- Research article
- Open Access
The adhesion molecule Necl-3/SynCAM-2 localizes to myelinated axons, binds to oligodendrocytes and promotes cell adhesion
© Pellissier et al; licensee BioMed Central Ltd. 2007
- Received: 23 May 2007
- Accepted: 29 October 2007
- Published: 29 October 2007
Cell adhesion molecules are plasma membrane proteins specialized in cell-cell recognition and adhesion. Two related adhesion molecules, Necl-1 and Necl-2/SynCAM, were recently described and shown to fulfill important functions in the central nervous system. The purpose of the work was to investigate the distribution, and the properties of Necl-3/SynCAM-2, a previously uncharacterized member of the Necl family with which it shares a conserved modular organization and extensive sequence homology.
We show that Necl-3/SynCAM-2 is a plasma membrane protein that accumulates in several tissues, including those of the central and peripheral nervous system. There, Necl-3/SynCAM-2 is expressed in ependymal cells and in myelinated axons, and sits at the interface between the axon shaft and the myelin sheath. Several independent assays demonstrate that Necl-3/SynCAM-2 functionally and selectively interacts with oligodendrocytes. We finally prove that Necl-3/SynCAM-2 is a bona fide adhesion molecule that engages in homo- and heterophilic interactions with the other Necl family members, leading to cell aggregation.
Collectively, our manuscripts and the works on Necl-1 and SynCAM/Necl-2 reveal a complex set of interactions engaged in by the Necl proteins in the nervous system. Our work also support the notion that the family of Necl proteins fulfils key adhesion and recognition functions in the nervous system, in particular between different cell types.
- Green Fluorescent Protein
- Myelinated Axon
- Mammalian Expression Vector
- Aggregation Assay
- Central Pontine Myelinolysis
Multicellular organization entails cell-cell recognition and adhesion. The cell adhesion molecules (CAMs) are among the specialized plasma membrane proteins that carry out these functions. The mechanisms of recognition and adhesion are of particular relevance in the nervous system whose operation heavily relies on cell-cell communication, and whose many cell types acting in concert are capable of extensive re-organization in development, learning and memory. Recently two related CAMs, Necl-2-SynCAM [1–4] and Necl-1 , were shown to fulfill important functions in the central nervous system (CNS). In addition to acting as a CAM in other tissues [6–11], SynCAM can induce presynaptic differentiation in co-cultured neurons [1, 4], whereas Necl-1 is expressed specifically in brain and localizes at contact sites between neurons and glial cells . These two CAMs are Ig superfamily members and genomic analysis predicts that they are part of a set of four closely related proteins [1, 12–15] for which different nomenclatures have been proposed, in particular nectin-like 1 to 4 (Necl-1 to -4), and synaptic CAM 1 to 4 (SynCAM-1 to -4), each with its merits [1, 13, 15, 16].
Here we describe Necl-3/SynCAM-2, a previously uncharacterized member of the family, which we term Necl-3 throughout for simplicity and because the term is neutral with respect to function. Necl-3 shares with the other Necls/SynCAMs a conserved modular organization comprising three Ig domains, a single trans-membrane pass and a short cytoplasmic region containing 4.1 and PDZ binding motifs [1, 12–15]. Necl-3 accumulates in several tissues, including those of the nervous system, where it localizes to myelinated axons and in ependymal cells. We also demonstrate that Necl-3 engages in homo- and heterophilic interactions leading to cell aggregation and discuss its possible implication in processes dependent on neural cell adhesion.
Using the Necl-3 antibody, we ran western blots on extracts from a series of different organs. The Necl-3 protein was detected in brain structures, but also in kidney, heart and liver (Fig. 2B). Necl-3 migrates with an apparent mass of approximately 52 kDa, which is slightly more than its calculated molecular weight of 48 kDa. This is due at least in part to N-linked glycosylation since treatment with the N-glycosidase PNGaseF converts Necl-3 to a faster migrating form, consistent with the presence of N-glycosylation consensus sites in the extracellular domain (data not shown). We also monitored the accumulation of Necl-3 in the postnatal brain and found that the protein is barely detectable at P1 or P12 and has reached a plateau by P60 (Fig. 2C).
