InteractiveFly: GeneBrief

Contactin: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - Contactin

Synonyms -

Cytological map position - 82A6

Function - receptor

Keywords - septate junctions, CNS, neural insulation

Symbol - Cont

FlyBase ID: FBgn0037240

Genetic map position - 3R

Classification - C-lectin domain, Ig domains, FNIII repeats, GPI-anchor sequence

Cellular location - surface



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

Septate junctions (SJs) in epithelial and neuronal cells play an important role in the formation and maintenance of charge and the size of selective barriers. They form the basis for the ensheathment of nerve fibers in Drosophila and for the attachment of myelin loops to axonal surface in vertebrates. The cell-adhesion molecules NRX IV/Caspr/Paranodin (NCP1), contactin and Neurofascin-155 (NF-155) are all present at the vertebrate axo-glial SJs. Mutational analyses have shown that vertebrate NCP1 and its Drosophila homolog, Neurexin IV (Nrx IV) are required for the formation of SJs. The Drosophila homolog of vertebrate contactin, Cont, has been characterized genetically, molecularly, and biochemically. Ultrastructural and dye-exclusion analyses of Cont mutant embryos show that Cont is required for organization of SJs and paracellular barrier function. Cont, Neuroglian (Nrg) (Drosophila homolog of NF-155) and Nrx IV are interdependent for their SJ localization and these proteins form a tripartite complex. Hence, these data provide evidence that the organization of SJs is dependent on the interactions between these highly conserved cell-adhesion molecules (Faivre-Sarrailh, 2004).

The evolution of a unique set of intercellular junctions in multicellular organisms allows their regulation of the solute/solvent exchange and the preservation of the microenvironment of epithelial and excitable cells, thus preventing paracellular diffusion. Invertebrates and vertebrates evolved septate or tight junctions, respectively, to perform this function. In Drosophila, apicolateral SJs between epithelial cells act as functional analogs to the vertebrate tight junctions. They are characterized by regular arrays of electron-dense transverse structures or septa that span the intermembrane space and form a physical barrier to diffusion. Besides serving as a paracellular barrier to diffusion, SJs play additional roles in the maintenance of epithelial polarity and cell adhesion in Drosophila. In the Drosophila nervous system, SJs are formed by the perineurial glial cells that ensheath the nerve fibers. In vertebrates, SJs are encountered at the axo-glial interface in the paranodal region of myelinated nerve fibers, acting as ionic barriers and molecular fences to maintain distinct molecular domains at the nodes of Ranvier (Faivre-Sarrailh, 2004 and references therein).

Several molecular components of the vertebrate axo-glial SJs have been identified, which include NCP1, contactin and NF-155 as the major cell-adhesion molecules (CAM). F3/F11/contactin is a GPI-anchored CAM that contains immunoglobulin domains linked to fibronectin type III (FNIII) repeats (Gennarini, 1989; Brümmendorf, 1996). In vertebrates, contactin is predominantly expressed by neurons and has been implicated in the control of axonal growth and fasciculation through heterophilic interactions with multiple ligands (Berglund, 1999; Falk, 2002), including the L1-type molecules, L1-CAM, NrCAM and neurofascin (Brümmendorf, 1996) (Faivre-Sarrailh, 2004 and references therein).

A major role of contactin in myelinated fibers is to organize axonal subdomains at the nodes of Ranvier. The nodal region is highly enriched in voltage-gated Na+ channels, thereby allowing the rapid saltatory conduction of the action potential. At either end of the node, in the paranodal region, a series of septate-like junctions anchors the myelin terminal loops to the axolemma. Contactin is an essential axonal component of the paranodal SJs, where it forms a cis-complex with NCP1 (Menegoz, 1997; Peles, 1997). Deficiency in either NCP1 or contactin results in a loss of SJs and an aberrant organization of the paranodal region, which causes a severe reduction in nerve conduction velocity (Bhat, 2001; Boyle, 2001). The glial ligand NF-155 was shown to form a ternary complex with NCP1 and contactin at the axon-glial interface of the paranodes (Charles, 2002) (Faivre-Sarrailh, 2004 and references therein).

Nerve ensheathment in Drosophila and myelination in vertebrates share important common features, including process extension around axons and formation of SJs, which isolate the nerve fibers from the extracellular fluid. Nrx IV (Nrx: FlyBase), the fly homolog of NCP1, plays a crucial role in the formation of SJs in perineurial glial cells in the peripheral nervous system (PNS) and is required for the integrity of the blood-nerve barrier and the proper conduction of nerve impulses. NF-155 belongs to the L1-type family which in Drosophila is encoded by a single gene, nrg. Recent studies have suggests the role of Nrg along with other components such as gliotactin and Na+K+ATPase in the formation of SJs. In order to better understand the formation and function of SJs and to investigate the parallels that exist between Drosophila and vertebrate SJs, additional components of SJs need to be identified. The physiological interaction between vertebrate NCP1 and contactin suggested that a similar molecular complex might exist at the invertebrate SJs. The Drosophila homolog of vertebrate contactin, Drosophila Contactin (Cont), co-localizes with Nrx IV and Nrg at epithelial SJs and in perineurial glial cells of the PNS. Biochemical data indicate that they form a tripartite complex. Ultrastructural and dye-exclusion analyses demonstrate that Cont plays an important role in the organization of SJs and in the maintenance of a functional paracellular barrier. These studies thus provide evidence that the paranodal SJs of myelinated fibers in vertebrates may share their evolutionary origin with invertebrate SJs, thus making them amenable to comparable genetic and molecular analysis (Faivre-Sarrailh, 2004).

These studies agree with the vertebrate studies where homologous cell-adhesion molecules, NCP1 and contactin are required to establish paranodal SJs in complex with NF-155 (Girault, 2002; Bhat, 2003). In addition, NCP molecules interact with scaffolding proteins of the FERM (4.1, Ezrin, Radixin, Moesin) family (see Drosophila Moesin), 4.1B and coracle (see Drosophila Coracle) in vertebrate and invertebrate species, respectively. Therefore, the molecular composition of Drosophila SJs and vertebrate axo-glial SJs is highly conserved (Faivre-Sarrailh, 2004).

A single representative of the contactin gene family is present in the Drosophila genome, whereas six genes have been identified to date in vertebrates. A phylogenetic analysis indicates that Cont is not a specific ortholog of any one of the vertebrate contactin-type proteins. Since Cont is a component of SJs, it appears to be the functional counterpart of vertebrate contactin, which is a component of paranodal SJs. In vertebrates, contactin interacts with NCP1/caspr in cis at the paranodal SJs (Peles, 1997), whereas a contactin-related protein TAG-1 has been identified as a glial component of the juxta-paranodes (Traka, 2002) and interacts with NCP2/caspr2 (Poliak, 1999; Traka, 2003). The binding partners for other NCP members, which may include other members of the contactin family, are still not known. The Drosophila genome encodes only one contactin, which interacts with the only member of the Drosophila NCP family, Nrx IV. Therefore, it seems that heterophilic binding between contactin and NCP molecules has occurred early during evolution and has been conserved following gene duplication in both gene families (Faivre-Sarrailh, 2004).

In vertebrates, efficient transport of NCP1 from the endoplasmic reticulum to the surface of transfected cells requires association with the GPI-linked contactin (Faivre-Sarrailh, 2000; Bonnon, 2003). NCP1 accumulates intracellularly and fails to be recruited to paranodes in the nerves of contactin-deficient mice demonstrating that contactin is essential for axonal sorting of NCP1 in vivo (Boyle, 2001). It came as a surprise that in Drosophila, a different mode of operation appears to control the membrane expression of the contactin-NCP complex and, conversely, Nrx IV is required for the distribution of Cont at the cell surface. In nrx IV mutant embryos, Cont is not efficiently expressed at the plasma membrane, but rather appears to accumulate in vesicular organelles in the cytoplasm of epithelial cells. In transfected N2a cells, Cont remains associated with the ER and is only efficiently transported to the cell surface upon co-transfection with Nrx IV. Such a mechanism of co-targeting would allow the controlled sorting of Cont to the cell surface, where it may essentially be present in a cis-complex with Nrx IV (Faivre-Sarrailh, 2004).

