InteractiveFly: GeneBrief

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

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).

REGULATION

Heterotrimeric G proteins regulate a noncanonical function of septate junction proteins to maintain cardiac integrity in Drosophila

The gene networks regulating heart morphology and cardiac integrity are largely unknown. The heterotrimeric G protein gamma subunit 1 (Ggamma1) has been shown to mediate cardial-pericardial cell adhesion in Drosophila. This study shows that G-oalpha47A and Gbeta13F cooperate with Ggamma1 to maintain cardiac integrity. Cardial-pericardial cell (CC-PC) adhesion also relies on the septate junction (SJ) proteins Neurexin-IV (Nrx-IV), Sinuous, Coracle, and Nervana 2, which together function in a common pathway with Ggamma1. Furthermore, Ggamma1 signaling is required for proper SJ protein localization, and loss of at least one SJ protein, Nrx-IV, induces cardiac lumen collapse. These results are surprising because the embryonic heart lacks SJs and suggest that SJ proteins perform noncanonical functions to maintain cardiac integrity in Drosophila. These findings unveil the components of a previously unrecognized network of genes that couple G protein signaling with structural constituents of the heart (Yi, 2008).

The results of this study show that the heterotrimeric G proteins G-oα47A, Gβ13F, and Gγ1 function together to maintain CC-PC adhesion during the late stage of heart formation in Drosophila. By mapping a new broken hearted (bro) mutant (Nrx-IV) and characterizing additional candidate genes, a noncanonical role was discovered for SJ proteins in mediating CC-PC and CC-CC adhesion outside SJs. Four SJ proteins, Nrx-IV, Sinu, Cora, and Nrv2, operate in a common pathway with Gγ1 to maintain cardiac integrity; these proteins require Gγ1 for proper subcellular localization in the heart. Mechanistically, the presence of SJ proteins in both CCs and PCs suggests that these proteins act in trans to maintain cell-cell adhesion in the dorsal vessel. A model is favored in which the extracellular domain of Nrx-IV engages in heterophilic interactions with SJ-proteins such as Neuroglian or Contactin, and that these interactions would be stabilized by ECM proteins such as Pericardin (Prc). Alternatively, the SJ proteins may directly interact with ECM proteins to provide a structural basis for cardiac integrity (Yi, 2008).

Heterotrimeric G proteins G-oα47A/G-iα65A, Gβ13F, and Gγ1 function with the GPCR moody and the RGS protein loco to regulate SJ formation in the Drosophila brain-blood barrier (Schwabe, 2005). Although loco mutant embryos show the bro heart phenotype, moody mutations do not induce a heart phenotype. A search of the Drosophila protein interaction map reveals that the GPCR CG32447 interacts with both the SJ protein Sinu and the RGS Kermit. Kermit also interacts with Loco, suggesting that the CG32447 GPCR participates in the control of cardiac integrity. However, a deficiency uncovering CG32447 does not induce the bro phenotype. Since the screen for bro mutants, visualized as a perturbation in the ordered expression pattern of Hand-GFP in cardial and pericardial cells, did not identify a GPCR that maintains cardiac integrity, it is concluded that the GPCR regulating cardiac integrity is either pleiotropic, with an early embryonic function that precludes its identification as a regulator of cardiac integrity, or is redundant to a second GPCR in the dorsal vessel (Yi, 2008).

Alternatively, cardiac integrity may be regulated by a GPCR-independent mechanism. In neuroblasts, G-iα65A, Gβ13F, Gγ1, and loco regulate mitotic spindle orientation, protein localization, and ultimately asymmetric cell division via a GPCR-independent signaling pathway. During neuroblast cell division, heterotrimeric G proteins are activated by the GTPase exchange factor (GEF) Ric-8, but not by GPCRs (see David, 2005). However, the lethal mutation ric-8^G0397 does not induce the bro phenotype (Yi, 2008).

During blood-brain barrier formation, sequestering Gβγ or hyperactivating G-oα47A signaling in glial cells leads to SJ defects, whereas hyperactivating G-iα65A signaling does not affect SJ function. A similar relationship exists among heterotrimeric G proteins during asymmetric cell division in neuroblasts. In contrast, sequestering Gβγ in the dorsal vessel has no effect on cardiac integrity, while hyperactivating G-oα47A in the embryonic heart induces the bro phenotype. It is concluded that the bro phenotype in Gβ13F or Gγ1 mutants is caused by misregulation of G-oα47A signaling. This is in sharp contrast to the G proteins regulating blood-brain barrier formation and asymmetric cell division where Gβγ dimers activate a set of downstream effectors distinct from that of G-oα47A signals (Yi, 2008).

G protein signaling regulates SJ formation in Drosophila and tight junction formation in mammalian cells. Even though SJs are analogous to vertebrate tight junctions, it is striking that G protein signaling components colocalize with both SJ and tight junction proteins. In addition, Gα_s interacts with the tight junction protein ZO-1 throughout junction formation, suggesting that Gα subunits physically regulate tight junction assembly. Thus, septate/tight junction proteins appear to be direct targets of G proteins in both flies and vertebrates (Yi, 2008 and references therein).

Although the embryonic heart lacks SJs, the current results are consistent with the idea that SJ proteins are direct targets of G proteins in the dorsal vessel. G protein mutants phenocopy SJ-protein mutants and G proteins operate in a common pathway with SJ proteins to maintain cardiac integrity. In addition, proper localization of SJ proteins in the embryonic heart requires G protein signaling, and G proteins regulate at least one SJ protein at the posttranscriptional level. Finally, loss of G-oα47A signaling (G-oα47A mutants) and hyperactivation of G-oα47A signaling (overexpressing G-oα47A) both result in the bro phenotype; thus Gα signaling is localized to specific foci in cells of the dorsal vessel. It is proposed that an appropriate level of Gα signaling mediates SJ-protein localization, whereas loss or hyperactivation of Gα signaling mislocalizes SJ proteins leading to a loss in cardiac integrity (Yi, 2008).

Cell-cell adhesion plays an essential role during organ morphogenesis. In the Drosophila heart, cell-cell adhesion along three distinct CC membrane domains is required to maintain cardiac integrity. Medioni (2008) provide a detailed description of two CC domains participating in cell-cell adhesion: the adherent domain, positioned immediately dorsal and ventral to the cardiac lumen, promotes cell-cell adhesion between CCs on opposing sides of the heart, and the basal-lateral adherent domain, positioned along the lateral CC membrane, promotes cell-cell adhesion between neighboring CCs on one side of the heart. These studies suggest that a third CC membrane domain, referred to as the pericardial adherent domain, is positioned opposite to the luminal domain and promotes PC-CC adhesion. The loss of cell-cell adhesion along each of the three CC domains gives rise to a unique phenotype: luminal collapse (referred to hereafter as type-1), breaks between neighboring cardial cells (type-2), and loss of PC-CC adhesion (type-3), respectively. The unique nature of these three phenotypes can provide insight into the molecular pathways regulating cardiac integrity (Yi, 2008).

Loss of heterotrimeric G proteins or SJ proteins induces the type-3 (bro) phenotype, and mutations in at least one SJ-protein gene, Nrx-IV, leads to the type-1 phenotype. In addition, Sinu, Cora, and Nrv2 localize to the luminal and perhaps the adherent domains, suggesting that loss of these proteins will also cause the type 1 phenotype. The type 2 phenotype is observed in a subset of Gγ1 embryos, but not in any other heterotrimeric G protein or SJ-protein mutants. Thus, the pathways regulating cell-cell adhesion along the CC basal-lateral membrane may be distinct from those identified in this study (Yi, 2008).

The guidance ligand Slit has been shown to regulate multiple aspects of cardiogenesis in Drosophila, and mutations in slit induce luminal collapse (referred to hereafter as type-1), breaks between neighboring cardial cells (type-2), and likely loss of PC-CC adhesion (type-3) phenotypes. In addition, slit mutant embryos show mesoderm migration and CC polarity defects, however these defects are genetically separable from cardiac integrity defects. Slit signals through the Robo receptors and mutations in genes encoding downstream components of the Robo signaling pathway do not dominantly enhance slit mutations. In contrast, mutations in genes encoding integrins or integrin ligands, such as scab, mys, and Lan-A, dominantly enhance slit mutations and transheterozygous embryos show the type-2 phenotype. This study suggests that Slit activates two pathways during cardiogenesis: one pathway utilizes typical Robo signaling to regulate mesoderm migration and CC polarity while a second pathway uses atypical, or Robo-independent, signaling to regulate cell adhesion between neighboring CCs and likely between opposing CCs to promote lumen formation. Although the role of Slit in regulating PC-CC adhesion has not been studied in detail, one possibility is that Slit signals through G-oα47A/Gβ13F/Gγ1 to regulate CC-CC and even PC-CC adhesion (Yi, 2008).

SJ proteins are functionally interdependent and localization of Sinu to SJs requires Nrx-IV, Cora, and Nrv2 (Wu, 2004), while Nrx-IV, Cora, Cont, and Nrg are equally interdependent for localization to SJs. In addition, both Nrv2 and Nrx-IV are transmembrane proteins, and the extracellular domain of Nrv2 at least is required for SJ function. Since every SJ-protein mutant examined showed PC-CC adhesion defects, SJ proteins likely form interdependent complexes in PCs and CCs. The extracellular domains of SJ proteins may act in trans, either through direct interactions with SJ proteins along opposing membranes or through indirect interactions with ECM proteins such as Pericardin, to maintain cardiac integrity. A search of the Drosophila protein interaction map reveals an interaction between Pericardin and Sinu, supporting the latter possibility. Alternatively, SJ proteins could be required for the formation or function of adherens junctions in the dorsal vessel (Yi, 2008).

