Neurexin IV: Biological Overview | Evolutionary Homologs | Protein interactions | Developmental Biology | Effects of Mutation | References

Gene name - Neurexin IV

Synonyms -

Cytological map position - 68F

Function - multifunctional transmembrane protein

Keyword(s) - ectoderm, cns and septate junctions

Symbol - Nrx-IV

FlyBase ID:FBgn0013997

Genetic map position -

Classification - EGF domain, laminin G motif, discoidin motif, glycophorin C motif

Cellular location - surface - transmembrane protein



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

Neurexins are components of septate junctions playing a role in cell adhesion; in vertebrates they are components of synapses, playing a potential role in axon guidance and innervation. Neurexin functions in the establishment of an association of the internal cytoskeleton with the cell surface, and can function, at least in vertebrates, in transducing extracellular events to the inside of the cell via a specialized intracellular kinase.

Drosophila Neurexin, termed Neurexin IV (NRX) because of its homology to a human homolog, is the first member of the neurexin family isolated in a nonmammlian species. Three Neurexins in vertebrates have large extracellular domains with multiple laminin G motifs (see Drosophila Laminin A) and epidermal growth factor repeats, and are expressed exclusively in the brain.

Neurexin functions in association with septate junctions. Epithelial cells contain specialized junctions that have been classified at the ultrastructural level. Different types of junctions fulfill a variety of functions: some mediate cell communication (gap junctions and chemical synapses), others anchor cells to the extracellular matrix or adjacent cells (adherens junctions, focal contacts, and desmosomes), and still others serve as selective-permeability barriers, separating apical and basal boundaries (tight junctions and septate junctions). Septate junctions (SJs) in Drosophila are common to all epithelia and can be subdivided into two types: smooth SJs (sSJs) found in gut endoderm and Malpighian tubules, and pleated SJs (pSJs) found in ectodermally derived epithelia, i.e., glial sheaths, epidermis, trachea, salivary glands, ectodermal parts of the alimentary canal, and imaginal discs. Neurexin is involved in formation of pSJs, and appear to be responsible for distinguishing sSJs and pSJs (Tepass, 1994 and Baumgartner, 1996).

Other molecules found at septate junctions include the cell adhesion protein Fasciclin III, as well as Expanded, and Coracle. Both Expanded and Coracle are members of the 4.1 family of proteins that includes mammalian ezrin, radixin and moesin. The 4.1 family proteins physically bind cytoskeletal elements (Fehon, 1994 and Woods, 1996). The protein Discs-large contains PDZ-domains, and a domain with homology to guanylate kinases, suggesting a role in cell signaling. DLG protein is required for junction structure, cell polarity, and proliferation control in Drosophila epithelia. (Woods, 1996).

In vertebrates, glial tight junctions serve the purpose of providing a selective-diffusion barrier to ions, protecting the nervous system from contact with the environment. Such junctions are virtually absent in insects, and it is thought that pleated septate junctions serve this barrier function. Each SJ may confer partial impermeability, so that together they form a barrier that protects neurons from high potassium ion concentration in the hemolymph of insects. Neurexin IV mutants are defective in providing the protective barrier shielding the fly's nervous system from high potassium ion levels. Nrx mutants are paralyzed, and electrophysiological studies indicate that the lack if NRX iin glial-glial SJs causes a breakdown in the blood-brain barrier. Electron microscopy demonstrates that Nrx mutants lack the ladder-like intracellular septa characteristic of pleated septate junctions. Similar breakdown occurs with gliotactin mutation (Baumgartner, 1996 and Auld, 1995).

In addition, mutation of Drosophila Nrx results in mislocalization of Coracle at SJs and causes dorsal closure defects similar to those observed in coracle mutants.

Drosophila NRX does not localize to synapses as does vertebrate Neurexins, and Drosophila NRX does not play a role in synaptic function. It is clear that vertebrate Neurexins have assumed a role not found in Drosophila. Alternative splicing of vertebrate neurexins represents a potential mechanism for creating a large number of cell surface receptors expressed by specific subsets of neurons, perhaps functioning to determine highly specific axon guidance and innervation (Puschel, 1995). Neurexins are also involved in cell signaling. The mammalian protein CASK is an intracellular protein kinase that interacts with different neurexins. CASK is composed of an N-terminal Ca2+, calmodulin-dependent protein kinase and a C-terminal region that is similar to the intercellular junction proteins homologous to Drosophila Discs large 1. CASK is enriched in synaptic plasma membranes in the brain, but is also detectable at low levels in other tissues (Hata, 1996).