Cont, Nrx IV and Nrg are interdependent for their restricted distribution at the apicolateral membrane, but the molecular arrangement of this ternary complex is still unknown. Nrx IV does not mediate homophilic adhesion. Since Nrx IV is required for transport of Cont to the cell surface, it can be assumed that the two molecules interact with each other in a cis configuration, within the same plane of the membrane. In the vertebrate paranodal junctions, a ternary complex of NCP1/contactin interacting with NF-155 is crucial for mediating the axo-glial heterotypic contact. A different context is encountered in Drosophila, in which SJs occur via the homotypic adhesion of two epithelial or glial cells. Nrg is a potent homophilic adhesion molecule. It is currently unknown whether the Cont/Nrx IV complex interacts with Nrg in a cis- or in a trans-manner (Faivre-Sarrailh, 2004).

Nrg is a component of the Drosophila SJs in epithelial cells and is involved in the apicolateral restricted distribution of Nrx IV and Cont. The ultrastructural analysis reveals that small strands of septa can be formed in the absence of Nrg. This is in agreement with the studies of Genova (2003) that indicated that mutation in nrg disrupts the paracellular barrier in the embryonic salivary gland, and induce alteration of the SJs. Similarly, small strands of septa are occasionally formed in the Cont mutant, although the organization of these septa in the apical membrane is severely impaired. The transverse septa, which are characteristic of the pleated SJs, are missing in nrx IV mutants and, therefore, Nrx IV is crucial for the assembly of SJ strands. This activity may rely on its interaction with the scaffolding protein coracle, which is also strictly required for septa formation (Faivre-Sarrailh, 2004).

Since Nrx IV does not display homophilic binding, its role in septa formation may also rely on binding with a still unidentified adhesion molecule. Molecular interactions between Nrx IV, Cont and Nrg are likely to be involved in the organization and lateral positioning of the junctional strands. Therefore, the molecular requirement for septa formation in Drosophila is somewhat similar to what has been reported for the vertebrate paranodal SJs. In the mouse, disruption of contactin and NCP1 genes both result in the disappearance of intermembranous septa and disorganization of paranodal junctions (Bhat, 2001; Boyle, 2001). The loss-of-function analysis of the glial NF-155 should reveal whether NF-155 is required for the formation of paranodal SJs (Faivre-Sarrailh, 2004).

Invertebrate SJs display similar molecular composition and morphology to the vertebrate paranodal junctions, but also display functional analogy with the vertebrate tight junctions by forming a diffusion barrier. A remarkable difference between the septate and tight junctions resides in their ultrastructural morphology. SJs are characterized by rows of intermembrane septa, whereas tight junctions have membrane-kissing points resulting from the sealing of claudin strands. Recent studies report the identification of two claudins, Megatrachea (Behr, 2003) and Sinuous (Wu, 2004), as components of the Drosophila SJs that are essential for the barrier function. This is an indication that the two types of junctions display some molecular similarities in addition to their functional analogy. The question of whether the vertebrate paranodal junctions also contain claudins is still unresolved (Faivre-Sarrailh, 2004).

In addition, an increasing number of adhesion proteins have been recently reported to be essential for the organization of invertebrate SJs and formation of the paracellular diffusion barrier, including the Ig-CAM Lachesin, and the cholinesterase-like molecule Gliotactin. Strikingly, all these components are interdependent for their distribution at SJs (e.g. Nrx IV is mislocalized to the basolateral membrane in megatrachea, sinuous, gliotactin, or lachesin mutant embryos). A lack of intermembrane septa has been observed in lachesin and megatrachea mutants, whereas sinuous mutant embryos display a phenotype similar to the Cont mutant, showing some strands of septa that are disorganized. A unique function has been proposed for Gliotactin that colocalizes with other SJ markers at the tricellular junctions. Ultrastructural analysis has indicated that septa are present in the gliotactin mutant but the rows of septa are not tightly arrayed, and gliotactin may serve as an anchor at the tricellular corners and induce apical compaction of the SJ strands. Now, the question that arises is what is molecular interplay between all these SJ markers? The future challenges would be to molecularly dissect the structural elements forming the intermembrane septa, and finding out how the distinct SJ components determine elongation of the strands and compaction of the parallel rows in the apical half of epithelial cell membrane, that is central to establishing an effective diffusion barrier (Faivre-Sarrailh, 2004).


REGULATION

Protein Interactions

Analyses of cont, nrx IV and nrg mutants have shown that these genes display a common alteration of SJ phenotype and thus are required for the formation and/or organization of SJs. In addition, the immunohistochemical analysis of these mutants shows that Cont, Nrx IV and Nrg are dependent on each other for SJ localization. The phenotypic similarities raise the interesting issue of whether these proteins are part of a macromolecular protein complex that exists at SJs, and thus become dependent on each other for their localization. To determine whether these three proteins form a biochemical complex, microsomal NP-40 extracts were prepared from Drosophila embryos and used for co-immunoprecipitation experiments. Cont was efficiently co-immunoprecipitated with Nrx IV using either anti-Nrx IV or anti-Cont antibodies. Cont and Nrx IV were not co-precipitated by monoclonal anti-Nrg antibodies, owing to weak ability to immunoprecipitate Nrg complexes. However, by probing Western blots with the 3C1 anti-Nrg mAb, Nrg was detected in anti-Cont immunoprecipitates. Only the lower molecular weight Nrg167 isoform, which is expressed in epithelial cells, was co-immunoprecipitated with Cont. By contrast, the neuronal Nrg180 isoform could only be detected in the total NP-40 lysate. These results demonstrate that Cont, Nrx IV and Nrg are part of a protein complex and that the molecular interactions between these proteins may underlie the organization of the SJs (Faivre-Sarrailh, 2004).

Thus biochemical and immunohistochemical data show that Cont is part of a protein complex that includes Nrx IV and that Cont is mislocalized in nrx IV mutants. Furthermore, in these mutants, Cont protein is not expressed properly on the plasma membrane and is seen in intracellular vesicles in the epithelial cells. This raised the possibility that Nrx IV might be involved in the cell surface targeting or stabilization of Cont (Faivre-Sarrailh, 2004).

Since Drosophila S2 cells constitutively express Nrx IV and Cont, this question was addressed using neuroblastoma N2a cells as a heterologous expression system. In transfected N2a cells, Nrx IV is efficiently expressed at the cell surface. By contrast, in cells expressing Cont, the Cont protein appears to remain localized intracellularly and double-staining with the ER marker BiP indicates that Cont is retained in the ER. Immunofluorescence staining under permeabilizing conditions of N2a cells, which were co-transfected with Cont and Nrx IV, revealed that Cont is transported to and co-localized with Nrx IV at the plasma membrane. Similar results were obtained with intact living cells. Thus, these results indicate that Nrx IV mediates the cell-surface targeting of Cont. Next, Cont was co-immunoprecipitated with Nrx IV from co-transfected COS cell lysates, as further evidence that these proteins interact in cis. Such a cis-interaction has been reported for their vertebrate counterparts contactin and NCP1 (Peles, 1997) (Faivre-Sarrailh, 2004).


DEVELOPMENTAL BIOLOGY

Embryonic

To determine the expression pattern of the Cont protein, 0- to 16-hour-old embryos were immunostained with anti-Cont antibodies. Both guinea pig and rat anti-Cont antibodies revealed that Cont is expressed in ectodermally derived epithelial cells from stage 12. All these tissues, such as epidermis, hindgut, foregut, salivary glands and trachea, have been shown to contain pleated SJs. To establish the subcellular localization of Cont more precisely and to compare its distribution with that of Neurexin, a known SJ specific protein, and Neuroglian, a homolog of the vertebrate axo-glial SJ component NF-155, co-immunolocalization studies of wild-type embryos were carried out using guinea pig anti-Cont. This triple labeling showed that Cont expression overlaps with that of Nrx IV and Nrg in most epithelial tissues in which these molecules are co-expressed. Furthermore, the onset of Cont and Nrx IV expression detected by immunostaining is identical and, like Nrx IV, Cont does not have any maternal contribution. Nrx IV is expressed in perineurial glial cells, which insulate peripheral nerves, and in the midline glial cells that ensheath anterior and posterior commissures. Contactin colocalizes with Nrx IV in perineurial glial cells of peripheral nerves, whereas it shows a distribution clearly distinct from Nrx IV in the midline glial cells. Since Nrg is strongly expressed in the peripheral nerves, regions of overlap between the three markers can be shown in a merged image. In the midline, some of the glial cells express Cont, Nrx IV and Nrg, as indicated by intense white color (Faivre-Sarrailh, 2004).