All of the bro genes have close vertebrate orthologs. Since the function of mevalonate pathway genes in heart development is conserved from Drosophila to vertebrates (D'Amico, 2007; Edison, 2005; Yi, 2006), it is speculated that G protein-mediated regulation of SJ proteins is also evolutionarily conserved. To date, the role of heterotrimeric G proteins in regulating vertebrate heart development has not been identified, but heterotrimeric G proteins do play a role in heart disease. In contrast, Sinu is a member of the Claudin protein family and even though this protein family is rather divergent (Wu, 2004), vertebrate Claudin-1 is required for normal heart looping in the chick. In addition, Claudin-5 localizes to the lateral membrane of cardiomyocytes and is associated with human cardiomyopathy. Lastly, mutations in the prc ortholog, collagen alpha-1(IV), cause vascular defects in mice and humans. Taken together, these studies raise the possibility that heterotrimeric G proteins and tight junction proteins ensure proper vertebrate cardiovascular morphogenesis (Yi, 2008).

Crooked, coiled and crimpled are three Ly6-like proteins required for proper localization of septate junction components

Cellular junction formation is an elaborate process that is dependent on the regulated synthesis, assembly and membrane targeting of constituting components. This study reports on three Drosophila Ly6-like proteins essential for septate junction (SJ) formation. SJs provide a paracellular diffusion barrier and appear molecularly and structurally similar to vertebrate paranodal septate junctions. Crooked (Crok), a small GPI-anchored Ly6-like protein, is required for septa formation and barrier functions. In embryos that lack Crok, SJ components are produced but fail to accumulate at the plasma membrane. Crok is detected in intracellular puncta and acts tissue-autonomously, which suggests that it resides in intracellular vesicles to assist the cell surface localization of SJ components. In addition, this study found that two related Ly6 proteins, Coiled (Cold) and Crimpled (Crim), are required for SJ formation and function in a tissue-autonomous manner, and Cold also localizes to intracellular vesicles. Specifically, Crok and Cold are required for correct membrane trafficking of Neurexin IV, a central SJ component. The non-redundant requirement for Crok, Cold, Crim and Boudin (Bou; another Ly6 protein that was recently shown to be involved in SJ formation) suggests that members of this conserved family of proteins cooperate in the assembly of SJ components, possibly by promoting core SJ complex formation in intracellular compartments associated with membrane trafficking (Nilton, 2010).

Ly6 proteins constitute large protein families in both vertebrates and insects. In mammals, they are expressed in cells of hematopoetic origin, the brain, vascular epithelium, kidney tubular epithelium, lung, keratinocytes, stomach, testis and prostate. Reflecting their differential expression, Ly6 proteins are used in diverse biological processes. Apart from acting as GPI-linked cell accessory proteins of the immune system, vertebrate Ly6 proteins function in the modulation of nicotinic acetylcholine receptors, remodelling of the extracellular matrix during skeletal muscle regeneration, self-renewal of erythroid progenitors, and lipolytic processing of triglyceride-rich lipoproteins by binding lipoprotein lipase. The presence of a GPI-anchor in Ly6 molecules, a lipid anchor that tethers the proteins on the outer leaflet of the membrane, also suggests that Ly6 proteins can aggregate in lipid rafts to alter the activity of associated proteins (Nilton, 2010).

This study found that the Drosophila Ly6-like genes also exhibit a diverse tissue-specific distribution during development, with subsets of genes showing similar tissue expression, suggesting that they participate in similar biological processes. Indeed, five of the 18 Drosophila Ly6-like genes are expressed in a similar pattern in the developing ectoderm, and at least four are required for SJ formation, including bou (Hijazi, 2009), crok, crim and cold. The only other GPI-linked Drosophila Ly6 protein studied so far is quiver (qvr; also known as sleepless; Koh, 2008), which is required for sleep and appears to affect the levels of the voltage-dependent potassium channel Shaker (Nilton, 2010).

Phenotypic analyses of crok mutants show that Crok is required for plasma membrane accumulation of SJ components. Since SJ components were detected in the tracheal cytoplasm of stage 15 crok mutant embryos and protein analyses on immunoblots show that ATPα and Nrx-VI are present in crok mutants, it is concluded that Crok is not required for the synthesis of SJ components. Moreover, elevated levels of ATPα and Nrx-VI were found in mutant embryonic extracts, despite an apparent reduction of immunofluorescence staining for these proteins, suggesting that these components are more easily solubilized in the mutant embryos. It thus appears that Crok is required for the formation of SJ complexes at the correct plasma membrane domain (Nilton, 2010).

The function of both Crok and Cold appears to be necessary for the efficient incorporation of Nrx-IV into a stable SJ-associated complex. Upon loss of Crok and Cold, Nrx-IV-GFP accumulates in large intracellular vesicles. A similar situation occurs upon loss of Cora, which interacts with the cytoplasmic tail of Nrx-IV. The presence of fluid-phase dextran in these vesicles suggests that the Nrx-IV-GFP puncta in crok and cold mutants represent endocytosed protein, possibly on route to degradation in lysosomal compartments. Consistent with this idea, subsets of the Nrx-IV vesicles in cold mutants colocalize with endocytic markers, including Rab5 (early endosomes), Rab11 (recycling endosomes), Hrs (late endosomes) and Dor (lysosomes). As other known SJ components were not detected in these vesicles, it appears that they contain SJ subcomplexes that include Nrx-IV and Cora. This specificity could suggest either that Nrx-IV and interacting proteins are more sensitive than are other components to disruption in SJ assembly, or that Crok and Cold are specifically involved in the stable integration of Nrx-IV and interacting proteins into the SJ complex. Further experiments will be required to discern these alternative possibilities (Nilton, 2010).

Bou, Crok and Cold all appear to accumulate in intracellular membrane compartments. Previous studies have shown that urokinase/plasminogen activator receptor uPAR, an extensively studied GPI-linked protein with two LU domains, is endocytosed and recycled, and that this trafficking is essential for its function. Consistent with this idea, it was found that Cold colocalizes with dextran-labelled vesicles in cultured cells. In addition, although Bou appears to accumulate in the perinuclear cytoplasm (Hijazi, 2009), the non-autonomous behaviour of Bou suggests that it travels to the plasma membrane. Like Bou, Crok shows an apparent association with internal membrane compartments, particularly the ER. The Crok antiserum does not detect endogenous Crok at high levels, and it is possible that Crok in addition associates transiently with the cell surface. Possible colocalization of Nrx-IV with Crok or Cold, or with markers for endosomal compartments, has not been addressed and it is currently unclear whether the Ly6 proteins act in the sorting, trafficking or pre-assembly of SJ components prior to their transport to the site of junction assembly, or in an endocytic recycling of the components (Nilton, 2010).

The non-redundant requirement for four Ly6-like proteins in SJ assembly is intriguing and coherent with the need for multiple Ly6-like proteins in the allosteric modulation of nicotinic acetylcholine receptor (nAChR) functions. These Ly6-like proteins include Lynx1 and possibly Lypd6 in neurons, and the secreted proteins Slurp1 and Slurp2 in the ectoderm. It has further been suggested that the lynx-nAChR interactions occur during receptor biosynthesis and maturation in the endoplasmic reticulum, a main site for assembly of the multi-subunit membrane proteins of nAChRs. In analogy, the Drosophila Ly6-like proteins might assume roles as allosteric modulators of multiprotein SJ complexes to promote their functional association (Nilton, 2010).

Together, the analyses of Crok, Crim, Cold and Bou highlight a central task for Ly6 proteins in SJ formation that might also be essential for vertebrate paranodal junction assembly. Identification of their ligands and the subcellular site of action should contribute further understanding of how highly ordered, multi-protein complexes form along precise subdomains of the plasma membrane (Nilton, 2010).

The regulation of glial-specific splicing of Neurexin IV requires HOW and Cdk12 activity

The differentiation of the blood-brain barrier (BBB) is an essential process in the development of a complex nervous system and depends on alternative splicing. In the fly BBB, glial cells establish intensive septate junctions that require the cell-adhesion molecule Neurexin IV. Alternative splicing generates two different Neurexin IV isoforms: Neurexin IV^exon3, which is found in cells that form septate junctions, and Neurexin IV^exon4, which is found in neurons that form no septate junctions. This study shows that the formation of the BBB depends on the RNA-binding protein HOW (Held out wings), which triggers glial specific splicing of Neurexin IV^exon3. Using a set of splice reporters, it was shown that one HOW-binding site is needed to include one of the two mutually exclusive exons 3 and 4, whereas binding at the three further motifs is needed to exclude exon 4. The differential splicing is controlled by nuclear access of HOW and can be induced in neurons following expression of nuclear HOW. Using a novel in vivo two-color splicing detector, a screened was carried out for genes required for full HOW activity. This approach identified Cyclin-dependent kinase 12 (Cdk12) and the splicesosomal component Prp40 as major determinants in regulating HOW-dependent splicing of Neurexin IV. Thus, in addition to the control of nuclear localization of HOW, the phosphorylation of the C-terminal domain of the RNA polymerase II by Cdk12 provides an elegant mechanism in regulating timed splicing of newly synthesized mRNA molecules (Rodrigues, 2012).