Human Neurexin IV, in contrast to Neurexins I, II and III, contains an N-terminal discoidin domain. In human NRX IV and Drosophila Neurexin, the discoidin domain is found in place of the N-terminal laminin domain and an EGF-like repeat found in the other human Neurexin proteins. The discoidin motif is thought to be a carbohydrate binding domain characteristic of lectins. Thus it is thought that Neurexin plays a role in cell adhesion or adherence to extracellular matrix, binding carbohydrates on the surface of adjacent cells or constituting a part matrix components surrounding cells (Valencia, 1989).

The human Neurexin IV is expressed in brain, kidney and lung. The Neurexins, including the Drosophila protein, contain a cytoplasmic domain homologous to glycophorin C, a protein required for anchoring protein 4.1 (Drosophila homolog: Coracle) to the inner aspect of cell surfaces (Baumgartner, 1996 and references).

Septate junctions are required for ommatidial integrity and blood-eye barrier function in Drosophila

The anatomical organization of the Drosophila ommatidia is achieved by specification and contextual placement of photoreceptors, cone and pigment cells. The photoreceptors must be sealed from high ionic concentrations of the hemolymph by a barrier to allow phototransduction. In vertebrates, a blood-retinal barrier (BRB) is established by tight junctions (TJs) present in the retinal pigment epithelium and endothelial membrane of the retinal vessels. In Drosophila ommatidia, the junctional organization and barrier formation is poorly understood. This study reports that septate junctions (SJs), the vertebrate analogs of TJs, are present in the adult ommatidia and are formed between and among the cone and pigment cells. The localization of Neurexin IV (Nrx IV), a SJ-specific protein, coincides with the location of SJs in the cone and pigment cells. Somatic mosaic analysis of nrx IV null mutants shows that loss of Nrx IV leads to defects in ommatidial morphology and integrity. nrx IV hypomorphic allelic combinations generated viable adults with defective SJs and displayed a compromised blood-eye barrier (BEB) function. These findings establish that SJs are essential for ommatidial integrity and in creating a BEB around the ion and light sensitive photoreceptors. These studies may provide clues towards understanding the vertebrate BEB formation and function (Bannerjee, 2008).

This study analyzed developing and adult Drosophila ommatidia for the presence and location of SJs. SJs are shown to be present in the retinal epithelial cells in the imaginal discs, pupal eye discs and the adult ommatidia. In the adult ommatidia these junctions are specifically located in the apical and basal regions between the CCs, between the PCs and between the CCs and PCs. The localization of Nrx IV, a SJ specific protein, coincides with the location of SJs during key stages of eye development. Taking advantage of the nrx IV null and hypomorphic mutants, it was shown that SJs are essential for ommatidial integrity and BEB function. Studies in the future will allow a structure/function analysis of the organization and function of SJs in an adult Drosophila organ that provides a read out of the signaling abnormalities during its development and is not crucial for the survival of the organism (Bannerjee, 2008).

Previous studies on the eye development have established the fate map of various cell types that form an adult ommatidium. While the anatomy and placement of cell types that include neuronal photoreceptors (PRs), four cone cells (CCs) and three types of pigment cells (PCs) is well worked out, the junctional organization between these cell types remains to be elucidated. Specialized areas of contacts between adjacent cell membranes are extremely important for proper cellular assembly and tissue organization. Among the junctional components typical of any polarized epithelia, AJs have been characterized in greater detail in the Drosophila ommatidia. The AJs demarcate the apical and basal domains, contribute to intercellular adhesion and are coupled with the cytoskeleton. The AJs also serve as sites for many signal transduction pathways (Bannerjee, 2008).

It is hypothesized that the presence of SJs between and among the CCs and PCs in the adult Drosophila ommatidia function to maintain proper adhesion between many cell types. The presence of rhabdomeres in the lamina in large nrx IV clones suggests that the ommatidia fail to hold together either due to loss of adhesion at the apical and basal ends of the ommatidia or due to defective stretching during ommatidial development. These misplaced rhabdomere phenotypes are only observed in large clones. Small nrx IV mutant clones display fused or abnormally structured rhabdomeres suggesting that proper junctional organization between the cells is essential for ommatidial integrity. The immunofluorescence analysis of Nrx IV revealed that within the eye discs Nrx IV is enriched in the CC and PC precursors and found at much reduced levels in the PR precursors. This unequal distribution in different cell types in the eye imaginal tissues correlates with the distribution pattern of SJs in the Drosophila ommatidia. In Musca, SJs have been located between CCs and between the CCs and PCs. Although a SJ marker in Musca has not been molecularly identified, homologs of Drosophila SJ proteins are likely to localize at the Musca eye SJs. Thus, the current findings indicate that the dynamic profile of Nrx IV localization corresponds to the location of SJs in the developing and adult ommatidium (Bannerjee, 2008).