The expression pattern of Cont is similar to that of other septate junction markers such as Coracle, which is expressed by ectodermally derived epithelial cells and along peripheral nerves although it is not present in midline glial cells. Unlike Nrx IV, Cont is not expressed in midline glial cells, indicating that Nrx IV may have additional partners in the midline glial cells (Faivre-Sarrailh, 2004).

In Drosophila, epithelial tissues of the epidermis and hindgut contain SJs along the apicolateral domain below the zonula adherens or adherens junctions. To determine whether Cont precisely localizes to SJs in the epidermis and the hindgut, confocal microscopy images of these tissues, which focus on the epithelial cells, were captured at high magnification. Cont, Nrx IV and Nrg display almost complete colocalization at the SJs of epidermal cells. Although, Nrg is clearly enriched at SJs, it exhibits a broader distribution and is also expressed along the basolateral membrane. In addition, all three proteins colocalize along the peripheral nerves where SJs have been identified. Similarly, the distribution of Cont in hindgut epithelial cells, which are more columnar than epidermal epithelial cells, was determined. Cont, Nrx IV and Nrg colocalize at SJs, and here Nrg also shows an additional basolateral localization. Thus, the immunohistochemical analysis shows that Cont, Nrx IV and Nrg are co-expressed at SJs (Faivre-Sarrailh, 2004).


EFFECTS OF MUTATION

To determine the in vivo function of Cont, a genetic analysis of the Cont locus was initiated. Based on the genomic sequence information, the Cont gene maps to 82A6-B1 and spans ~5.7 kb with eight exons. A deficiency chromosome that uncovered Cont locus was examined by Southern blot analysis to determine whether the Cont locus was deleted and also by immunohistochemical analysis using anti-Cont antibodies to determine whether the deficiency homozygous embryos were lacking the Cont protein. Southern blot analysis using a region of the Cont cDNA as a probe showed half-signal intensity in Df(3R)XM3 when compared with wild-type signals confirming the deletion of the Cont gene in Df(3R)XM3 chromosome. Immunohistochemistry using anti-Cont antibodies further established that Df(3R)XM3 homozygous embryos did not produce any Cont protein (Faivre-Sarrailh, 2004).

As there are no chemically or P element-induced mutations in Cont gene, loss-of-function mutants had to be generated. To initiate the mutational analysis in the Cont gene, a viable P element KG9756 was obtained that had inserted in the neighboring gene, CG11739. This gene encodes a mitochondrial tricarboxylate-carrier activity and is located in the 5' end of the Cont locus. The P-element insertion site is ~2.5 kb from the predicted 5' end of Cont and ~3.2 kb from its ATG start codon. The transcription start site (predicted by FlyBase) of the Cont locus is just 90 bp from the 3' end of CG11739. Standard P element mobilization procedures were followed to obtain imprecise excisions. Approximately 3000 individual chromosomes were screened and 13 lethal lines over Df(3R)XM3 deficiency were obtained, 11 of which showed complete loss of Cont. The breakpoints in one of the excision lines (contex956) started from the P insertion site and deleted ~5 kb towards the Cont locus. This excision causes embryonic lethality. To rescue the lethality of the contex956 excision, transgenic lines were generated using a 7 kb SpeI-SpeI genomic fragment, which breaks in the middle of CG11739 (deleting its promoter region and the first 144 amino acids out of a total of 321 amino acids) and has intact Cont locus. This transgene fully rescues the lethality of homozygous contex956 excisions. The rescued adults are fully viable, fertile and are indistinguishable from those of the wild-type flies, and have been maintained as a stable stock. Therefore, the lack of CG11739 gene product is not associated with a lethal phenotype. Two additional genes in Drosophila, CG5254 and CG6782, which also encode a mitochondrial tricarboxylate-carrier activity, might substitute for CG11739 function. These results indicate that the lethality in contex956 is due to the loss of Cont, and that this excision represents a null allele of Cont (Faivre-Sarrailh, 2004).

Vertebrate NCP1, contactin and NF-155 co-localize at the paranodal SJs (Bhat, 2001) and each of these proteins are found to be mislocalized in the mutant backgrounds of one another (Bhat, 2001; Boyle, 2001). In order to further investigate the analogy between the paranodal and invertebrate SJs, whether Cont, Nrx IV and Nrg are mutually dependent on each other for their SJ localization was examined. Whether the loss of Nrx IV would result in the mislocalization of Cont and Nrg was analyzed; nrx IV-null mutant embryos exhibit a diffuse distribution of Cont along the basolateral cell membrane as opposed to its sharp localization at the SJs, as seen in the wild-type embryos. In addition, Cont immunoreactivity is often associated with intracellular vesicles, suggesting that Cont may not be either efficiently transported or stabilized at the cell surface in nrx IV mutants. In addition, Nrg, which is present at high levels at the SJs in the wild-type embryos is mislocalized in nrx IV mutant embryos and shows a more basolateral localization. These results indicate that Nrx IV is required for proper SJ localization of Cont and Nrg (Faivre-Sarrailh, 2004).

Similarly, triple immunostaining was carried out of nrg1-null mutants for Nrx IV and Cont localization. In wild-type embryos, Nrg is enriched at SJs with a lower level of expression along the basolateral membrane. In epithelial cells of nrg mutant embryos, the distribution of Nrx IV and Cont becomes basolateral indicating that Nrx IV and Cont are dependent on Nrg for their SJ localization (Faivre-Sarrailh, 2004).

Cont-null mutant embryos were analyzed for SJ localization of Nrx IV and Nrg. With the loss of Cont, Nrx IV and Nrg are mislocalized to the basolateral membrane. Taken together, the immunohistochemical analyses of nrx IV, nrg and Cont-null mutants demonstrate that these proteins are mutually dependent on each other for their localization to SJs (Faivre-Sarrailh, 2004).

Thus, the loss of Cont results in the mislocalization of Nrx IV and Nrg to the basolateral membrane. To further demonstrate that the mislocalization phenotype observed in Cont mutants is due to the loss of Cont only, and not due to any other gene expression disrupted by the P element excision, 4- to 10-hour-old embryos obtained from hs-cont/hs-cont; contex956/TM3,Sb,dfd-lacZ parental genotype were heat-shocked at 37°C for 60 minutes to induce Cont. After 7 hours incubation at room temperature, the embryos were processed for immunostaining using antibodies against Cont, Nrx IV or Nrg. hs-cont/hs-cont; contex956/contex956 homozygous embryos were identified by the absence of ß-galactosidase. Induction of Cont expression restores the localization of Nrx IV to SJs. Similarly, localization of Nrg was analyzed in these embryos. Cont expression results in the enrichment of Nrg at the SJs. In addition, the SJ localization of Nrx IV and Nrg was restored in homozygous contex956 embryos by expression of wild-type Cont from the SpeI-SpeI genomic rescue fragment. Taken together, the rescue experiments demonstrate that Cont is required for the localization of Nrx IV and Nrg at epithelial SJs, and further strengthen the conclusion that the mislocalization phenotype of Nrx IV and Nrg is solely due to the loss of Cont in Cont-null embryos (Faivre-Sarrailh, 2004).