Cdk12 protein is a nuclear localized kinase that phosphorylates the C-terminal domain (CTD) of the RNA polymerase II during transcriptional elongation (Bartkowiak, 2010). The phosphorylated CTD is bound by Prp40, a subunit of the U1 snRNP. Prp40, in turn, has been shown to interact with the HOW-binding protein Crn/Clf1. Thus, Cdk12 is in a position to facilitate splicing of pre-mRNAs that have bound the HOW protein (Rodrigues, 2012).

Differential splicing is a key element in generating the amazing complexity of higher nervous systems. Through relatively few regulatory elements, a single gene can generate several different isoforms with potential distinct cellular functions. In Drosophila, differential splicing is required for the correct glial development. This study has dissected the role of the STAR-family member HOW in controlling such a differential splicing event at the Nrx-IV locus, which is pivotal for the generation of the BBB (Rodrigues, 2012).

Nrx-IV exons 3 and 4 are spliced in a mutually exclusive manner. They share DNA sequence identity of 60% and encode related Discoidin domains, which provide distinct adhesive properties. Within glial cells, expression of Nrx-IV^exon3 predominates and participates in the formation of septate junctions. Interestingly, the binding partner of Nrx-IV at the Drosophila septate junctions, Neuroglian, or the Caspr-binding partner at the septate-like junctions in vertebrates, Neurofascin, are also subject to cell-type specific, differential splicing (Rodrigues, 2012).

Differential splicing appears to be of more general relevance during the formation of septate junctions. The fly homologue of the membrane-skeleton protein 4.1, Coracle, binds to Nrx-IV and mediates the linkage of the septate junctions to the cytoskeleton. Differential splicing of coracle generates at least four different splice variants that encode four distinct proteins. RT-PCR experiments indicate that the Coracle-PB isoform is generated in a HOW-dependent manner (Rodrigues, 2012).

STAR proteins, like HOW, bind sequence motifs in the pre-mRNA of their targets. Following site-specific mutation of all HOW response elements (HREs), it was shown that HRE1 may be needed for general exon definition. The mutation of this sequence motif leads to increased exon skipping of both exon 3 and exon 4, suggesting a crucial role for HRE1 in general splicing, possibly affecting the branch point of this intron. The HRE2, HRE3 and HRE4 elements influence mutually exclusive splicing. Upon mutation of these motifs, both exons are left in the mRNA more frequently, which suggests their function in exon selection. Such an effect was not observed in neurons. Thus, these HREs seem to play a role in exon selection (Rodrigues, 2012).

The HOW isoforms share an identical KH RNA-binding domain. HOW(S) predominantly localizes to the cytoplasm and HOW(L) is found mostly in the nucleus of glial cells. This study showed that nuclear HOW is sufficient for the induction of glial-specific splicing in neurons. Interestingly, both HOW isoforms can partially rescue the how mutant phenotype. HOW(S) appears to have higher rescuing abilities. As both transgenes are inserted in the same chromosomal landing site, resulting in identical expression levels, it is assumed that HOW(S) must be efficiently transported into the nucleus to promote Nrx-IV splicing. Because, following overexpression of the HOW(S), most of the protein stays in the cytoplasm, the shuttle mechanism(s) directing HOW(S) into the nucleus must be very tightly regulated. Possibly, HOW(S) has better rescuing abilities as HOW(S), but not HOW(L), can facilitate the nuclear import of the splice factor Crn (Rodrigues, 2012).

STAR family proteins are phosphorylated on several residues. In the past, it has been established that the HOW homolog Sam68 is phosphorylated by MAPK the regulation of which is controlled by Raf. Indeed, expression of a dominant-negative Raf protein in glia shifted the splicing pattern towards the neuronal form, suggesting a role for receptor tyrosine kinase signaling for glial differentiation as it has been demonstrated at several other instances (Rodrigues, 2012).

In addition, it is noted that silencing of Cdk12 resulted in a shift of the splicing pattern towards the neuronal form. Cdk12 is a broadly expressed serine/threonine kinase that also contains stretches of arginine- and serine-rich sequences (SR domains) known to be present in RNA-processing proteins, which regulate splicing, nuclear export and stability of the mRNA. Drosophila Cdk12 is associated with the C-terminal domain (CTD) of the RNA polymerase II (RNAPII) and phosphorylates Ser2 (Bartkowiak, 2010). The CTD of RNAPII acts as an assembly platform that controls transcription and pre-mRNA processing. Phosphorylated CTD in turn is recognized by Prp40, which belongs to the U1 snRNP. Moreover, a direct interaction between PrP40 and Crocked neck like factor 1 (Clf1), which binds HOW, has been demonstrated. Thus, phosphorylation of CTD by Cdk12 (Bartkowiak, 2010) recruits the assembly of the spliceosome at specific pre-mRNA targets defined by binding of HOW. In line with this model, it is noted that silencing of Prp40 also alters Nrx-IV splicing (Rodrigues, 2012).

In Drosophila, Cdk12 associates with Cyclin K (Bartkowiak, 2010), which is required for its catalytic activity. The activity of cyclins can be regulated by RTK signaling and thus might present a link that connects the Raf/MAPK pathway with a direct control of splicing activity. Additionally, CTD phosphorylation could be linked to MAPK activity in former studies. Cdk12 is expressed in the nucleus of almost all cells. To further decipher the role of Cdk12 during splicing, a loss-of-function allele was used. Homozygous mutant animals are lethal at the beginning of larval development. However, these mutants show no splicing defects, most probably owing to strong maternal contributions (Rodrigues, 2012).

The formation of the BBB implies the maturation of septate junctions only in fully differentiated subperineurial glial cells. Thus, the timing of splicing of pre-mRNAs encoding septate junction proteins is crucial and most likely regulated by two independent signaling cascades. It is proposed that the mRNA-binding protein HOW integrates these signaling events and is key in determining cellular differentiation (Rodrigues, 2012).

Protein Interactions

Mammalian protein 4.1 is the prototype of a family of proteins that include ezrin, talin, brain tumor suppressor merlin, and tyrosine phosphatases. Protein 4.1 functions to link transmembrane proteins with the underlying spectrin/actin cytoskeleton. To permit a genetic analysis of the developmental role and cellular functions of this membrane-skeletal protein, its Drosophila homolog (termed D4.1) has been identified and characterized. D4.1 is localized to the septate junctions of epithelial cells and is encoded by the coracle gene, a new locus whose primary mutant phenotype is a failure in dorsal closure. In addition, coracle mutations dominantly suppress Ellipse, a hypermorphic allele of the Drosophila EGF-receptor homolog. These data indicate that D4.1 is associated with the septate junction, and suggest that it may play a role in cell-cell interactions that are essential for normal development (Fehon, 1994).

The protein 4.1 superfamily comprises a diverse group of cytoplasmic proteins, many of which have been shown to associate with the plasma membrane via binding to specific transmembrane proteins. Coracle, a Drosophila protein 4.1 homolog, is required during embryogenesis and is localized to the cytoplasmic face of the septate junction in epithelial cells. Using in vitro mutagenesis, it has been demonstrated that the amino-terminal 383 amino acids of Coracle define a functional domain that is both necessary and sufficient for proper septate junction localization in transgenic embryos. Genetic mutations within this domain disrupt the subcellular localization of Coracle and severely affect its genetic function, indicating that correct subcellular localization is essential for Coracle function. The localization of both Coracle and the transmembrane protein Neurexin to the septate junction displays an interdependent relationship, suggesting that Coracle and Neurexin interact with one another at the cytoplasmic face of the septate junction. Consistent with this notion, immunoprecipitation and in vitro binding studies demonstrate that the amino-terminal 383 amino acids of Coracle and the cytoplasmic domain of Neurexin interact directly. Together these results indicate that Coracle provides essential membrane-organizing functions at the septate junction, and that these functions are carried out by an amino-terminal domain that is conserved in all protein 4.1 superfamily members (Ward, 1998).

The interdependence between Coracle and NRX for proper localization suggests that at least one other protein in the presumptive septate junction serves as the initial target for both proteins to be properly localized. Based on the protein 4.1 paradigm of a ternary complex consisting of protein 4.1, glycophorin C, and p55 , it is predicted that a PDZ repeat-containing protein is a part of the complex containing Coracle and NRX. The most likely candidate for this additional protein is DLG, based on its extensive sequence similarity to p55. DLG is expressed maternally, and initially is uniformly distributed along the lateral membrane (and to a lesser extent throughout the cytoplasm). Coincident with the expression of Coracle and NRX, this subcellular localization is refined to the presumptive septate junction. This expression pattern might be expected of a protein that serves to "prepattern" the septate junction. However, attempts to detect any interaction between Coracle and DLG by immunoprecipitation have failed; no genetic interaction between coracle and dlg mutant alleles have been detected. The embryonic defects associated with dlg mutants are different from those of coracle and Nrx. These results suggest that DLG is not involved in a ternary complex together with Coracle and NRX, despite its structural similarity with p55. The question of whether there is another PDZ repeat-containing protein that functions to stabilize Coracle-NRX interactions remains to be answered, although the structural similarities between the respective Drosophila and human proteins strongly suggest that one exists. The recent identification of EBP50 as a PDZ repeat-containing protein that associates with ERM proteins suggests that an interaction with a PDZ repeat-containing protein may be a ubiquitous feature of protein 4.1 members. Regardless, the results described here strongly suggest that at least one other component is involved in Coracle/NRX localization and function (Ward. 1998).