Necl-3 is targeted to the plasma membrane and engages in homo- and heterophilic interactions
The distribution of Necl-3 in the nervous system
Necl-3 selectively binds to oligodendrocytes
Several laboratories have predicted the existence of Necl-3, based on bioinformatic considerations [1, 12–15]. Biederer has done a particularly interesting analysis with respect to splice variants, gene size, and protein structure . Here we describe several of Necl-3's biological features. We demonstrate that it is a bona fide adhesion molecule able to engage in homo- and heterophilic interactions. The contacts Necl-3 makes with itself and with Necl-1, -2 and -4 are strong enough to withstand co-precipitation and cause cell aggregation. Likewise, Kakunaga et al. have shown that Necl-1 interacts with itself, Necl-2, nectin-1 and nectin-3 , whereas Necl-2/SynCAM binds to itself and to Necl-1, nectin-3 and CRTAM [1, 8–10, 12, 20, 27, 28]. It was also recently reported that Necl-1 and Necl-4 binds homo- and heterophilically and that Necl-2 and Necl-3 bind to Necl-1 and Necl-4 . In this latter report, the authors use a different technology than ourselves and reach similar conclusions, i.e. that Necl proteins engage in homo- and heterophilc interaction. The degree of promiscuity of the Necl proteins is an important biological question. It is unlikely that they only interact with the partners so far documented, but do they interact with many other Ig domain proteins and with proteins of other superfamilies? Here we show that Necl-3 does not react in trans with the Ig folds in IgG's and in ALCAM, but much more work will be needed to sort out the range of these interactions and clarify their biological relevance.
Using highly specific antibodies, we show that Necl-3 is enriched in myelinated axons in rat CNS and PNS. Our immuno-electron microscopy data indicate that Necl-3 accumulates at contact sites between neurons and oligodendrocytes. In addition, initial investigations suggest that Necl-3, similarly to Necl-1 [5, 26], is located at internodes on myelinated axons (data not shown). A function of Necl-3 compatible with these data, as well as with the fact that its maximal expression coincides with mature myelination, would be that Necl-3 participates in the myelin ensheathment of the axon [29, 30]. It could do so by "gluing" the oligodendrocytic processes to the axon shaft, a proposition that should be investigated in the future. Our adhesion assays show that Necl-3 preferentially adheres to oligodendrocytes. Since it is mostly expressed in neurons, this suggests that although capable of forming homophilic trans-interactions, Necl-3 preferentially forms heterophilic interactions. It raises the question of the identity of the Necl-3 interaction partner(s) in the oligodendrocyte. A good candidate for such a partner is Necl-1, as it is present in oligodendrocytes [5, 31] and binds to Necl-3 (this work). A recent report showed very convincingly that in the PNS Necl-1 is axonal and segregates preferentially to the internodal membrane whereas Necl-4 is expressed in Schwann cells and heterodimerizes with Necl-1, thereby forming a pair mediating axon-glia contact . Necl-4 is therefore a likely partner for Necl-3 in the CNS, as these two Necl proteins are able to make heterophilic interactions (our work and ). Indeed Spiegel et al. also report, as data not shown, that in the CNS Fc-Necl-3 bind to oligodendrocytes . Consequently, Necl-3 (this report and ) and Necl-1 [5, 26] are likely involved in the establishment axon-glia contacts. However, numerous other oligodendrocytic proteins could also serve as plausible Necl-3 partners [31, 32].