To demonstrate the role of Cont in SJ organization, ultrastructural analysis of Cont mutant embryos was carried out in the epidermis. In wild-type animals, the pleated SJs are characterized by rows of septa running in the apical half of the lateral membranes, below the adherens junctions. Ultrastructural analysis revealed a complete loss of transverse septa in nrx IV mutants. By contrast, strands of septa are encountered in 92% of Cont embryos at stage 15. However, the wild-type pattern of SJs, which show long stretches of ladder-like structure, was not seen in Cont mutants and only small clusters of septa were observed occasionally. A quantitative analysis was performed by selecting intercellular junctions that showed strands of septa in the epidermis of Cont homozygous and Cont/GFP embryos. In control Cont/GFP embryos, septa alignments are mainly found below adherens junctions where the intercellular membranes are very convoluted because of interdigitations of the two opposing cells. In Cont/GFP embryos, the mean number of septa in an average SJ is 44.7±1.9 whereas in Cont mutant embryos, intercellular junctions showing such large SJ strands are never found. Whenever any septa are present, they are often more basal in their position, and the mean number of septa is significantly reduced (to 27.1±3). At higher magnification, electron dense intramembranous structures on both sides of the junction display a scalloped appearance in Cont/GFP embryos. These intramembranous particles are not distinctly visible in Cont mutant embryos, suggesting that Cont may be involved in the formation and/or organization of these electron-dense structures. Interestingly, the regular spacing between septa (~20-22 nm) does not seem to be affected in Cont mutants (Faivre-Sarrailh, 2004).

Similarly, ultrastructural analysis of nrg mutant embryos was carried out to determine whether Nrg is required for the formation of SJs. This analysis revealed that occasionally clusters of septa are formed, often basal in their position, in 79% of nrg1 embryos at stage 15 . These observations are consistent with the recent study of Genova (Genova, 2003) that reports a reduced number of septa in nrg mutants. In conclusion, these results indicate that Cont and Nrg are not required for the formation of intermembrane septa, but that they may play a role in organizing the junctional strands of septa in the apicolateral domain to form an effective transepithelial barrier (Faivre-Sarrailh, 2004).

Next, whether Cont plays a role in the transepithelial barrier formation was examined by a method that relies on the ability of the salivary gland epithelia to exclude rhodamine-dextran (~10 kDa) after its injection into the body cavity of live embryos during late embryogenesis. After the dye injection, confocal sections were acquired on live embryos at time intervals ranging from 10 to 50 minutes. In control heterozygous Cont/GFP embryos, the dye remained excluded from the salivary gland lumen after 50 minutes of injection. In Cont mutants, diffusion of the dye into the lumen of salivary glands was observed within 20 minutes of the injection. Similar kinetics of dye diffusion into salivary gland lumen have been observed for gliotactin and megatrachea mutants, which display disorganization or absence of septal clusters (Behr, 2003; Schulte, 2003). None of the Cont mutants excluded the dye from their lumen. The dye also entered freely in the tracheal lumen of the Cont mutant embryos in addition to the lumen of the salivary glands. Thus, the dye exclusion data, in combination with the ultrastructural analysis, demonstrate that Cont is required for the organization and function of the SJs (Faivre-Sarrailh, 2004).

Axonal ensheathment and septate junction formation in the peripheral nervous system of Drosophila

Axonal insulation is critical for efficient action potential propagation and normal functioning of the nervous system. In Drosophila, the underlying basis of nerve ensheathment is the axonal insulation by glial cells and the establishment of septate junctions (SJs) between glial cell membranes. However, the details of the cellular and molecular mechanisms underlying axonal insulation and SJ formation are still obscure. This study reports the characterization of axonal insulation in the Drosophila peripheral nervous system (PNS). Targeted expression of tau-green fluorescent protein in the glial cells and ultrastructural analysis of the peripheral nerves allow visualization the glial ensheathment of axons. Individual or a groups of axons are ensheathed by inner glial processes, which in turn are ensheathed by the outer perineurial glial cells. SJs are formed between the inner and outer glial membranes. Neurexin IV, Contactin, and Neuroglian are coexpressed in the peripheral glial membranes and these proteins exist as a complex in the Drosophila nervous system. Mutations in neurexin IV, contactin, and neuroglian result in the disruption of blood-nerve barrier function in the PNS, and ultrastructural analyses of the mutant embryonic peripheral nerves show loss of glial SJs. Interestingly, the murine homologs of Neurexin IV, Contactin, and Neuroglian are expressed at the paranodal SJs and play a key role in axon-glial interactions of myelinated axons. Together, these data suggest that the molecular machinery underlying axonal insulation and axon-glial interactions may be conserved across species (Banerjee, 2006a).

The localization of Nrx IV, Cont, and Nrg in the embryonic nervous system was studied. Nrx IV, Nrg, and Cont show colocalization at the nerve glial membranes. Nrx IV, Cont, and Nrg are interdependent for their epithelial SJ localization. Having established that Nrx IV, Cont, and Nrg colocalize in the peripheral nerves, whether these proteins are interdependent for their localization in the peripheral nerves was studied. The effect of the absence of each of these three proteins on the localization of the other two was studied in the embryonic peripheral nerves using nrx IV, cont, and nrg null mutants. nrx IV mutant embryos were stained with anti-Nrx IV, anti-Cont, and anti-Nrg. Cont and Nrg proteins showed a rather diffused localization when compared with their wild-type localization. In addition, Cont is present as puncta in the cytoplasm of the glial cells because of its failure to get targeted properly to the membrane in the absence of Nrx IV. Similarly, cont null mutant embryos also showed less defined distribution and reduction of Nrx IV and Nrg in the glial membrane. nrg null mutant embryos display significant reduction in Nrx IV and Cont localization in the glial membranes. These results demonstrate that Nrx IV, Cont, and Nrg are interdependent for their proper localization in the embryonic peripheral nerves (Banerjee, 2006a).

Ultrastructural analyses of the embryonic epithelia in nrx IV, cont, and nrg mutants showed that these genes are required for the formation and/or organization of epithelial SJs. Nrx IV, Cont, and Nrg are interdependent for their proper localization both in the epithelia and in the peripheral nerves. These phenotypic similarities raised an interesting possibility that these proteins are part of a macromolecular protein complex that exists in the nervous system. To determine whether these three proteins form a biochemical complex in the nervous system, Drosophila heads, which are a rich source of both neurons and glial cells, were used. Coimmunoprecipitation experiments using Nrx IV, Cont, and Nrg antibodies efficiently coprecipitated Nrx IV, Cont, and Nrg. Interestingly, both isoforms of Nrg (180 kDa neuronal and 167 kDa epithelial) were immunoprecipitated by Nrx IV and Cont antibodies, suggesting that both isoforms are part of a protein complex that includes Nrx IV and Cont. However, at this point, it is not possible to differentiate whether isoform-specific complexes are formed or whether both isoforms are in the same complex. In addition, sucrose density gradient analysis of the fly head lysates was performed to determine how Nrx IV, Cont, and Nrg distribute in buoyant density gradients. Nrx IV and Cont cosediment in overlapping fractions. Nrg shows distribution in the lighter sucrose density fractions that do not overlap with Nrx IV and Cont but partially overlaps with that of Nrx IV and Cont, indicating that these proteins are associated with subcellular structures of the same density and may associate into a biochemical complex that is partially maintained during subcellular fractionation (Banerjee, 2006a).

The immunofluorescence analysis of nrx IV, cont, and nrg null mutants showed that in each of these mutants, the other two proteins show qualitatively reduced fluorescence intensities under identical confocal settings. The possible explanation for reduced fluorescence intensity could be that loss of any of these proteins affects the localization or stability of the other proteins. Whether the levels of the other two proteins had changed in nrx IV, cont, and nrg null mutants was examined using immunoblot analysis. Nrx IV protein levels did not seem to be affected in cont and nrg mutants when compared with wild type. Cont protein levels were severely affected in nrx IV mutants compared with wild-type and nrg mutant embryos. Nrg protein levels were also affected in nrx IV mutants but showed no change in cont mutants. At this stage, it cannot be rule out whether the change in Cont and Nrg protein levels in nrx IV mutants are attributable to reduced stability or less synthesis of these proteins. Together, the biochemical data indicate that Nrx IV, Cont, and Nrg form a protein complex in the nervous system and that, in the absence of Nrx IV, the stability of Cont and Nrg is severely affected (Banerjee, 2006a).