NRX is localized apicolaterally, adjacent to Crumbs, which delimits the zonula adherens. These two proteins are not coexpressed, placing NRX apicolaterally. Both Fasciclin3 and NRX colocalize at salivary gland synaptic junctions. NRX precisely colocalized with D4.1/Coracle except in the PNS and CNS where D4.1/Coracle is only expressed in a few cells.

No defects in the localization of Discs large protein is detected in Nrx mutants. However, D4.1/Coracle is not restricted to septate junctions in Nrx mutants. These results suggest that the short cytoplasmic portion of NRX that shows homology to glycophorin C is required to localize D4.1/Coracle to septate junctions, creating a parallel with red blood cell cytoskeletal anchoring proteins (Baumgartner, 1996).

Drosophila Discs large 1 interacts with protein 4.1 homologs. All members of the protein 4.1 superfamily share a highly conserved N-terminal 30-kDa domain whose biological function is poorly understood. It is believed that the attachment of the cytoskeleton to the membrane may be mediated via this 30-kDa domain, a function that requires formation of multiprotein complexes at the plasma membrane. Synthetically tagged peptides and bacterially expressed proteins were used to map the protein 4.1 binding site on human erythroid glycophorin C, a transmembrane glycoprotein, and on human erythroid p55, a palmitoylated peripheral membrane phosphoprotein. The 30-kDa domain of protein 4.1 binds to a 12-amino acid segment within the cytoplasmic domain of glycophorin C and to a positively charged, 39-amino acid motif in p55. Sequences similar to this charged motif are conserved in other members of the p55 superfamily, including the Drosophila Discs-large tumor suppressor protein. Thus protein 4.1, known to interact with the cytoskeleton, also interacts with DLG family members (Marfatia, 1995). A Drosophila protein with homology to protein 4.1 is the recently discovered Inscuteable (Kraut, 1996).

Distribution of two family 4.1 proteins, Expanded and Coracle, are disrupted in dlg mutants. Loss of Discs large also affects the distribution of Fasciclin III and neuroglian, two transmembrane proteins thought to be involved in cell adhesion (see DLG: Biological overview). These results suggest that DLG serves as a binding protein linking cell surface receptors with the cytoskeleton via family 4.1 proteins (Woods, 1996).

Neuroglian and Nrx are interdependent for their SJ localization and these proteins form a tripartite complex

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 are required for the formation of SJs. The Drosophila homolog of vertebrate contactin, Contactin, has been genetically, molecularly, and biochemically characterized. 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).

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).

On the role of the MAGUK proteins encoded by Drosophila varicose during embryonic and postembryonic development

Membrane-associated guanylate kinases (MAGUKs) form a family of scaffolding proteins, which are often associated with cellular junctions, such as the vertebrate tight junction, the Drosophila septate junction or the neuromuscular junction. Their capacity to serve as platforms for organising larger protein assemblies results from the presence of several protein-protein interaction domains. They often appear in different variants suggesting that they also mediate dynamic changes in the composition of the complexes. This study shows by electron microscopic analysis that Drosophila embryos lacking varicose function fail to develop septate junctions in the tracheae and the epidermis. In the embryo and in imaginal discs varicose expresses two protein isoforms, which belong to the MAGUK family. The two isoforms can be distinguished by the presence or absence of two L27 domains and are differentially affected in different varicose alleles. While the short isoform is essential for viability, the long isoform seems to have a supportive function. Varicose proteins co-localise with Neurexin IV in pleated septate junctions and are necessary, but not sufficient for its recruitment. The two proteins interact in vitro by the PDZ domain of Varicose and the four C-terminal amino acids of Neurexin IV. Postembryonic reduction of varicose function by expressing double-stranded RNA affects pattern formation and morphogenesis of the wing and the development of normal-shaped and -sized eyes. Expression of two Varicose isoforms in embryonic epithelia and imaginal discs suggests that the composition of Varicose-mediated protein scaffolds at septate junctions is dynamic, which may have important implications for the modulation of their function (Bachmann, 2008).

This study demonstrates that two out of three predicted Vari proteins are in fact expressed in the embryo. Both proteins are also present in imaginal discs of third instar larvae and heads of adult flies, while in ovaries only Vari-short could be detected. In the embryo, the two isoforms are differentially expressed, the smaller one being expressed earlier and much more abundant than the larger form. Localised Varicose protein is detected even later, after stage 12. This, together with the analysis of vari transcripts and proteins in the different mutants suggests that there are several levels of gene expression regulation. For example, in vari^03953b the longer transcript is highly upregulated, which is not reflected at the protein level. Here, the near absence of the small isoform seems to have an effect on the synthesis and/or stability of the longer isoform. In vari^38EFa2 mutant embryos the truncated vari-long transcript is very abundant, which is not associated with more (truncated) Vari-long protein, pointing to less efficient translation an/or reduced stability of the mutant protein. In contrast, the truncated vari-short transcript is strongly reduced in abundance, which is not paralleled by a reduction in protein levels. In embryos mutant for this allele, properly localised protein can be detected, though in lower amounts. Since the Vari antibody used does not allow discriminating between the two proteins, it cannot be determined whether both isoforms are correctly localised. Although in vari^MD109, vari³²⁷ or vari^R979 one or both isoforms are synthesised in normal size and amount, in no case these proteins are correctly localised at the membrane (Bachmann, 2008).

Although not predicted by commonly used domain prediction programs, sequence comparison of Vari-long with its closest vertebrate orthologues, MPP2_b, MPP6_c, and other related MAGUKs, makes the presence of a second, more divergent N-terminal L27 domain in the longer Vari isoform very likely. This situation is similar to the MAGUK Stardust, the L27N domain of which also fits less to the canonical sequence. The expression of two isoforms of a MAGUK protein, which differ by the presence or absence of the L27 domain(s) is not unique. For example, one close vari orthologue, human MPP6, also encodes two isoforms, one of which, MPP6_a, lacks the two L27 domains. The human postsynaptic density (PSD)-95 protein and DrosophilaDiscs large also come in two variants, one with and one without a L27 domain, respectively. In the case of Discs large, the DlgA isoform is predominantly expressed in the embryo and Dlg-S97 in the adult brain, but both isoforms are co-expressed in the larval NMJ. In the case of Vari, both isoforms are expressed in embryonic epithelia. Since the antibody used in this study recognises an epitope common to both Vari isoforms, the possibility that different embryonic epithelia express different Vari isoforms cannot be rule out, although this seems unlikely due to the interdependence between Vari-long and Vari-short. The data further suggest that they are localised at the septate junction, since both co-immunoprecipitate with NrxIV-GFP, which is localised in the septate junction. Targeting and/or stabilisation of Neurexin IV are probably mediated by direct interaction between the PDZ domain of Vari and the C-terminal amino acids of Neurexin IV. However, other partners of Vari, particularly those interacting with the L27 domains, are still elusive. Similar as human VAM-1/MPP6_c, which binds human Veli-1 in vitro (Tseng, 2001), the L27 domain of Drosophila Vari can interact with the L27 domain of DLin-7 in vitro. The different localisation of the two proteins, at least in epithelia of the Drosophilaembryo, however, makes their in vivo interaction in these cells very unlikely (Bachmann, 2008).

What could be the significance of expressing two Vari isoforms? L27 domains can mediate the targeting of the respective protein to a particular membrane compartment, such as the synapse or the adherens junction, or stabilise interacting proteins by directly binding to the L27 domain of the respective partner. In contrast, MAGUK proteins without L27 domains can efficiently be brought to their proper site, using other targeting mechanisms, for example palmitoylation. This raises the question as to 1) whether the two Vari proteins rely on different mechanisms for targeting to the septate junctions and 2) whether the two Vari isoforms have specific functions in the Drosophilaembryo. Using Gal4-mediated overexpression, either of them is capable to rescue vari^F033 mutant embryos to viability. This allele has been classified as a null allele, based on its genetic behaviour (Wu, 2007) and the complete and nearly complete lack of Vari-long and Vari-short, respectively. The rescuing capability of Vari-short indicates that the L27 domains are not essential for viability, but does not exclude any non-essential function, in the embryo or at later stages. Strikingly, the hypomorphic allele vari^38EFa2, which still expresses the short Vari isoform, but a modified Vari-long protein, gives rise to weak, but viable and fertile adults. The deletion in this allele removes 51 amino acids in the N-terminus, which affects both L27 domains. The fact that the escapers do not exhibit any mutant eye or wing phenotype similar to that obtained upon RNAi-mediated knockdown of vari in imaginal discs suggests a more supportive function for the larger isoform. The predominant role of Vari-short is further highlighted by the fact that vari^MD109, in which Vari-short is absent, is lethal, despite expression of normal Vari-long proteins. Hence, physiological amounts of only Vari-long are not sufficient for viability, but excess levels and/or earlier expression (using daG32/daughterlessGal4) can restore viability in the absence of Vari-short (Bachmann, 2008).