The complex set of interactions engaged in by the Necl proteins suggests that they fulfill important cell adhesion and recognition functions. Our results reinforce the notion that the Necl family of proteins is involved in cell adhesion and recognition in the nervous system, as recently proposed . There, Necl-1 and Necl-3 would favor neuron-glia contact (this work and ) through heterophilic binding to Necl-4 ; and Necl-2 would enable neuron-neuron contact [1, 15]. Intriguingly, Necl-2 also promotes interactions between the nervous and immune systems [20, 27, 28, 33]. It will be of interest to test whether other Necl family members fulfill similar neuro-immune functions. It also points to a possible mechanism for the interaction between lymphocytes and myelinated axons as it occurs in demyelinating diseases such as multiple sclerosis, leukodystrophy, central pontine myelinolysis and others. Here, Necl-2 on lymphocytes would interact with Necl-4 on oligodendrocytes or Necl-1, Necl-2, or Necl-3 on the axon shaft. The Necl proteins may also be involved in certain aspects of nervous system regeneration and repair. In a PNS remyelination model, Necl-4 appears to be important for this process , and Necl-2 (SgIGSF) mRNA and protein are upregulated in a model of olfactory epithelium nerve transection . Since Necl-3 is not present exclusively in the nervous system, it will be important to assess its cellular localization in the other tissues that express it.
In conclusion, our data describe some of the properties of the novel adhesion molecule Necl-3 and highlight its implication in the nervous system. This work also supports the notion that the Necl family of proteins fulfills important adhesion and recognition functions in the nervous system.
PCR reactions were done with Pfu Turbo polymerase (Stratagene) according to the supplier's instructions. The mammalian expression vectors pNecl-3-GFP and pNecl3-DsRed2 were constructed as follows: the cDNA DKFZp761G128 (accession AL834270) was used as a template for PCR amplification with the primers 5'-GGAATTCATGATTTGGAAACGC-3' and 5'-CCCCAATTGCAATGAAATACTCTTT-3. The resulting amplicon was digested with EcoR1 and Mfe1 and ligated into the EcoR1 site of pEGFP-N1 and pDsRed2-N1 (BD Biosciences Clonetech). The mammalian expression vector pNecl-3-myc was constructed as follows: the cDNA DKFZp761G128 was used as a template for PCR amplification with the primers 5'-GGAATTCATGATTTGGAAACGC-3' and 5'-CCCCAATTGCAATGAAATACTCTTT-3. The resulting amplicon was digested with EcoR1 and Mfe1 and ligated into the EcoR1 site of pcDNA3.1/myc-His (Invitrogen). The mammalian expression vector pFc-Necl-3 was constructed as follows: the plasmid pNecl-3-myc was used as a template for PCR amplification with the primers 5'-AGGTCTATATAAGC-3' and 5'-GGGGTACCAGGGCCATTCTGGCC-3. The resulting amplicon was digested with Nhe1 and Kpn1 and ligated into the plasmid pIgplus (kindly provided by Patrick Doherty) digested with the same enzymes. The insect cell expression vector pMT-Fc-Necl-3 was constructed as follows: the pFc-Necl-3 vector was digested with Nhe1 and Apa1 and the resulting insert was ligated into the plasmid pMT (Invitrogen), digested with Spe1 and Apa1. The insect cell expression vector pMT-Necl-3-GFP and pMT-Necl-3-DsRed2 were constructed as follows: the pNecl-3-GFP and pNecl-3-DsRed2 vectors were digested with EcoR1 and Not1 and the resulting inserts were ligated into the plasmid pMT (Invitrogen), digested with the same enzymes. The bacterial expression vector pQE-Necl-3(aa169-367) was constructed as follows: the plasmid pNecl-3-myc was used as a template for PCR amplification with the primers 5'-AGGTCTATATAAGC-3' and 5'-GGGGTACCAGGGCCATTCTGGCC-3. The resulting amplicon was digested with Dra1 and Kpn1 and ligated into the blunted BamH1 and Kpn1 sites of the plasmid pQE-30 (Qiagen). The insect cell expression vectors pMT-Necl-1-GFP was constructed as follows: the cDNA IMAGE:5199627 (accession BC033819) was used as a template for PCR amplification with the primers 5'-CCCCAATTGATGGGGGCCCCAGCCGC-3' and 5'-CCCCAGATCTAAGATGAAATATTCCTTCTTGTCGTCCCC-3. The