Inner glial membrane processes are involved in the ensheathment of either an individual axon or a group of axons. This glial ensheathment not only provides insulation of axons but also generates unique junctions between either glial membranes or between axons and glial membranes. Nrx IV, Cont, and Nrg are involved in the establishment of glial–glial SJs. Although additional components involved in these interactions need to be identified, these findings provide a basis for additional analysis of neuronal SJs, which would be relevant to the understanding of their vertebrate counterparts: the paranodal axo-glial SJs (Bhat, 2003; Hortsch, 2003; Salzer, 2003; Banerjee, 2006a and references therein).

The fundamental basis of axonal ensheathment in any species is to faithfully transmit neuronal signals along the nerve fibers and optimize desired cellular responses. To maximize the speed of conduction and/or to minimize the loss of nerve signals, many species evolved mechanisms in which axonal lengths remained short (as seen in insects) by increasing the diameter of the axons or by clustering voltage-gated Na+ channels to discrete unmyelinated regions of the axon, the node of Ranvier, as seen in myelinated nerve fibers of vertebrates. Most invertebrate species use some type of glial cells to ensheath their axons without generating a myelin sheath. The insulation is contiguous without any breaks, suggesting that primitive nodal structures or clustering of voltage-gated Na+ channels may not exist in invertebrates. However, recent reports have challenged some of these notions. In copepod crustaceans, ultrastructural analysis of the first antenna and the CNS has revealed extensive myelination of sensory and motor axons. These studies have raised some fundamental questions about axonal insulation and origins of myelination (Banerjee, 2006a and references therein).

In invertebrates like Drosophila, two types of glial cells are involved in insulation. The inner peripheral glial cells are involved in axonal ensheathment, and the outer (perineurial) glial cells wrap around the inner glial cells to provide another level of ensheathment. This two-cell ensheathment in Drosophila peripheral nerves may be advantageous to ensure that high K+ containing hemolymph does not interfere with action potential propagation. Although the cellular aspects of axonal insulation are being unraveled, the molecular mechanisms underlying the axonal ensheathment remain to be investigated. Most importantly, what are the protein constituents of the insulating membranes, and whether some of the vertebrate myelin proteins have their homologs in invertebrates? A detailed molecular analysis of the nature of the glial cells that ensheath axons as in Drosophila or produce myelin-like structures and the type of myelin in copepods may provide insights into whether myelinating glial cells arose from a common ancestor. However, a genetic dissection of the axonal ensheathment in Drosophila will uncover some of the basic aspects of the neuron-glial interactions that lead to ensheathment of nerve fibers across species (Banerjee, 2006a).

Cell adhesion molecules play a pivotal role in establishing intercellular junctions [e.g., cadherins and associated catenins form a protein scaffold that establish adhesion contacts at the adherens junctions and link them to the actin cytoskeleton. Similarly, claudins and associated cellular scaffolding proteins are required for establishing TJs. In both of these examples, transmembrane proteins bring two opposing membranes together to establish junctions through homophilic and/or heterophilic interactions (Banerjee, 2006a and references therein).

The finding that nrg null mutant nerves display increased spacing between the outer and inner glial membranes suggest that Nrg may be involved in cell–cell interactions and cell–cell adhesion between glial membranes. In contrast, the observation that loss of nrx IV and cont does not affect the membrane spacing between the inner and outer glial membranes suggests that Nrx IV and Cont are not involved in membrane adhesion or bringing the glial membranes in close apposition. Together, these data suggest that Nrg is critical for both the adhesion and SJ formation, whereas Nrx IV and Cont are critical for the formation of the septa at SJs. In addition, the missing axonal fascicles in nrg mutant nerves could result from axon fasciculation defects or axonal degeneration as a secondary consequence resulting from the loss of glial support. Axonal fasciculation defects have been observed in nrg mutants. Alternatively, axonal loss in nrg mutants might result from a disruption in axonal cytoskeleton, because Nrg possesses domains that could potentially interact with and stabilize the axonal cytoskeleton. Based on Nrg expression in S2 cells, Nrg is predicted to recruit membrane skeleton assembly within specialized domains of neurons in response to cell adhesion. Both Nrg protein forms Nrg167 and Nrg180 contain a short cytoplasmic domain as a binding site for ankyrin. Ankyrins are linker proteins that connect various membrane proteins with the actin-spectrin network in the cell. Loss of axons observed in nrg mutants is clearly suggestive of axon-glial interdependence that may alter axonal survival. In vertebrates, axolemmal-myelin interactions are critical for the formation of the paranodal axo-glial SJs (see Garcia-Fresco, 2006). This raises an interesting possibility that axon-glial interactions in Drosophila may use similar interactive mechanism for establishing axo-glial SJs. Thus, the current studies on Nrx IV, Cont, and Nrg suggest that these proteins are critical for the formation and/or organization of the SJs between either glial cells and possibly between axons and glial cells, which remains to be further investigated (Banerjee, 2006a).

Invertebrate axons are insulated from their salt-rich environment through a glial-dependent BBB, which plays a crucial role in electrical and chemical insulation. Ultrastructural studies have demonstrated SJs between perineurial glial cells and inner glial cell membrane to form the structural basis of BBB of some insects. Absence of SJs in nrx IV, cont, and nrg mutants, and a compromised BNB in the PNS as evidenced by dye exclusion analyses, provide additional confirmation in support of SJs as a prerequisite for blood-nerve barrier (BNB) formation. Not just in the PNS, recent reports on the BBB formation and function also in the CNS of Drosophila underscore the importance of SJs in proper sealing and insulation of the nerve cord (Schwabe, 2005), thereby supporting that insulation and establishment of functional SJs in both PNS and CNS go hand in hand. Although, G-protein-coupled receptor signaling pathway members have been identified to establish the BBB in Drosophila CNS, the signaling mechanisms that operate in the PNS still remain to be established (Banerjee, 2006a).

The axo-glial SJs in the myelinated axons share many anatomical features similar to those of invertebrate SJs, especially the electron-dense ladder-like transverse septa (Bhat, 2003; Banerjee, 2006a). Ensheathment of Drosophila axons by perineurial glial cells in the absence of myelin-producing glial cells would have predicted that the molecular components of the invertebrate SJs would be different from those of the vertebrate axo-glial SJs. Surprisingly, the fly SJ molecular components are present at the vertebrate axo-glial SJs and not at the TJs, which serve similar functions. Thus, the molecular similarities reflect an evolutionarily conserved function of creating an ionic barrier both at Drosophila SJs and at axo-glial SJs in the paranodal region. More importantly, Drosophila nerves contain a large number of axons, which are collectively held together by glial membranes not only to maintain nerve fasciculation but also to maintain the insulation of individual axons. It would be of significant interest to establish whether axo-glial SJs are present in Drosophila nerves and to identify downstream components of these junctions in Drosophila that link these junctions to the glial or axonal cytoskeleton. Identification of such molecules will provide insights into whether these junctions play a much broader role in axon-glial signal transduction. In summary, the molecular and functional similarities between the Drosophila SJs and vertebrate axo-glial SJs should allow a genetic and molecular dissection to be undertaken of the formation and function of these junctions in Drosophila (Banerjee, 2006a).