Mutational analysis of vari³²⁷ uncovered a five amino acid deletion in the SH3-domain of Vari, which almost completely removes one of the four core β-strands present in all SH-3 domains, thus completely abolishing Vari function. Although both Vari isoforms are expressed in vari³²⁷ in wild-type amounts, this allele is a functional null and no localised protein could be detected. The fact that the mutant proteins are not localised suggests that either the SH3 domain is necessary for targeting, or that the overall structure of the protein is affected, preventing proper localisation. Structural and functional analysis of other MAGUKs, e. g., PSD-95 or hCASK, point to either intra- or intermolecular interactions between the SH3 and the GUK domain. In the MAGUK PSD-93, binding of a ligand to the PDZ domain releases intramolecular inhibition of the GUK domain by the SH3 domain (Bachmann, 2008).

A strong reduction of varicose function by RNAi in postembryonic stages also affects the normal development of eyes and wings. It is interesting, however, to note that the consequences of RNAi-mediated knockdown of vari in wing and eye imaginal discs seem to be different. Reduced vari function in wing imaginal discs attenuates N signaling, as revealed by lowered activity of a N reporter gene construct and disrupted expression of the N target gene wingless at the prospective margin of wing imaginal discs. In wild-type wing imaginal discs N is activated on both sides of the dorsal/ventral compartment boundary by its two ligands, dorsally expressed Serrate and ventrally expressed Delta. N activity in the wing margin activates downstream genes, like cut or wingless, which are involved in the regulation of growth and patterning. Reduction of N activity results in the formation of notches in the margin, as observed in this study. In contrast, the N reporter seems to be normally activated in eye imaginal discs upon vari RNAi induction. Eye imaginal discs with reduced vari function display abnormal folding and adult eyes are smaller and misshapen. Additionally, ocelli and bristles are sometimes replaced by bare head cuticle. This phenotype is reminiscent of eye phenotypes observed in certain allelic combinations of coracle, which exhibit roughening of the eyes due to abnormally spaced ommatidia, but without affecting the patterning of the photoreceptor cells, and often lack ocelli and bristles. In addition, some coracle mutations suppress the effects of hypermorphic mutations in the EGF-receptor (Bachmann, 2008).

This suggests that SJs are differentially required for normal signalling at various developmental stages, but may affect different signalling pathways in a tissue dependent way. Given that SJs are required throughout imaginal disc development as suggested by the results, their lack may affect various signalling pathways, which are spatially and developmentally regulated and integrated as shown for the EGF-receptor and Notch pathway. So far, nothing is known how SJs may affect signalling. They could be involved in the correct localisation of receptors, membrane-bound ligands and/or components involved in signal transduction. The stage and tissue-dependent differential contribution of various signalling pathways may explain the different phenotypes obtained upon RNAi-mediated vari knockdown in eye and wing discs (Bachmann, 2008).

Septate junctions in larval eye imaginal discs have been well documented before, but their exact role during postembryonic development is largely unknown. NrxIV has recently been shown to be required for septate junction formation between and among the cone and pigment cells and for ommatidial morphology and integrity. Some of the phenotypes observed in NrxIV mutant clones in the developing eye, which are reminiscent to those obtained by eyGal4-mediated overexpression of vari-RNAi, have been interpreted as the result of compromised adhesion. Based on the molecular and genetic interaction between NrxIV and Vari, it is tempting to speculate that vari has a comparable role during eye development. In addition, during morphogenetic movements, cell divisions and cell rearrangements, SJs have to be redistributed. Loss of vari may thus interfere with these processes, which could explain the abnormal folding of eyGal4>UAS vari-RNAi eye imaginal discs (Bachmann, 2008).

The current data suggest that the final vari mutant phenotype is the consequence of compromised SJ function at different stages of development, which, in turn, may affect several cell-cell signalling and adhesion processes. A detailed dissection of the complexity of the mutant phenotype will provide a well-suited system to study the postembryonic function of SJs (Bachmann, 2008).

Implications from these findings are threefold: (1) varicose is required for septate junction development in Drosophila embryos; (2) expression of two Varicose isoforms in embryonic epithelia and imaginal discs suggests that the composition of Varicose-mediated protein scaffolds at septate junctions is dynamic; (3) varicose is required for normal wing and eye development (Bachmann, 2008).

Drosophila Neurexin IV stabilizes neuron-glia interactions at the CNS midline by binding to Wrapper

Glia play crucial roles in ensheathing axons, a process that requires an intricate series of glia-neuron interactions. The membrane-anchored protein Wrapper is present in Drosophila midline glia and is required for ensheathment of commissural axons. By contrast, Neurexin IV is present on the membranes of neurons and commissural axons, and is highly concentrated at their interfaces with midline glia. Analysis of Neurexin IV and wrapper mutant embryos revealed identical defects in glial migration, ensheathment and glial subdivision of the commissures. Mutant and misexpression experiments indicated that Neurexin IV membrane localization is dependent on interactions with Wrapper. Cell culture aggregation assays and biochemical experiments demonstrated the ability of Neurexin IV to promote cell adhesion by binding to Wrapper. These results show that neuronal-expressed Neurexin IV and midline glial-expressed Wrapper act as heterophilic adhesion molecules that mediate multiple cellular events involved in glia-neuron interactions (Wheeler, 2009).

A sim-Gal4 UAS-tau-GFP transgenic strain and confocal microscopy have been used to study the development of Drosophila CNS midline cells. In sim-Gal4 UAS-tau-GFP embryos, GFP is localized to the cytoplasm of all midline cells - both neurons and glia. When examined in sagittal views, this allows visualization of the morphology of each midline cell type during development. Identification of specific midline cell types employed immunostaining or in situ hybridization with more than 90 validated cell type-specific reagents. The current study used this system to investigate the dynamics of MG development during embryonic stages 12-17 in both wild-type and mutant embryos. MG were identified by their distinct shape, relatively dorsal position within the CNS and expression of wrapper, which is high in AMG, low in PMG, and absent from neurons. PMG were additionally identified by expression of engrailed (en). Antibodies recognizing all neurons (anti-Elav), their axons (MAb BP102), and the midline precursor 1 (MP1) neurons (anti-Lim3) provided a comprehensive view of MG interactions with nearby neurons and axons. At the beginning of CNS axonogenesis {stages 12/3 to 12/0), commissural axons initially converge into a single axon bundle. At stage 12/3, the AMG reside in the anterior of the segment and have an elongated morphology as they migrated toward the axon commissure. Approximately three AMG make contact with the anterior surface of the commissure while the remaining AMG undergo apoptosis. At stage 12/0, the AMG send processes across both the dorsal and ventral surfaces of the commissure. As the commissure separates into AC and PC, the AMG membranes completely ensheath the AC. AC ensheathment concludes with the movement of an AMG cell body between the commissures. After the AC is completely ensheathed, a single, dorsally located AMG migrates across the dorsal surface of the PC during stages 15-16. This AMG extends processes posteriorly to ensheath the PC. During stages 15-17, the AMG also sends cytoplasmic projections into both the AC and PC that become more elaborate as development proceeds. Using electron microscopy, it has been shown that these cytoplasmic projections divide each commissure into distinct subdomains (Wheeler, 2009).

Both the MP1 neurons and PMG are also in proximity to AMG and the commissures. Their positions are constant relative to the migrating AMG. The MP1 neurons remain in close contact with the ventral-most AMG from stages 11-17, and prior to commissure separation the MP1 neurons are closely associated with the commissure along its ventral side. The PMG migrated dorsally and in an anterior direction during stage 12, and at least one PMG abuts the posterior side of the PC from stages 12-16 before undergoing apoptosis. The cell bodies of non-midline-derived neurons also make extensive contacts with the AMG and, together with the PMG and MP1 neurons, they might play important roles in AMG development (Wheeler, 2009).

This paper provides genetic, cellular and biochemical evidence that Nrx-IV and Wrapper physically interact to mediate cell adhesion between MG and neurons. The data presented here on Nrx-IV and wrapper mutant phenotypes are in close agreement with results previously published for wrapper (Noordermeer, 1998), and show that both genes have identical MG phenotypes, a likely outcome for two genes involved in heterophilic cell adhesion. The Nrx-IV-Wrapper interactions occur between MG and three neuronal cell types or structures: (1) MP1s, (2) cell bodies of lateral CNS neurons, and (3) commissural axons. Together, these interactions control the position of MG and the ensheathment and subdivision of commissures (Wheeler, 2009).

This paper provides direct evidence that MP1 neurons closely interact with AMG, suggesting an important role in their development. Beginning at late stage 12, there is a strong accumulation of Nrx-IV at the interface between the MP1s and a subset of AMG, and the Nrx-IV concentration is maintained as the AMG migrate and ensheath the commissures. Nrx-IV accumulation at the MG-MP1 boundaries is abolished in wrapper mutants, and both Nrx-IV and wrapper mutants have gaps between the AMG and MP1s. These results indicate that the MP1s physically adhere to the AMG, and this adhesive interaction is required for proper positioning of MG and ensheathment of the commissures (Wheeler, 2009).

Nrx-IV protein is present in most, if not all, CNS neurons. However, protein levels are generally low. The exceptions are the neurons that flank the MG. These cells show a strong accumulation of Nrx-IV at the interfaces with MG. This indicates an aspect of midline cell biology not commonly considered - that MG interact closely with adjacent lateral CNS neurons. This might act to physically constrain migrating MG at the midline and restrict their lateral movement. In this sense, the lateral CNS neurons, the MP1 neurons and axon commissures work together to construct the MG cytoarchitectural scaffold. Alternatively, the adhesion between lateral CNS neurons and MG might allow developmental signals to pass between these cell types (Wheeler, 2009).

