a density of 250,000 cells/ml, and 10 ml were added to each plate. The cultures were incubated in a 10% CO2 and 37°C tissue culture incubator for 90 min. The plates were then gently washed with dPBS twice, and cells were fixed with 4% PFA. Phase-contrast digital images of the spots (10× fields) were captured with a microscope (Eclipse TE2000-U; Nikon), and the number of cells per field was determined using ImageJ software (National Institutes of Health).
RNAi of Necl proteins
To generate shRNAs, we used the pLentiLox (pLL3.7) vector in which the U6 promoter drives shRNA expression and GFP is expressed under separate promoter control (provided by L. Van Parijs; Rubinson et al., 2003
; Dillon et al., 2005
). Two 21 nucleotide shRNAs (#1: nt 79–99, GTGCAGACAGAGAATGTGACG; #2: nt 151–171, GGGTCTATAGTCGTCATTCAG) that targeted sequences within the first IgG domain of Necl-4 were designed using Easy siRNA (ProteinLounge), BLOCK-iT RNAi designer (Invitrogen), and siRNA sequence selector (CLONTECH Laboratories, Inc.). The shRNA stem loops for pLL3.7 vector were designed to contain a sense shRNA sequence followed by a short (9 nt) nonspecific loop sequence and an antisense shRNA sequence followed by six thymidines, which serve as a stop signal for RNA polymerase III. The 5′-phosphorylated PAGE-purified oligonucleotides were annealed and subcloned into HpaI–XhoI sites of pLL3.7. The lentiviral vector was transfected into 293FT cells together with packaging plasmids Δ8.9 and pCMV-VSVG (provided by J. Milbrandt, Washington University, St. Louis, MO) using LipofectAMINE 2000 (Invitrogen). As controls, we used the empty pLL3.7 vector or a vector encoding shRNA to a nonspecific (luciferase) sequence. Viral supernatants were collected 72 h after transfection, centrifuged at 3,000 rpm for 15 min, aliquoted for one-time use, and frozen at −80°C. An shRNA targeting a 21-nt sequence (nt 733–753; GTGCAGACAGAGAATGTGACG) in the third IgG domain of Necl-1 was designed using the same approach.
For rescue experiments, a codon-modified Necl-4 construct was generated using the QuikChange XL Site-Directed Mutagenesis kit (Stratagene). The sequence GTGCAGACAGAGAATGTGACG (nt 79–99) was replaced with sequence GTACAAACGGAAAACGTAACA, which did not change the amino acid composition of Necl-4 but rendered the construct insensitive to the Necl-4–specific shRNA #1. The modified Necl-4 was subcloned into the pLenti6/V5 Directional Topo vector (Invitrogen), and viral production was performed as per the pLL3.7 constructs.
Freshly plated Schwann cells (106 cells per 100-mm plate) were incubated for 3 d with viruses at a 2/3 dilution (vol/vol) in D media (DME, 10% FBS, and 2 mM l-glutamine) supplemented with forskolin and rhNRG-β1 (EGF domain). Cells were expanded for an additional week and maintained for 3 d in D media before use. Protein knockdowns were confirmed by Western blotting and by immunohistochemistry.
Northern blot analysis
RNA was isolated from rat sciatic nerves and Schwann cells by CsCl2
gradient centrifugation (Chirgwin et al., 1979
). Equal amounts (10 μg) of total RNA were electrophoresed in 1% agarose and 2.2 M formaldehyde gels, transferred to nylon membranes (Duralon; Stratagene) in 6× SSC, and UV cross-linked (0.12 J). Blots were prehybridized, hybridized, and washed using standard techniques; the final stringency of the wash was 0.2× SSC at 65°C for 30 min (Sambrook et al., 1989
). cDNAs corresponding to nt 1–981 of rat Necl-1 and nt 1–966 of rat Necl-4 were used as probes. The probes were generated by PCR, isolated by agarose gel electrophoresis, and purified with the QIAquick Gel Extraction kit (QIAGEN). 32
P-labeled cDNA probes with specific activities of 2–5 × 109
cpm/μg were prepared by primer extension with random hexamers using the Prim-a-gene kit (Promega) according to the manufacturer's instructions
Online supplemental material
Fig. S1 presents amino acid sequences of the Necl proteins. Fig. S2 shows Necl expression CHO cell lines and specificity of anti-Necl antibodies. Fig. S3 shows PCR of Necl-2 isoforms and Northern analysis of Necl-1. Fig. S4 presents the binding of Necl-Fc fusion proteins to Necl-expressing cells. Fig. S5 shows that the knockdown of Necl-4 inhibits myelination in cocultures. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200705132/DC1