The lateral mobility of cell adhesion molecules is highly restricted at septate junctions in Drosophila

A complex of three cell adhesion molecules (CAMs) Neurexin IV (Nrx IV), Contactin (Cont) and Neuroglian (Nrg) is implicated in the formation of septate junctions between epithelial cells in Drosophila. These CAMs are interdependent for their localization at septate junctions. For example, null mutation of nrx IV or cont induces the mislocalization of Nrg to the baso-lateral membrane. These mutations also result in ultrastructural alteration of the strands of septate junctions and breakdown of the paracellular barrier. Varicose (Vari) and Coracle (Cora), that both interact with the cytoplasmic tail of Nrx IV, are scaffolding molecules required for the formation of septate junctions. Photobleaching experiments were conducted on whole living Drosophila embryos to analyze the membrane mobility of CAMs at septate junctions between epithelial cells. GFP-tagged Nrg and Nrx IV molecules were shown to exhibit very stable association with septate junctions in wild-type embryos. Nrg-GFP is mislocalized to the baso-lateral membrane in nrx IV or cont null mutant embryos, and displays increased mobile fraction. Similarly, Nrx IV-GFP becomes distributed to the baso-lateral membrane in null mutants of vari and cora, and its mobile fraction is strongly increased. The loss of Vari, a MAGUK protein that interacts with the cytoplasmic tail of Nrx IV, has a stronger effect than the null mutation of nrx IV on the lateral mobility of Nrg-GFP. It is concluded that the strands of septate junctions display a stable behavior in vivo that may be correlated with their role of paracellular barrier. The membrane mobility of CAMs is strongly limited when they take part to the multimolecular complex forming septate junctions (see Organization of adhesion complexes at epithelial cell contacts in the wild-type and mutant embryos). This restricted lateral diffusion of CAMs depends on both adhesive interactions and clustering by scaffolding molecules. The lateral mobility of CAMs is strongly increased in embryos presenting alteration of septate junctions. The stronger effect of vari by comparison with nrx IV null mutation supports the hypothesis that this scaffolding molecule may cross-link different types of CAMs and play a crucial role in stabilizing the strands of septate junctions (Laval, 2008. Full text of article).


EVOLUTIONARY HOMOLOGS

Cloning and initial characterization of Contactin homologs

Several members of the Ig superfamily are expressed on neural cells where they participate in surface interactions between cell bodies and processes. Their Ig domains are more closely related to each other than to Ig variable and constant domains and have been grouped into the C2 set. Another member of this group, the mouse neuronal cell surface antigen F3, has been cloned and characterized. The F3 cDNA sequence contains an open reading frame that could encode a 1,020-amino acid protein consisting of a signal sequence, six Ig-like domains of the C2 type, a long premembrane region containing two segments that exhibit sequence similarity to fibronectin type III repeats and a moderately hydrophobic COOH-terminal sequence. The protein does not contain a typical transmembrane segment but appears to be attached to the membrane by a phosphatidylinositol anchor. Antibodies against the F3 protein recognize a prominent 135-kD protein in mouse brain. In fetal brain cultures, they stain the neuronal cell surface and, in cultures maintained in chemically defined medium, most prominently neurites and neurite bundles. The mouse f3 gene maps to band F of chromosome 15. The gene transcripts detected in the brain by F3 cDNA probes are developmentally regulated, the highest amounts being expressed between 1 and 2 wk after birth. The F3 nucleotide and deduced amino acid sequence show striking similarity to the recently published sequence of the chicken neuronal cell surface protein contactin. However, there are important differences between the two molecules. In contrast to F3, contactin has a transmembrane and a cytoplasmic domain. Whereas contactin is insoluble in nonionic detergent and is tightly associated with the cytoskeleton, about equal amounts of F3 distribute between buffer-soluble, nonionic detergent-soluble, and detergent-insoluble fractions. Among other neural cell surface proteins, F3 most resembles the neuronal cell adhesion protein L1, with 25% amino acid identity between their extracellular domains. Based on its structural similarity with known cell adhesion proteins of nervous tissue and with L1 in particular, it is proposed that F3 mediates cell surface interactions during nervous system development (Gennarini, 1989).

A zebrafish homolog of the F3/F11/contactin (F3) recognition molecule has been identified. The gene shares 55% amino acid identity with F3 molecules in other vertebrates. Expression of F3 mRNA is first detectable at 16 h post-fertilization (hpf) in trigeminal and Rohon-Beard neurons. At 18-24 hpf, additional weaker expression is present in discrete cell clusters in the hindbrain, in the anterior lateral line/acoustic ganglion and in spinal motor neurons. Transcription factors of the LIM homeodomain class (LIM-HD) and their associated cofactors CLIM/NLI/Ldb (CLIM) have been implicated in the development of peripheral axons of trigeminal and Rohon-Beard neurons. Ectopic overexpression of a dominant-negative CLIM molecule early during zebrafish development strongly reduces expression of F3 mRNA in these neurons indicating regulation of F3 by the LIM-HD protein network. These results and the spatiotemporal correlation of F3 expression with axonal differentiation in a subset of primary neurons suggest an important role of F3 for axon growth (Gimnopoulos, 2003).

Timing of Contactin expression

F3/contactin (CNTN1) and TAG-1 (CNTN2) are closely related axonal glycoproteins that are differentially regulated during development. In the cerebellar cortex TAG-1 is expressed first as granule cell progenitors differentiate in the premigratory zone of the external germinal layer. However, as these cells begin radial migration, TAG-1 is replaced by F3/contactin. To address the significance of this differential regulation, transgenic mice were generated in which F3/contactin expression is driven by TAG-1 gene regulatory sequences, which results in premature expression of F3/contactin in granule cells. These animals (TAG/F3 mice) display a developmentally regulated cerebellar phenotype in which the size of the cerebellum is markedly reduced during the first two postnatal weeks but subsequently recovers. This is due in part to a reduction in the number of granule cells, most evident in the external germinal layer at postnatal day 3 and in the inner granular layer between postnatal days 8 and 11. The reduction in granule cell number is accompanied by a decrease in precursor granule cell proliferation at postnatal day 3, followed by an increase in the number of cycling cells at postnatal day 8. In the same developmental window the size of the molecular layer is markedly reduced and Purkinje cell dendrites fail to elaborate normally. These data are consistent with a model in which deployment of F3/contactin on granule cells affects proliferation and differentiation of these neurons as well as the differentiation of their synaptic partners, the Purkinje cells. Together, these findings indicate that precise spatio-temporal regulation of TAG-1 and F3/contactin expression is critical for normal cerebellar morphogenesis (Bizzoca, 2003).

Transgenic transient axonal glycoprotein (TAG)/F3 mice, in which the mouse axonal glycoprotein F3/contactin was misexpressed from a regulatory region of the gene encoding the transient axonal glycoprotein TAG-1, exhibit a transient disruption of cerebellar granule and Purkinje cell development. The neurobehavioural consequences of this mutation have been explored. This study reports on assays of reproductive parameters (gestation length, litter size and offspring viability) and on somatic and neurobehavioural end-points (sensorimotor development, homing performance, motor activity, motor coordination and motor learning). Compared with wild-type littermates, TAG/F3 mice display delayed sensorimotor development, reduced exploratory activity and impaired motor activity, motor coordination and motor learning. The latter parameters, in particular, are affected also in adult mice, despite the apparent recovery of cerebellar morphology, suggesting that subtle changes of neuronal circuitry persist in these animals after development is complete. These behavioural deficits indicate that the finely coordinated expression of immunoglobulin-like cell adhesion molecules such as TAG-1 and F3/contactin is of key relevance to the functional, as well as morphological maturation of the cerebellum (Coluccia, 2004).

Trafficking of Contactin

During myelination, membrane-specialized domains are generated by complex interactions between axon and glial cells. The cell adhesion molecules caspr/paranodin and F3/contactin play a crucial role in the generation of functional septate-like junctions at paranodes. Association with the glycosylphosphatidylinositol-linked F3/contactin is required for the recruitment of caspr/paranodin into the lipid rafts and its targeting to the cell surface. When transfected alone in neuroblastoma N2a cells, caspr/paranodin is retained in the endoplasmic reticulum (ER). Using chimerical constructs, it has been shown that the cytoplasmic region does not contain any ER retention signal, whereas the ectodomain plays a crucial role in caspr/paranodin trafficking. A series of truncations encompassing the extracellular region of caspr/paranodin was unable to abolish ER retention. N-glycosylation and quality control by the lectin-chaperone calnexin are required for the cell surface delivery of caspr/paranodin. Cell surface transport of F3/contactin and caspr/paranodin is insensitive to brefeldin A and the two glycoproteins are endoglycosidase H-sensitive when associated in complex, recruited into the lipid rafts, and expressed on the cell surface. These results indicate a Golgi-independent pathway for the paranodal cell adhesion complex that may be implicated in the segregation of axonal subdomains (Bonnon, 2003).