During ommatidial development, the earliest cell types that are specified are the PRs during the larval third instar. The accessory CC and PC specification follows that of the PRs. During pupal development, the newly established cellular units stretch longitudinally to become an ommatidium. All cells establish a unique relationship with each other and with the PRs encased in the core of the ommatidium. The CC and PCs transform into long, slender chord-like cellular extensions that descend down to the retinal floor providing a vertical framework around the ommatidium. This network of lateral processes mechanically strengthens the unit, imparting a three-dimensional stability and integrity and optically insulates the PR rhabdomeres. In general terms, the entire ommatidium must be well suspended from the lens and the lateral processes of the accessory cells to help maintain constant spacing of the PR unit, ensuring their optical alignment. Not just vertically, the different types of PC membranes are connected horizontally as well, reinforcing support and stability (Bannerjee, 2008).

The CC and PCs have long been classified as accessory cell types of the ommatidial unit. Studies on Sparkling function hypothesized that CCs might be considered as glial cells that support the PR neurons. The present study further strengthens that CCs and PCs share similarities with the glial cells of the Drosophila nervous system as they strongly express SJ-specific glial protein, Nrx IV, and these cell types have SJs similar to the glial cells in the nervous system. Finally, CCs and PCs are non-neuronal accessory cells to the PR neurons and perform an ensheathment function around the PRs. This ensheathment function may be critical to the physiological changes that occur during phototransduction in the adult ommatidium (Bannerjee, 2008).

At the bottom of the ommatidium, the retinal floor has specialized structures. The CC processes end into bulb-shaped endfeet and the basal most ends of the secondary and tertiary PCs transform into a unique anatomical structure, the fenestrated membrane. In between the CC feet and the fenestrated membranes, distinct holes or ports are created through which the PR axon bundles project into the lamina area. Similar to AJs, focal adhesions are reported to surround the axon-exiting areas at the retinal floor. It is possible that the fenestrated regions function as an anchor at the base holding the elongated ommatidial unit upright and together with CC feet seal the optical unit at the base of the ommatidia. The finding that Nrx IV is localized to CC feet and the fenestrated membrane and the presence of SJs near the CC feet, further strengthens the idea that SJs are required to create an ionic microenvironment to protect the light and ion sensitive PR rhabdomeres in the adult ommatidia (Bannerjee, 2008).

The findings that the PR rhabdomeres fuse and that PRs degenerate in the absence of Nrx IV raises several interesting issues concerning the direct and indirect consequences of the loss of Nrx IV and SJs in the CCs and PCs. Since Nrx IV is not expressed in the wild type PRs, the degeneration of PRs observed in nrx IV clones and their altered cellular and junctional morphology are probably due to secondary consequences. Loss of Nrx IV function from the non-neuronal CC and PCs may lead to structural alterations and lack of proper cell-cell adhesion leading to misalignment of PRs. Since the CC processes taper as they reach towards the base of the ommatidia and form rounded protrusions, the CC feet, disruption of this structure could also misplace the PRs leading to their degeneration. Alternatively, Nrx IV at the SJs may play additional non-structural roles like signaling between PRs and CC and PCs to maintain PR survival. Similar PR degeneration phenotypes have been reported in sparkling mutants when Sparkling is lost from the precursors of the CCs. A mutual dependence between neurons and non-neuronal glial cells is not uncommon in the nervous system. Not just in the context of the eye, but there are numerous examples of interdependence between neuronal and glial cells for mutual survival and bidirectional signaling in the nervous system (Bannerjee, 2008).