A key functional role of MG is their interaction with commissural axons, and these interactions require complex MG movements and morphological changes. The AMG extend cytoplasmic processes between the commissures, followed by an AMG cell body. These MG structures effectively partition the AC from the PC. Previously, it was proposed that commissure separation is caused by the interposition of MG into the unseparated commissure. However, it is noted that in wrapper mutants, the AC and PC were well separated, even though MG processes were commonly absent between the commissures. It is possible that in wrapper mutants, MG initially cause commissure separation and then quickly retract or undergo apoptosis, indicating that MG function is required only transiently. Alternatively, commissure separation could be independent of MG interposition and the MG partition already-separated commissures. In contrast to wrapper mutants, Nrx-IV mutants have poorly separated commissures. This difference is most likely to reflect an additional function of Nrx-IV because: (1) the MG phenotypes are similar between Nrx-IV and wrapper mutants, (2) the mutants of each gene are null, (3) neither has a recognizable maternal effect, and (4) Nrx-IV is more widely expressed (Wheeler, 2009).

Throughout commissure ensheathment, axons have strong accumulations of Nrx-IV along their interface with the AMG. This suggests a continual requirement of Nrx-IV and Wrapper to mediate MG-axonal adhesion and is consistent with the wide variety of MG-axon adhesion defects observed in both Nrx-IV and wrapper mutants and the inability of elav-Gal4 UAS-Nrx-IV to rescue late Nrx-IV mutant phenotypes. By contrast, MG remained relatively well associated with each other, suggesting that neither wrapper nor Nrx-IV plays an important role in MG-MG adhesion (Wheeler, 2009).

MG projections also subdivide each commissure into discrete compartments. Previous work employing electron microscopy proposed that the MG subdivided each commissure into three dorsoventral regions. This subdivision also requires Nrx-IV and wrapper function because Nrx-IV and Wrapper accumulated in the AMG commissural projections, and the projections are absent in both Nrx-IV and wrapper mutants. Both the organizing principles and the significance of these commissural subdomains are unknown, and it remains to be determined whether the MG are a cause of the subdivision or are filling in axonal regions that are already subdivided (Wheeler, 2009).

The view of MG migration presented in this study builds on previous work, but also differs in several aspects. These include nomenclature, MG-neuron interactions and PMG migration. Klambt (1991) proposed a model in which three pairs of MG (MGA, MGM and MGP) arise in the anterior of the segment and, during migration, separate and ensheath the AC and PC. The MGA and MGM migrate posteriorly and ensheath the AC; the MGA ultimately resides anterior to the AC and the MGM between the AC and PC. By contrast, the MGP migrate anteriorly from the adjacent posterior segment and partially ensheath the PC. More recent observations, including some from this paper, point toward a different view. Analysis of 52 genes expressed in MG indicates that only two distinct MG cell types can be identified, termed AMG and PMG. There are six AMG in the anterior of the segment (this class includes MGA and MGM, which cannot be distinguished molecularly) and four PMG that reside in the posterior of the segment and are identical to MGP in terms of gene expression. Of the six initial AMG, only three survive. These cells migrate posteriorly, ensheath both the AC and PC, and elaborate projections into the commissures. By contrast, all PMG die by stage 17, and therefore do not ensheath the PC. Initially, it was proposed that PMG/MGP migrate from the adjacent posterior segment. In the current experiments, no evidence for this for this. Instead, PMG arise in the En⁺ posterior of the segment and migrate anterodorsally toward the commissure. Before undergoing apoptosis, a single PMG abuts the PC from the posterior side. Thus, the PMG are positioned to influence commissure development (Wheeler, 2009).

The experiments described in this paper strongly support the view that Nrx-IV and Wrapper directly bind and mediate cell adhesion. By contrast, neither protein mediates homophilic cell adhesion. Wrapper is an Ig superfamily protein, and experiments in both flies and vertebrates indicate that Nrx-IV can bind to additional Ig superfamily proteins. In Drosophila septate junctions, Nrx-IV forms a complex with Contactin and Neuroglian, which are two Ig superfamily proteins. It was proposed that Nrx-IV binds to Contactin at the membrane in a cis configuration, and that Contactin is required for proper Nrx-IV membrane localization. Contactin is present in the CNS, and might play a similar role in neurons. It is unknown whether Nrx-IV binds Neuroglian or Contactin in trans, similar to the mechanism proposed here for Nrx-IV-Wrapper binding. However, at paranodal axo-glial junctions in mice, the Nrx-IV homolog Caspr binds in cis to contactin, and in trans to the Neuroglian homolog neurofascin. In summary, Nrx-IV binding to Wrapper indicates a general feature of Nrx-IV, which is its ability to bind diverse Ig superfamily proteins (Wheeler, 2009).

One of the remarkable aspects of Nrx-IV is its strong membrane accumulation at sites where neurons are apposed to MG. In one sense, this resembles the accumulation of Nrx-IV in septate junctions. However, from a mechanistic perspective the situation appears different. In septate junctions, Nrx-IV membrane localization is constrained by interactions with Contactin and Neuroglian, as well as with cytoskeleton-associated proteins important for membrane localization. By contrast, the localization of Nrx-IV in neurons appears relatively fluid and dispersed, only accumulating at high levels when in contact with a Wrapper⁺ membrane. It remains possible that once Wrapper and Nrx-IV bind, additional proteins might bind to Nrx-IV to stabilize its membrane localization. These interactions could further regulate the dynamics of MG-neuron interactions (Wheeler, 2009).

Drosophila Neurexin IV stabilizes neuron-glia interactions at the CNS midline by binding to Wrapper

Ensheathment of axons by glial membranes is a key feature of complex nervous systems ensuring the separation of single axons or axonal fascicles. Nevertheless, the molecules that mediate the recognition and specific adhesion of glial and axonal membranes are largely unknown. This study used the Drosophila midline of the embryonic central nervous system as a model to investigate these neuron glia interactions. During development, the midline glial cells acquire close contact to commissural axons and eventually extend processes into the commissures to wrap individual axon fascicles. This wrapping of axons depends on the interaction of the neuronal transmembrane protein Neurexin IV with the glial Ig-domain protein Wrapper. Although Neurexin IV has been described to be an essential component of epithelial septate junctions (SJ), its function in mediating glial wrapping at the CNS midline is independent of SJ formation. Moreover, differential splicing generates two different Neurexin IV isoforms. One mRNA is enriched in septate junction-forming tissues, whereas the other mRNA is expressed by neurons and recruited to the midline by Wrapper. Although both Neurexin IV isoforms are able to bind Wrapper, the neuronal isoform has a higher affinity for Wrapper. It is concluded that Neurexin IV can mediate different adhesive cell-cell contacts depending on the isoforms expressed and the context of its interaction partners (Stork, 2009).

This study shows that wrapping of commissural axons by midline glia is dependent on the interaction between the GPI-linked protein Wrapper expressed on glial cells and the neuronally expressed Neurexin IV protein. Mutants for Neurexin IV and wrapper show similar wrapping defects at the midline. In both mutants, the midline glia is unable to infiltrate the axonal neuropil of the commissures to ensure ensheathment of individual fascicles. Tissue-specific rescues of Neurexin IV mutants showed that Neurexin IV acts in neurons and functions as an axon-autonomous-specific recognition signal for midline glial processes, as only Neurexin IV-expressing fascicles show restoration of ensheathment. Ectopic expression of Neurexin IV was even able to recruit pronounced midline glial processes to ectopic places far away from their normal localization in wild-type embryos. This suggests that Neurexin IV is a key factor in the axon-glia recognition at the midline, and a model is proposed in which Neurexin IV attracts and stabilizes midline glial processes in a contact-dependent manner (Stork, 2009).

It has been shown that midline glia exhibit thin, highly dynamic cell processes that explore neighboring neuronal substrates. Upon binding to a Neurexin IV-expressing axon fascicle, these initially transient midline glial processes might then be stabilized. This stabilization itself may be required to establish a tight glial wrap or to promote the assembly of further signaling complexes that are required for glial cell development. One possible candidate for neuron-glia communication could be the EGF-receptor ligand Spitz, which is provided by the commissural neurons and needs to be transferred to the midline glia in order to promote their survival. A tight adhesion of the glial processes to the neuronal membranes might facilitate this transfer (Stork, 2009).

Genetic analysis in vivo strongly suggested that Wrapper might act as the glial binding partner for neuronal Neurexin IV in this recognition process. Indeed aggregation assays in S2 cells revealed specific heterophilic binding of Wrapper and Neurexin IV. The Neurexin IV gene generates two distinct isoforms through alternative splicing. This study has shown that the two different isoforms are differentially expressed in the nervous system. Whereas the Nrx-IV^exon3 isoform is predominantly expressed in glial cells that can form septate junctions, the Nrx-IV^exon4-specific isoform is enriched in neurons. Both proteins differ only in the sequence of their N-terminal Discoidin-like domain that mediates interaction with carbohydrates present on many adhesion proteins. Although each isoform alone is able to interact with Wrapper in S2 cell aggregation experiments, a much higher affinity of the neuronally enriched Nrx-IV^exon4 isoform to Wrapper is observed in a competitive aggregation assay. The in vivo rescue experiments corroborate the results obtained by the tissue-specific mRNA isolation and the cell culture experiments. Although both isoforms are able to at least partially rescue the Neurexin IV mutant midline glial wrapping phenotype in the embryo, the rescuing abilities of Neurexin IV^exon3 is less pronounced compared with Neurexin IV^exon4. The alternatively spliced exons 3 and 4 are conserved in all Drosophilidae and Anopheles, and, thus, probably have important functional purposes (Stork, 2009).