Contactin mutation

Rapid nerve impulse conduction depends on specialized membrane domains in myelinated nerve, the node of Ranvier, the paranode, and the myelinated internodal region. GPI-linked contactin enables the formation of the paranodal septate-like axo-glial junctions in myelinated peripheral nerve. Contactin clusters at the paranodal axolemma during Schwann cell myelination. Ablation of contactin in mutant mice disrupts junctional attachment at the paranode and reduces nerve conduction velocity 3-fold. The mutation impedes intracellular transport and surface expression of Caspr and leaves NF155 on apposing paranodal myelin disengaged. The contactin mutation does not affect sodium channel clustering at the nodes of Ranvier but alters the location of the Shaker-type Kv1.1 and Kv1.2 potassium channels. Thus, contactin is a crucial part in the machinery that controls junctional attachment at the paranode and ultimately the physiology of myelinated nerve (Boyle, 2001).

The neural recognition molecule NB-3, which belongs to the contactin subgroup of the immunoglobulin superfamily, is expressed exclusively in the nervous system and mainly upregulated at the early postnatal stage during mouse brain development. The expression of NB-3 in the cerebellum increases until adulthood. In contrast, the expression in the cerebrum declines to a low level after postnatal day 7. To characterize the functional roles of NB-3 in vivo, NB-3-deficient mice were generated by substituting a part of the NB-3 gene with the beta-galactosidase (Lac Z) gene. Complete overlap of the Lac Z expression in the heterozygous mouse brain with the NB-3 immunostaining pattern in the rat cerebellum and with the pattern of in situ hybridization of NB-3 transcripts indicates that Lac Z expression reflects the expression of NB-3 in the mouse brain. NB-3-deficient mice are viable and fertile. The formation and organization of all nuclei and layers throughout the brains of mutant mice appears normal. Behavioral tests to examine motor function show that the mice deficient for NB-3 are slow to learn to stay on the rotating rod in the rotorod test during repeated trials, and that they display dysfunction of equilibrium and vestibular senses in the wire hang and horizontal rod-walking tests. In contrast, the mutant mice showed no difference of grasp force from the wild-type mice. Thus, NB-3-deficient mice are impaired in motor coordination (Takeda, 2003).

Contactin protein interactions

In myelinated fibers of the vertebrate nervous system, glial-ensheathing cells interact with axons at specialized adhesive junctions, the paranodal septate-like junctions. The axonal proteins paranodin/Caspr and contactin form a cis complex in the axolemma at the axoglial adhesion zone, and both are required to stabilize the junction. There has been intense speculation that an oligodendroglial isoform of the cell adhesion molecule neurofascin, NF155, expressed at the paranodal loop might be the glial receptor for the paranodin/Caspr-contactin complex, particularly since paranodin/Caspr and NF155 colocalize to ectopic sites in the CNS of the dysmyelinated mouse Shiverer mutant. The extracellular domain of NF155 binds specifically to transfected cells expressing the paranodin/Caspr-contactin complex at the cell surface. This region of NF155 also binds the paranodin/Caspr-contactin complex from brain lysates in vitro. In support of the functional significance of this interaction, NF155 antibodies and the extracellular domain of NF155 inhibit myelination in myelinating cocultures, presumably by blocking the adhesive relationship between the axon and glial cell. These results demonstrate that the paranodin/Caspr-contactin complex interacts biochemically with NF155 and that this interaction is likely to be biologically relevant at the axoglial junction (Charles, 2002).

Three cell adhesion molecules are present at the axoglial junctions that form between the axon and myelinating glia on either side of the nodes of Ranvier. These include an axonal complex of contacin-associated protein (Caspr) and contactin, which has been proposed to bind NF155, an isoform of neurofascin located on the glial paranodal loops. NF155 is shown to bind directly to contactin and surprisingly, coexpression of Caspr inhibits this interaction. This inhibition reflects the association of Caspr with contactin during biosynthesis and the resulting expression of a low molecular weight (LMw), endoglycosidase H-sensitive isoform of contactin at the cell membrane that remains associated with Caspr but is unable to bind NF155. Accordingly, deletion of Caspr in mice by gene targeting results in a shift from the LMw- to a HMw-contactin glycoform. These results demonstrate that Caspr regulates the intracellular processing and transport of contactin to the cell surface, thereby affecting its ability to interact with other cell adhesion molecules (Gollan, 2003).

Voltage-gated sodium channels are composed of a pore-forming alpha subunit and at least one auxiliary beta subunit. Both beta1 and beta2 are cell adhesion molecules that interact homophilically, resulting in ankyrin recruitment. In contrast, beta1, but not beta2, interacts heterophilically with contactin, resulting in increased levels of cell surface sodium channels. Advantage was taken of these results to investigate the molecular basis of beta1-mediated enhancement of sodium channel cell surface density, including elucidating structure-function relationships for beta1 association with contactin, ankyrin, and Nav1.2. beta1/beta2 subunit chimeras were used to assign putative sites of contactin interaction to two regions of the beta1 Ig loop. Recent studies have shown that glutathione S-transferase fusion proteins containing portions of Nav1.2 intracellular domains interact directly with ankyrinG. Native Nav1.2 associates with ankyrinG in cells in the absence of beta subunits and this interaction is enhanced in the presence of beta1 but not beta1Y181E, a mutant that does not interact with ankyrinG. beta1Y181E does not modulate Nav1.2 channel function despite efficient association with Nav1.2 and contactin. beta1Y181E increases Nav1.2 cell surface expression, but not as efficiently as wild type beta1. beta1/beta2 chimeras exchanging various regions of the beta1 Ig loop were all ineffective in increasing Nav1.2 cell surface density. These results demonstrate that full-length beta1 is required for channel modulation and enhancement of sodium channel cell surface expression (McEwen, 2004a).

Voltage-gated sodium channels localize at high density in axon initial segments and nodes of Ranvier in myelinated axons. Sodium channels consist of a pore-forming alpha subunit and at least one beta subunit. beta1 is a member of the immunoglobulin superfamily of cell adhesion molecules, and interacts homophilically and heterophilically with contactin and Nf186. beta1 interactions with contactin and Nf186 have been characterized in greater detail and interactions of beta1 with NrCAM, Nf155, and sodium channel beta2 and beta3 subunits have been investigated. Using Fc-fusion proteins and immunocytochemical techniques, beta1 is shown to interact with the fibronectin-like domains of contactin. beta1 also interacts with NrCAM, Nf155, sodium channel beta2, and Nf186, but not with sodium channel beta3. The interaction of beta1 with beta2 requires the region T(169)EEEGKTDGEGNA(181) located in the intracellular domain of beta2. Interaction of beta1 with Nf186 results in increased Na(V)1.2 cell surface density over alpha alone, similar to that shown previously for contactin and beta2. It is proposed that beta1 is the critical communication link between sodium channels, nodal cell adhesion molecules, and ankyrin(G) (McEwen, 2004b).

The upregulation of voltage-gated sodium channel Na(v)1.3 has been linked to hyperexcitability of axotomized dorsal root ganglion (DRG) neurons, which underlies neuropathic pain. However, factors that regulate delivery of Na(v)1.3 to the cell surface are not known. Contactin/F3, a cell adhesion molecule, has been shown to interact with and enhance surface expression of sodium channels Na(v)1.2 and Na(v)1.9. Contactin is shown to coimmunoprecipitate with Na(v)1.3 from postnatal day 0 rat brain where this channel is abundant, and from human embryonic kidney (HEK) 293 cells stably transfected with Na(v)1.3. Purified GST fusion proteins of the N and C termini of Na(v)1.3 pull down contactin from lysates of transfected HEK 293 cells. Transfection of HEK-Na(v)1.3 cells with contactin increases the amplitude of the current threefold without changing the biophysical properties of the channel. Enzymatic removal of contactin from the cell surface of cotransfected cells does not reduce the elevated levels of the Na(v)1.3 current. Finally, similar to Na(v)1.3, contactin is shown to be upregulate in axotomized DRG neurons and accumulates within the neuroma of transected sciatic nerve. It is proposed that the upregulation of contactin and its colocalization with Na(v)1.3 in axotomized DRG neurons may contribute to the hyper-excitablity of the injured neurons (Shah, 2004).