resulting amplicon was digested with Bgl2 and Mfe1 and ligated into the EcoR1 and BamH1 sites of pMT-Necl-3-GFP. The mammalian expression vector pNecl-2-GFP was constructed as follows: the cDNA IMAGE:4701395 (accession BC035930) was used as a template for PCR amplification with the primers 5'-GGAATTCATGGCGAGTGTAGTG-3' and 5'-CCCCAATTGCGATGAAGTACTCTTT-3. The resulting amplicon was digested with EcoR1 and Mfe1 and ligated into the EcoR1 site of pEGFP-N1 (BD Biosciences Clonetech). The insect vector pMT-Necl-2-GFP was constructed as follows: the pNecl-2-GFP vector was digested with EcoR1 and Not1 and the resulting inserts were ligated into the plasmid pMT (Invitrogen), digested with the same enzymes. The insect vector pMT-ALCAM-GFP was constructed as follows: the cDNA DKFZp667I089 (accession AL833702) was used as a template for PCR amplification with the primers 5'-CCCGAATTCATGGAATCCAAGGGGGCC-3' and 5'-CCCGGATCCAAGGCTTCAGTTTTGTGATTGTTTTCT-3. The resulting amplicon was digested with EcoR1 and BamH1 and ligated into the EcoR1 and BamH1 sites of pMT-Necl-3-GFP. The insect vector pMT-Necl-4-GFP, will be described in a manuscript in preparation (details are available on request).
Cell transfection and aggregation assays
S2 Drosophila cells were maintained in Schneider's Drosophila Medium (Gibco) supplemented with 10% FCS. They were transfected with Lipofectamine 2000 (Invitrogen) in 6-well culture plates according to the manufacturer's recommendations. 24 hours after transfection the medium was changed and 0.5 mM CuSO4 was added for another 24 hours. For aggregation assays, relevant cells were mixed after the medium change, induced with CuSO4 and gently shaken for several hours to allow for plasmid transcription, protein accumulation and aggregate formation.
Hela cells were maintained in DMEM 10% FCS and transfected with Fugene (Roche) according to the manufacturer's recommendations.
The E. coli strain M15 was transformed with the bacterial expression vector pQE-Necl-3(aa169-367), and used to express the corresponding Necl-3 fragment. Bacteria were grown exponentially, IPTG was added to 1 mM, and the cells were allowed to grow for another 4 h at 37°C. Cells were then harvested and lysed. The cleared supernatants were loaded under denaturing conditions onto Ni2+-nitrilotriacetic acid agarose columns (Qiagen), according to the manufacturer's recommendations. The eluted proteins were then extensively dialyzed against PBS and used for immunization.
The Fc-Necl-3 fusion protein was prepared as follows: the insect cell expression vector pMT-Fc-Necl-3 was co-transfected in S2 cells along with the plasmid pCoBlast that carries a blasticidin S resistance gene (Invitrogen). Stable clones were selected with blasticidin S according to the manufacturer's recommendations. Of these, high expressers of the Fc-Necl-3 fusion were selected. One such clone was expanded and grown in a 150 ml flat bottom flask and induced with 0.5 mM CuSO4. After 4 days, the conditioned culture medium was loaded onto a Protein A Sepharose CL-4B columns (Amersham Biosciences), and the Fc-Necl-3 fusion protein was recovered according to the manufacturer's recommendations.
The Necl-3(aa169-367) fragment produced in E. coli was used by a commercial producer (Eurogentech, Belgium) to immunize two rabbits. The same Necl-3(aa169-367) fragment was coupled to NHS activated Sepharose 4 Fast Flow (Amersham Biosciences) according to the manufacturer's recommendations, and used for affinity purification of the anti Necl-3 antibodies from the rabbit sera. The antibody-containing fractions were then loaded onto a Protein A Sepharose CL-4B column (Amersham Biosciences). The beads were washed with 10 mM Tris pH 7.5, then 10 mM Tris pH 7.5, NaCl: 500 mM, and were then rocked for 2 hours at 4C as a suspension in 200 ml of 10 mM Tris pH 7.5, NaCl: 500 mM, in order to select only the antibodies with a very slow off-rate. The beads were then washed again with 10 mM Tris pH 7.5, and then 10 mM Tris pH 7.5, NaCl: 500 mM. Finally the IgGs were eluted according to the manufacturer's recommendations.