Neurexin IV and Wrapper interaction possibly has not only an impact on the midline glial cell but also on the commissural axon. Neurexin IV accumulates in commissural axons, and by recruiting additional adaptors through its cytoplasmic domain it could reorganize the cytoskeleton. In epithelia, Neurexin IV recruits Coracle (Cor), a member of the band 4.1 superfamily, to septate junctions. No expression of Coracle is found at the midline and no mutant midline phenotype is detected in coracle mutant embryos. However, enhanced levels of β-Spectrin and Discs large protein are observed at the midline, which might hint towards a specific cytoskeletal connection established in commissural axons at the CNS midline (Stork, 2009).

In addition to a pure adhesive function of the Neurexin IV-Wrapper complex, Wrapper may also exert signaling properties in the glia cell. However, as Wrapper is a GPI-linked protein it would require a still unknown co-receptor for this function. In this respect, it is also interesting to note that Wrapper is more generally expressed in cortex glia and its binding to Neurexin IV may be a more general property of neuron-glia interaction (Stork, 2009).

Obviously, neuron-glia interaction is not confined to the Drosophila CNS but is also of eminent importance during the insulation of all axonal trajectories in both invertebrates and vertebrates. In vertebrates, Schwann cells wrap axons by either forming a myelin sheath or Remak fibers. Similarly, oligodendrocytes form myelin in the CNS. During myelination, the glial cell membranes form special contact zones with the axon, the paranodes, abutting the nodes of Ranvier. These are characterized by septate-like junctions that prevent current leakage. The ultrastructural architecture of these cell-cell junctions and also the molecules establishing these junctions have been conserved between flies and mammals, suggesting an ancient evolutionary origin of this axonal insulation. As core components of septate or septate-like junctions, the Caspr/Paranodin, Contactin and Neurofascin/155 and their Drosophila counterparts Neurexin IV, Contactin and Neuroglian have been identified. Interestingly, Wrapper appears to be less conserved. Although it is present in all Drosophilidae and the Drosophila genome harbors a Wrapper-related protein, Klingon, no clear Wrapper orthologs can be identified in mammals. However, there are several GPI-linked Ig-superfamily proteins in the mouse genome whose expression profiles need to be determined (Stork, 2009).

Besides prior identification of direct cis-binding partners of Neurexin IV/Caspr, which act in the same cell, this study has identified Wrapper as the first factor that interacts with Neurexin IV in a trans fashion. Based on the tissue culture data, a direct interaction is anticipated but at present the involvement of additional complex partners cannot be excluded. In previous studies it has been shown that Neurexin IV is required to facilitate the secretion of Contactin to the membrane, thereby allowing the generation of adhesive septate junctions. This study shows that Neurexin IV can directly perform adhesive functions by binding to the Wrapper protein decorating opposing cell membranes. Interestingly, Contactin and Wrapper are both similar Ig-domain proteins linked via GPI anchors to the plasma membrane (Stork, 2009).

Within the nervous system, Neurexin IV has been extensively studied for its role in organizing the formation of septate junctions between glial cells, which constitute the major structural component of the Drosophila blood brain barrier. Unlike in the vertebrate paranodes, septate junctions are found extensively at glial-glial cell contacts in the Drosophila nervous system and are only rarely detected between glial cells and axons. The midline glia is not part of this subperineurial glial sheath but rather belongs to the class of wrapping glia that ensures normal insulation of axon fascicles at the midline. In line with this notion, midline glial cells do not form septate junctions visible at the electron-microscopic level. Additionally, major septate junction components such as Coracle, Neuroglian and Lachesin are not enriched at the midline glia, and the corresponding mutants show normal midline glial wrapping behavior. For some septate junction components, midline expression has been reported. This study found that, in these cases, expression is restricted to channel glia, which is part of the subperineurial sheath known to form epithelial-like pleated septate junctions and is not related to the midline glia (Stork, 2009).

The results show that at the Drosophila midline Neurexin IV acts in a novel, septate junction-independent way to ensure neuron-glia adhesion; it will be interesting to determine whether similar adhesive interactions can be attributed to the mammalian homolog Caspr or to other members of the Caspr protein family. Interestingly, it has been recently reported, that Neurexin IV and other canonical septate junction-associated proteins control the adhesive properties of cardial and pericardial cells in the embryonic heart of Drosophila without forming septate junctions. Additionally, these noncanonical adhesive properties of septate junction proteins in the heart, and also the assembly of canonical septate junctions in the Drosophila blood brain barrier, are controlled by different heterotrimeric G protein signaling pathways and possibly Wrapper-Neurexin-IV-mediated adhesion at the CNS midline is also influenced by G protein signaling pathways. In the future, it will be interesting to determine the different roles of the Neurexin IV-Wrapper complex and to dissect the cellular responses triggered by this neuron-glia interaction (Stork, 2009).

Drosophila neurexin IV interacts with Roundabout and is required for repulsive midline axon guidance

Slit/Roundabout (Robo) signaling controls midline repulsive axon guidance. However, proteins that interact with Slit/Robo at the cell surface remain largely uncharacterized. This study reports that the Drosophila transmembrane septate junction-specific protein Neurexin IV (Nrx IV) functions in midline repulsive axon guidance. Nrx IV is expressed in the neurons of the developing ventral nerve cord, and nrx IV mutants show crossing and circling of ipsilateral axons and fused commissures. Interestingly, the axon guidance defects observed in nrx IV mutants seem independent of its other binding partners, such as Contactin and Neuroglian and the midline glia protein Wrapper, which interacts in trans with Nrx IV. nrx IV mutants show diffuse Robo localization, and dose-dependent genetic interactions between nrx IV/robo and nrx IV/slit indicate that they function in a common pathway. In vivo biochemical studies reveal that Nrx IV associates with Robo, Slit, and Syndecan, and interactions between Robo and Slit, or Nrx IV and Slit, are affected in nrx IV and robo mutants, respectively. Coexpression of Nrx IV and Robo in mammalian cells confirms that these proteins retain the ability to interact in a heterologous system. Furthermore, the extracellular region of Nrx IV was shown to be sufficient to rescue Robo localization and axon guidance phenotypes in nrx IV mutants. Together, these studies establish that Nrx IV is essential for proper Robo localization and identify Nrx IV as a novel interacting partner of the Slit/Robo signaling pathway (Banerjee, 2010).

Recent studies have established strong expression of Nrx IV at the interface between neurons and midline glie (MG) that underlies the adhesive interactions between these cells in maintaining ML cytoarchitecture. This function of Nrx IV is Wrapper-dependent. It is believed that the axon guidance function of Nrx IV is Wrapper independent based on the following observations. First, the juxtaposition of strong neuronal Nrx IV and glial Wrapper in the ML, together with the absence of axon guidance phenotype in wrapper mutants strongly suggest that Nrx IV localization in ML neurons does not contribute to the axon guidance function of Nrx IV. This is further supported by the failure to rescue the axon guidance phenotypes in nrx IV mutant by expressing Nrx IV in all ML neurons and glia using sim-Gal4::UAS-nrx IV. Therefore, a significant contribution of the axon guidance phenotype seen in nrx IV mutants is unlikely to come from ML neurons or glia. Interestingly, the MG ensheathment defects seen in nrx IV mutants are rescued by expression of Nrx IV in neurons alone and not MG. Furthermore, no changes in Robo localization, or crossing of Robo positive axons were seen in wrapper mutants, thus providing additional evidence that alterations in MG/neuronal architecture in wrapper mutants does not significantly contribute to the axon guidance phenotype. Of note, older stage 16 robo and slit mutants show considerable disorganization of MG, as revealed by immunostaining with anti-Wrapper antibodies; however, these glial phenotypes are thought to be secondary to their axon guidance phenotypes. In addition, Contactin (Cont) and Nrg, two well-established binding partners of Nrx IV at SJs, do not contribute to Nrx IV in its CNS axon guidance function, as revealed by Fas II immunostaining of cont and nrg mutants. Based on these observations, it is believed that Nrx IV is a multifunctional protein that functions in a cell type specific manner. Therefore, the axon guidance function of Nrx IV is to ensure proper localization and stability of Robo in the lateral CNS soma and axons during ML axon repulsion (Banerjee, 2010).

Additional support for tissue specific functions of Nrx IV comes from recent reports that Drosophila cardiac development uses a non-canonical role of Nrx IV to maintain cardiac integrity, by coupling with G-protein signaling. Both Robo and Slit have previously been shown to control cardiac cell polarity and morphogenesis. Since the embryonic heart cells lack SJs, these findings further underscore the fact that Nrx IV and Robo/Slit coordinate diverse roles in different tissues involving multiple molecular partners. Thus, Nrx IV functions in Slit/Robo axon guidance pathway independently of its other known partners, such as Cont, Nrg and Wrapper (Banerjee, 2010).

