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 (Br¸mmendorf, 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).

GENE STRUCTURE

cDNA clone length - 4300 bp

Bases in 5' UTR - 36

Exons - 8

Bases in 3' UTR - 91

PROTEIN STRUCTURE

Amino Acids - 1390

Structural Domains

The vertebrate contactin subfamily of GPI-anchored Ig-CAMs comprises TAG-1/axonin-1, and several other related proteins, BIG-1, BIG-2, NB-2/FAR-2 and NB-3. A Blast search of the Drosophila genome using the human contactin sequence identified a single Drosophila gene, CG1084 (FlyBase), with high sequence homology to contactin family members. The Cont cDNA was amplified by RT-PCR from larval mRNA and three independent clones were sequenced. This revealed an open reading frame of 1390 amino acids residues. Northern blot hybridization using a Cont cDNA probe revealed a single band of ~4.3-4.5 kb. Further RT-PCR analysis gave no indication that Cont undergoes alternative splicing. The primary protein structure of Cont encompasses a N-terminal signal peptide, a C-lectin domain, six Ig domains, four FNIII repeats and a consensus sequence for GPI-anchor addition. Except for the presence of the C-lectin domain at the N terminus, the modular domain organization of Cont is similar to vertebrate contactin. A phylogenetic analysis indicates that Cont is the Drosophila representative of the vertebrate contactin family (Faivre-Sarrailh, 2004).

To determine the relative molecular mass of the Cont protein, polyclonal antibodies were generated in rat and guinea pig against two his-tagged recombinant proteins, which contained Ig domains 5 and 6 with the hinge region or the N-terminal region containing the C-lectin domain, respectively. Embryonic extracts from 18-hour-old Drosophila embryos were prepared to obtain microsomal fractions, which were solubilized in NP-40, followed by Western blot analysis. Both antisera detected a major single band of ~180 kDa compared with a predicted size of 158 kDa. Since Cont sequence contains 10 putative N-glycosylation sites, the larger than expected apparent size of Cont may be the result of an extensive glycosylation. To test this hypothesis, microsomal fractions were treated with N-glycosidase F. Deglycosylation of the 180 kDa band results in a ~155 kDa band, corresponding to Cont protein core. Cont, which contains a GPI-anchor motif is recovered in the detergent insoluble lipid raft fraction like the GPI-anchored cell adhesion molecule Fas1. In addition, Cont can be released from embryonic microsomal fraction by PI-PLC treatment. Only a fraction of Cont is sensitive to PI-PLC cleavage, whereas almost all Fas1 from the same microsomal fraction is cleaved by PI-PLC. These data show that Cont is a GPI-anchored membrane associated glycoprotein (Faivre-Sarrailh, 2004).

date revised: 30 November 2004

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