Crude lysates from transfected S2 cells, or rat tissues were heated and sonicated in Laemmli sample buffer. Proteins were separated on a 12% SDS-polyacrylamide gels, and transferred to nitrocellulose membranes. These were probed with the appropriate antibodies and revealed by chemiluminescence (ECL, Amersham).
S2 cells were transfected with the pMT-Necl-2-GFP plasmid in 6-well culture plates. After 48 hours the cells were washed in PBS and lysed in 400 μl RIPA buffer (Tris-HCl: 50 mM, pH 7.4, NP-40: 1%, NaCl: 150 mM, EDTA: 1 mM, and protease inhibitors). 100 μl of the resulting lysate was pre-cleared for 30 min at 4°C with 25 μl (bed volume) of Protein A Sepharose CL-4B resin. After pre-clearing, the lysates was rocked 45 min at 4°C with the Fc-Necl-3 fusion protein immobilized on Protein A Sepharose CL-4B resin. The beads were then washed 3 times with 500 μl RIPA buffer, and boiled in 1× Laemmli sample buffer for western blot analysis.
Petri adhesion assays were done as described by Milner et al. . Briefly, the indicated proteins at a concentration of 15 ng/μl were coated for 1 h at 37°C on a bacteriological grade Petri dish, after which unbound proteins were washed away with PBS. P1 rat cortices were dissected in ice cold HBSS, meninges were removed, and the tissue was minced with a fire-polished Pasteur pipette. A single cell suspension was prepared by passing the homogenate through a nylon mesh, and the cells were suspended in 10 ml Neurobasal medium (Gibco/Invitrogen) supplemented with 10% FCS and antibiotics. They were then spun for 5 min at 1500 g and resuspended at a concentration of 106 per ml. 50 μl of this suspension was added to the coated dish for 40 min at 37°C. Unbound cells were washed away by rinsing and gentle shaking in the same medium, and the bound cells were incubated for 24 h in fresh medium before imaging. Quantification was done manually on pictures taken with a microscope-mounted Leica digital camera at 200× magnification. Adhesion assay with Fc-Necl-3 added to the 14 DIV neonatal cortex cultures was done exactly as described , using 300 ng/100 μl of Fc-Necl-3 or human IgGs.
Confocal and wide field immunofluorescence microscopy
Immunohistochemistry in the nervous system was done as follows: two-month-old rats were anaesthetized, their brains were rapidly excised and immersed for 5 min in isopentane pre-cooled to -20°C. Brains were then embedded in OCT medium (Miles Inc., Elkhart, IN) and cryo-sectioned at 12-μm. Sections were exposed for 10 min to -20°C absolute ethanol, rinsed in PBS and incubated for 30 min in PBS containing 2% BSA. Sections were then incubated for 2 h at 37°C in PBS, 0.5% BSA, containing the affinity-purified anti Necl-3 antibody (diluted 1:100) and the appropriate mouse monoclonal antibody. After washes in PBS, highly cross-adsorbed Alexa Fluor secondary antibodies (Molecular Probes) were used to detect the mouse and rabbit primary antibodies (with Alexa 488 and Alexa 594, respectively). Sections were then rinsed again in PBS and stained with Hoechst. Confocal laser scanning was performed on a Leica SP2 microscope (Leica, Germany) using 20 mW blue diode (405 nm), 30 mW ArKr (488 nm), 1 mW HeNe (594 nm) lasers and a 63× plan Apo oil immersion objective. Section planes were collected in steps through the entire cell thickness. Images of each field were merged using the Leica Confocal software. Z-section analysis was done with the Imaris software.