The presence of SJs in the adult ommatidium can play dual functions: one to provide cell adhesion and thus mechanical strength and second to create an ionic or molecular barrier. Thus SJs can provide both the adhesive and barrier functions for the encased PRs from the top and the bottom. Although the functional correlates of this barrier are well known in the mammalian systems, the establishment or the maintenance of the blood-retinal barrier in the Drosophila eye remains to be investigated. The breach of the BEB in nrx IV hypomorphic allelic combinations suggests that a barrier system exists in the eye. How this barrier system allows the photosensitive rhabdomeres to function during phototransduction is not clear. Since Drosophila PR rhabdomeres trap axially directed light, a stable ionic microenvironment may help reduce the noise or variations in phototransduction and this can be only possible if a barrier is maintained around the rhabdomeres. As shown in this study, in the wild type heads, no dye seems to pass the lobular plate/lamina and lamina/retina boundaries and the levels of the dye present is below detection limits within the tissues. This suggests that a barrier system exists at these boundaries. Interestingly, in nrx IV hypomorphic mutant combinations, the dye breaches through the barrier at varying degrees with most severe hypomorphic combination displaying the highest level of the breach. These findings suggest that a barrier system exists at the bottom of the ommatidia but do not rule out the role of the apical junctions in the barrier function. Similarly, the presence of the dye in the ommatidia in the most severe hypomorphs shows that the dye has penetrated all the way into the apical regions of the ommatidia. This is consistent with the ultrastructural analysis of these mutants where normal cellular organization is seen but aberrant ultrastructure of the SJs, which leads to the breach of the barrier. Thus functional SJs are essential for BEB integrity and defects in SJ structure lead to a breakdown of the BEB system. Furthermore, the hypomorphic phenotypes suggest that the mutant Nrx IV proteins retain some of the functions at least the adhesive properties but fail to maintain the barrier function based on the fact that the ommatidial organization is not disrupted. These observations highlight important aspects of the SJ protein functions where individual domains of these proteins may be involved in specific protein-protein interactions during SJ biogenesis. Such phenotypes have been observed in moody flies, which also lack the barrier function of the SJs but the adhesive properties are retained (Bannerjee, 2008).

In insects, such as Musca, TJs are known to exist between marginal glia at the base of the lamina, which might be a basis of a lamina-medulla barrier. It is unlikely that TJs in the lamina layer play any appreciable role in the blood-eye barrier formation, since the barrier exists at the retinal floor where SJs are observed. The observations of this study suggest that a blood-eye barrier is established by the SJs in the insects. In Drosophila, there might also be additional barriers below the basement membrane down to the underlying lamina and medulla layers formed by a variety of glial cells that inhabit these layers and ensheath the PR axons. Existence of such a barrier below the basement membrane has been suggested in the housefly and locust eyes. In the future, manipulation of the junctional organization coupled with the electrophysiological measurements may provide insights into how SJs are involved in creating a functional blood-eye or blood-retinal barrier in the Drosophila eye. The classical and most widely studied role of SJs has been in the paracellular barrier formation and function during embryonic development. A compromise in the integrity of the vertebrate BRB due to breakdown of the occluding junctional architecture is the underlying basis of many pathologies, with still poorly understood cellular mechanisms. Understanding the molecular organization of a barrier system in the adult Drosophila eye may be more advantageous as a functional read out could be assessed by the dye-penetration assay as well as electrophysiological methods to monitor phototransduction. In conclusion, these studies establish the presence and novel functions of SJs in the Drosophila eye. The characterization of Nrx IV function during eye development has provided a glimpse into some of the important aspects of cellular events in ommatidial development. Future studies may provide additional insights into whether the SJs, in addition to providing structural integrity and barrier function, play a much broader role in intercellular communication between neuronal PRs and glia-like CCs and PCs (Bannerjee, 2008).


GENE STRUCTURE

genomic length - 12 kb

Exons - at least 4


PROTEIN STRUCTURE

Amino Acids - 1283

Structural Domains

The initiation methionine is followed by a secretory signal sequence. There is a transmembrane-spanning domain close to the C-terminus. The protein shows a domain structure like that of vertebrate neurexins. Drosophila NRX contains five laminin G domains (see Drosophila Laminin) and two EGF modules. The only difference between Drosophila and vertebrate proteins is that an N-terminal 6th laminin G domain and a third EGF module in vertebrate neurexins is replaced by a discoidin domain, thought to represent a lectin-binding domain. The similarity to neurexins is highest in the EGF modules and lower in the laminin G domain. In addition, the intracellular domain of NRX displays 68% similarity to the intracellular domain of glycophorin C. A human Neurexin found in brain, kidney and lung, humIV, contains an N-terminal DS domain. It is suggested that the Drosophila protein is a homolog of the human gene, and indicates the existence of a fourth neurexin in vertebrates whose expression is not limited to the nervous system. There is no indication of alternative splicing in the Drosophila gene (Baumgartner, 1996).


Neurexin IV : Evolutionary Homologs | Protein interactions | Developmental Biology | Effects of Mutation | References

date revised: 30 Dec 96 

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