Contactin proteins act as ligands for Notch

Axon-derived molecules are temporally and spatially required as positive or negative signals to coordinate oligodendrocyte differentiation. Increasing evidence suggests that, in addition to the inhibitory Jagged1/Notch1 signaling cascade, other pathways act via Notch to mediate oligodendrocyte differentiation. The GPI-linked neural cell recognition molecule F3/contactin is clustered during development at the paranodal region, a vital site for axoglial interaction. F3/contactin acts as a functional ligand of Notch. This trans-extracellular interaction triggers gamma-secretase-dependent nuclear translocation of the Notch intracellular domain. F3/Notch signaling promotes oligodendrocyte precursor cell differentiation and upregulates the myelin-related protein MAG in OLN-93 cells. This can be blocked by dominant negative Notch1, Notch2, and two Deltex1 mutants lacking the RING-H2 finger motif, but not by dominant-negative RBP-J or Hes1 antisense oligonucleotides. Expression of constitutively active Notch1 or Notch2 does not upregulate MAG. Thus, F3/contactin specifically initiates a Notch/Deltex1 signaling pathway that promotes oligodendrocyte maturation and myelination (Hu 2003).

Neurons and glia in the vertebrate central nervous system arise in temporally distinct, albeit overlapping, phases. Neurons are generated first followed by astrocytes and oligodendrocytes from common progenitor cells. Increasing evidence indicates that axon-derived signals spatiotemporally modulate oligodendrocyte maturation and myelin formation. F3/contactin is a functional ligand of Notch during oligodendrocyte maturation, revealing the existence of another group of Notch ligands. NB-3, a member of the F3/contactin family, acts as a novel Notch ligand to participate in oligodendrocyte generation. NB-3 triggers nuclear translocation of the Notch intracellular domain and promotes oligodendrogliogenesis from progenitor cells and differentiation of oligodendrocyte precursor cells via Deltex1. In primary oligodendrocytes, NB-3 increases myelin-associated glycoprotein transcripts. Thus, the NB-3/Notch signaling pathway may prove to be a molecular handle to treat demyelinating diseases (Cui, 2004).

Contactin and synaptic plasticity

Changes in synaptic efficacy are believed to mediate the processes of learning and memory formation. Accumulating evidence implicates cell adhesion molecules in activity-dependent synaptic modifications associated with long-term potentiation (LTP); however, there is no precedence for the selective role of this molecule class in long-term depression (LTD). The mechanisms that modulate these processes still remain unclear. A novel role is reported for glycosylphosphatidyl inositol (GPI)-anchored contactin in hippocampal CA1 synaptic plasticity. Contactin selectively supports paired-pulse facilitation (PPF) and NMDA (N-methyl-D-aspartate) receptor-dependent LTD but is not required for synaptic morphology, basal transmission, or LTP. Molecular analyses indicate that contactin is essential for the membrane and synaptic targeting of the contactin-associated protein (Caspr/paranodin) and for the proper distribution of a presumptive ligand, receptor protein tyrosine phosphatase beta (RPTPbeta)/phosphacan. These results indicate that contactin plays a selective role in synaptic plasticity and identify PPF and LTD, but not LTP, as contactin-dependent processes. Engagement of the contactin-Caspr complex with RPTPbeta may thus regulate cell-cell interactions contributing to specific synaptic plasticity forms (Murai, 2002).


REFERENCES

Search PubMed for articles about Drosophila Contactin

Banerjee, S., Pillai, A. M., Paik, R., Li, J. and Bhat, M. A. (2006a). Axonal ensheathment and septate junction formation in the peripheral nervous system of Drosophila. J. Neurosci. 26(12): 3319-29. 16554482

Banerjee, S., Sousa, A. D. and Bhat, M. A. (2006b). Organization and function of septate junctions: an evolutionary perspective. Cell Biochem. Biophys. 46(1): 65-77. 16943624

Behr, M., Riedel, D. and Schuh, R. (2003). The claudin-like megatrachea is essential in septate junctions for the epithelial barrier function in Drosophila. Dev. Cell 5: 611-620. 14536062

Berglund, E. O., Murai, K., Fredette, B., Sekerkova, G., Marturano, B., Weber, L., Mugnaini, E. and Ranscht, B. (1999). Ataxia and abnormal cerebellar microorganization in mice with ablated contactin gene expression. Neuron 24: 739-750. 10595523

Bhat, M. A., Rios, J. C., Lu, Y., Garcia-Fresco, G. P., Ching, W., Martin, M. S., Li, J., Einheber, S., Chesler, M., Rosenbluth, J. et al. (2001). Axon-glia interactions and the domain organization of myelinated axons require neurexin IV/caspr/paranodin. Neuron 30: 369-383. 11395000

Bhat, M. A. (2003). Molecular organization of axo-glial junctions. Curr. Opin. Neurobiol. 13: 552-559. 14630217

Bizzoca, A., et al. (2003). Transgenic mice expressing F3/contactin from the TAG-1 promoter exhibit developmentally regulated changes in the differentiation of cerebellar neurons. Development 130(1): 29-43. 12441289

Bonnon, C., Goutebroze, L., Denisenko-Nehrbass, N., Girault, J. A. and Faivre-Sarrailh, C. (2003). The paranodal complex of F3/contactin and caspr/paranodin traffics to the cell surface via a non-conventional pathway. J. Biol. Chem. 278: 48339-48347. 12972410

Boyle, M. E., Berglund, E. O., Murai, K. K., Weber, L., Peles, E. and Ranscht, B. (2001). Contactin orchestrates assembly of the septate-like junctions at the paranode in myelinated peripheral nerve. Neuron 30: 385-397. 11395001

Brümmendorf, T. and Rathjen, F. (1996). Structure/function relationships of axon-associated adhesion receptors of the immunoglobulin superfamily. Curr. Opin. Neurobiol. 6: 584-592. 8937821

Charles, P., et al. (2002). Neurofascin is a glial receptor for the paranodin/Caspr-contactin axonal complex at the axoglial junction. Curr Biol. 12(3): 217-20. 11839274

Coluccia, A., et al. (2004). Transgenic mice expressing F3/contactin from the transient axonal glycoprotein promoter undergo developmentally regulated deficits of the cerebellar function. Neuroscience 123(1): 155-66. 14667450

Cui, X. Y., et al. (2004). NB-3/Notch1 pathway via Deltex1 promotes neural progenitor cell differentiation into oligodendrocytes. J. Biol. Chem. 279(24): 25858-65. 15082708

Faivre-Sarrailh, C., et al. (2004). Drosophila contactin, a homolog of vertebrate contactin, is required for septate junction organization and paracellular barrier function. Development 131: 4931-4942. 15459097

Falk, J., Bonnon, C., Girault, J. A. and Faivre-Sarrailh, C. (2002). F3/contactin, a neuronal cell adhesion molecule implicated in axogenesis and myelination. Biol. Cell. 94: 327-334. 12500940

Garcia-Fresco, G. P., et al. (2006). Disruption of axo-glial junctions causes cytoskeletal disorganization and degeneration of Purkinje neuron axons. Proc. Natl. Acad. Sci. 103(13): 5137-42. 16551741

Gennarini, G., Cibelli, G., Rougon, G., Mattei, M. and Goridis, C. (1989). The mouse neuronal cell surface protein F3, a phosphatidylinositol-anchored member of the immunoglobulin superfamily related to the chicken contactin. J. Cell Biol. 109: 775-788. 2474555

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Biological Overview

date revised: 10 November 2017

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