For immunofluorescence on cell cultures, Hela cells were grown on glass coverslips and transfected as indicated above. They were then fixed in PBS 3% paraformaldehyde for 5 min and quenched 5 min in PBS 50 mM NH4Cl. The cells were then blocked with PBS 10% FCS for 20 min, and incubated in PBS, 0.5% BSA, containing the affinity-purified anti Necl-3 antibody (diluted 1:200). After washes in PBS, highly cross-adsorbed Alexa Fluor secondary antibodies were as above. Coverslips were then rinsed again in PBS, and stained with Hoechst. Widefield microscopy was performed on a Zeiss AXIOZ1 microscope with a 63× oil immersion objective. Mouse mAbs were obtained from the following suppliers: DSHB (University of Iowa), neurofilament and Rip; Roche, GFAP and GFP; Serotec, CD11b; Chemicon, MBP and Upstate, Myc tag.
Anesthetized rats were perfused with PBS containing 2% paraformaldehyde, 0,1% glutaraldheyde. Brain were dissected and put overnight in PBS with 2% paraformaldehyde, 25% sucrose for cryoprotection, then embedded in O.C.T. medium (Miles Inc., Elkhart, IN) and cryo-sectioned at 12 μm thickness. Sections were put on superfrost slides (Menzel GmbH, Braunschweig, Germany) and incubated for 30 min with PBS plus 0,1 % BSA (New England Biolabs, USA). Sections were incubated overnight in a humid chamber at 4°C with 40 μl of anti-Necl-3 antibody diluted (1:50) in PBS plus 0,1% BSA. They were then washed 5 times for 20 min with PBS plus 0,1 % BSA and incubated in a humid chamber with 40 μl of anti-rabbit IgG FAB fluoronanogold Alexa 488 (Nanoprobes Inc., Yaphank, NY, USA) diluted (1:10) in PBS (0,1% BSA, 0,1% FSK gelatine) for 2 h at room temperature. Sections were first washed with PBS plus 0.1% BSA (3 times 20 min) and then 3 times with 0,1 M sodium cacodylate (pH 7.2). After fixation with 2% glutaraldheyde in 0,1 M sodium cacodylate (30 min at room temperature) specimen were further washed with 0,1 M sodium cacodylate and distilled water. Gold enhancement of the nanogold particles was performed for 4 min according to the manufacturers' recommendations (Nanoprobes, Inc.). After enhancement, sections were washed 4 times with water and 3 times 10 min with 0,1 M sodium cacodylate. Sections were post-fixed in 0,1 M OsO4 for 1 h, washed 3 times 5 min with 0,1 M sodium cacodylate and 5 times 5 min with water and stained with uranyl acetate. Finally sections were dehydrated and embedded in resin (Agar 100 Resin, Agar Scientific, UK). Sections of 70 nm were collected on 200 slot grids, counterstained with half saturated uranyl acetate then 2.5 % lead citrate and analyzed using either a TEM Philips 410 or a Tecnai G2 electron microscope.
Real time PCR analysis
Real time PCR analysis were prepared with SYBR green (Molecular Probes) as described  on an iCycler iQ Bio-Rad station, with quantitation, melt curve, and PCR efficiency analysis derived by the station's software. RNA purification and cDNA synthesis were prepared as described . The primers used for amplifying Necl-3 were 5'-TGACCATGCTCTCATAGG-3' and 5'-TGCCAGATATCGACCAAG-3', those for GAPDH were 5'-TGATTCTACCCACGGC-3' and 5'-TGATGGGTTTCCCATTGATGA-3'.
We are grateful to Estelle Jorand, Michèle Geindre, Carmela Villani, Daria Gavriouchkina, Nathalie Girardin, and Iona Garcia for help at various stages of this project. We thank Jozsef Kiss and Jean Gruenberg for critical reading of the manuscript. We are indebted to Jorge Ritz and to the NCCR Frontiers in Genetics imaging platform for technical assistance. Our laboratory is supported by the Swiss NSF, the State of Geneva and the ProVisu Foundation.
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