The findings strongly support the existence of Nrx IV, Robo and Slit as a molecular complex. Although Nrx IV function appears interwoven with Robo and Slit, the phenotypes displayed by nrx IV mutants do not completely phenocopy either slit or robo mutants, suggesting that Nrx IV plays a modulatory role in Slit/Robo signaling. The biochemical analyses suggest that Robo is required for Nrx IV stability, as the levels of Nrx IV are significantly reduced in robo mutants. Slit, on the other hand, showed a modest stimulatory effect on Robo and Nrx IV association and expression levels, further confirming that these three proteins are functionally interlinked. Furthermore, the Slit/Robo complex is less efficiently immunoprecipitated from nrx IV mutant. Thus, while loss of Nrx IV does not abolish interactions between Robo and Slit, it could potentially affect proper functioning of the Robo/Slit signaling complex. Similarly, reduced association of Slit and Nrx IV in robo mutants suggests that Robo is also important for efficient complex formation between these three proteins (Banerjee, 2010).

Both Nrx IV and Robo are transmembrane proteins that colocalize in longitudinal axons. Most of the known Nrx IV interacting proteins, such as Cont, Nrg and Wrapper belong to Ig superfamily of cell adhesion molecules (CAM). Therefore, it is conceivable that Nrx IV associates with Robo (an Ig CAM) in neurons which do not express detectable levels of Cont or Wrap. Furthermore, nrx IV mutant phenotypes resemble those of robo mutant, and Nrx IV interacts with Robo/Slit, suggesting that Nrx IV functions in the Robo/Slit pathway (Banerjee, 2010).

Recent studies support a model where Slit stimulation recruits cytoplasmic Sos to Robo receptor via Dock to activate Rac-dependent cytoskeletal changes within the growth cone during repulsion. This study shows that Nrx IV and Robo retain their ability to colocalize and interact when co-expressed in a heterologous system, and indicate that Slit is dispensable for their interaction. In addition, slit RNAi experiments in S2 cells reveal that Nrx IV and Robo associate in the absence of Slit. However, Slit stimulation of nrx IV/robo cotransfected CHO cells caused enhanced colocalization of Nrx IV and Robo in intracellular compartments and membrane ruffles, further supporting a functional relationship between Nrx IV and Robo/Slit. Together, these in vivo and in vitro findings indicate that Nrx IV and Robo interact in the absence of Slit, and in the presence of Slit ligand the molecular interactions between Nrx IV and Robo are strengthened. Formation of this larger molecular complex at the axonal surface thus ensures proper ML axon guidance (Banerjee, 2010).

The phenotypic similarities, dose-dependent genetic interactions and the in vivo biochemical data suggest that Nrx IV acts as a modulator in Slit/Robo signaling pathway. One of the key reasons for this conclusion is the fact that the axon guidance phenotypes in nrx IV mutants is rescued by the expression of the extracellular region of Nrx IV (Nrx IV^mycΔCT), where as the phenotype is not rescued by the intracellular region of Nrx IV (Nrx IV^mycΔNT). For Nrx IV to act as an independent signal transducer, it would need an intact cytoplasmic region. Since the axon guidance phenotypes and Robo localization are both rescued by Nrx IV^mycΔCT, the downstream signaling controlling axon repulsion is controlled by Robo or an as yet unidentified protein. Therefore, the data support a role for Nrx IV in the proper localization and stabilization of Robo at axonal membrane, where it interacts with Slit, to regulate downstream axon guidance signaling. Although the exact domains regulating Nrx IV-Robo interactions are unknown at this point, it is predicted that they occur via the Ig or FNIII domains, as these domains regulate Nrx IV-Cont interactions. The data rule out the possibility that Nrx IV interactions with Robo occur via a large cytoskeletal scaffolding complex, as Nrx IV^mycΔCT lacks the cytoplasmic region. This led to the conclusion that Nrx IV and Robo interact in cis through their extracellular regions, and therefore eliminate the possibility of a parallel Nrx IV signaling pathway (Banerjee, 2010).

One of the interesting in vivo observations is the association between Nrx IV and Slit still occurs in robo null mutant embryos, indicating that Slit/Nrx IV can interact in the absence of Robo. Although it remains to be seen if Nrx IV and Slit can associate in the absence of all Drosophila proteins in a heterologous system, based on the existing findings it is tempting to speculate that Nrx IV may function as a co-receptor for Slit, and together with Robo, they stabilize the complex to ensure proper presentation or retention at the axonal surface. A similar role has been assigned to Sdc, which is thought to be critical for the fidelity of Slit repellent signaling, as sdc mutants exhibit consistent defects in ML axon guidance. Thus, a multitude of mechanisms seem to operate at the axonal surface and growth cones to ensure that axons reach their correct targets. The Slit-independent interactions between Nrx IV and Robo, but seemingly enhanced colocalization and interactions in the presence of Slit, point to an interesting mechanism where signaling molecules use accessory proteins to ensure their proper localization and stability. This mechanism ensures checks and balances at several molecular levels to allow navigating axons to reach their final destinations. Very few proteins have been implicated in Slit/Robo signaling at the axonal surface, and additional yet unidentified proteins may be involved. With the identification of Nrx IV as an essential component of the Slit/Robo complex, new insights into this highly sophisticated molecular pathway are opened, and may allow for future studies aimed at identifying the modulatory proteins that coordinate and/or control axon guidance (Banerjee, 2010).

Canoe functions at the CNS midline glia in a complex with Shotgun and Wrapper-Nrx-IV during neuron-glia interactions

Vertebrates and insects alike use glial cells as intermediate targets to guide growing axons. Similar to vertebrate oligodendrocytes, Drosophila midline glia (MG) ensheath and separate axonal commissures. Neuron-glia interactions are crucial during these events, although the proteins involved remain largely unknown. This study shows that Canoe (Cno), the Drosophila ortholog of AF-6, and the DE-cadherin Shotgun (Shg) are highly restricted to the interface between midline glia and commissural axons. cno mutant analysis, genetic interactions and co-immunoprecipitation assays unveil Cno function as a novel regulator of neuron-glia interactions, forming a complex with Shg, Wrapper and Neurexin IV, the homolog of vertebrate Caspr/paranodin. These results also support additional functions of Cno, independent of adherens junctions, as a regulator of adhesion and signaling events in non-epithelial tissues (Slováková, 2011).

The midline constitutes a key boundary of bilateral organisms. In vertebrates, it is the floorplate and the functionally equivalent structure in Drosophila is the mesectoderm, which gives rise to all midline cells, neurons and glia, in the most ventral part of the embryo. MG are of great relevance at the midline as an intermediate target during axonal pathfinding, providing both attractive and repulsive guidance cues. These signals allow contralateral axons to cross the midline but never to recross, and they also keep ipsilateral axons away from the midline. In addition to this early function in guiding commissural axons towards the midline, MG are also fundamental later on to separate the commissures by enwrapping and subdividing them. This study shows that the PDZ protein Cno and the DE-cadherin Shg participate in, and contribute to, the regulation of these later stage neural differentiation events, in which neuron-glia interactions play a central role (Slováková, 2011).

In Drosophila, Wrapper and Nrx-IV physically interact to promote glia-neuron intercellular adhesion at the MG. This study proposes that Cno and Shg are important components of this adhesion complex and key to its function. Both Cno and Shg are present at the MG, being highly restricted to the interface between MG and commissural axons. Cno and Shg were detected in a complex in vivo with Wrapper at the CNS MG. Nrx-IV, which is located on the surface of commissural axons, was also consistently found in a complex with Cno, although the amount of Cno protein that was co-immunoprecipitated was much lower than that present in Cno-Wrapper complexes. One plausible explanation is that whereas Cno and Wrapper are present in the same cell (MG), Cno and Nrx-IV are in different cell types (MG and neurons, respectively) and, in addition, Cno is a cytoplasmic protein that is indirectly linked to Nrx-IV through other proteins in the same complex (i.e., Shg and Wrapper). Intriguingly, stronger genetic interactions were found between Cno and Nrx-IV than between Cno and Wrapper (double heterozygote analysis). A possible explanation for this is that Nrx-IV is not only acting through Wrapper-Shg-Cno in the MG but also through other partners. In this way, when the dose of Cno and Wrapper was halved, Nrx-IV could still function fully through these other, putative partners. However, halving the dose of Cno and Nrx-IV would impair not only the Nrx-IV-Wrapper-Cno signal but also the other potential pathways. In vertebrates, the ortholog of Nrx-IV, termed contactin-associated protein (Caspr or Cntnap) or paranodin, is located at the septate-like junctions of the axonal paranodes, where it interacts in cis with contactin (at neurons) and in trans with neurofascin (at the glia). The Drosophila homologs of these Ig superfamily proteins, Contactin and Neuroglian, interact in the same way with Nrx-IV at the septate junctions. However, there are no septate junctions at the neuron-MG interface. Hence, other, as yet unknown partners of Nrx-IV might exist at this location (Slováková, 2011).

Cno and its vertebrate orthologs afadin/AF-6/Mllt4 have been shown to localize at epithelial adherens junctions (AJs), where they regulate the linkage of AJs to the actin cytoskeleton by binding both actin and Nectin family proteins. However, Cno is not exclusively present at the AJs of epithelial tissues. Indeed, it was found that Cno is also expressed in mesenchymal tissues, where it dynamically regulates three different signaling pathways required for muscle/heart progenitor specification. The asymmetric division of these muscle/heart progenitors and of CNS progenitors also requires an AJ-independent function of Cno to asymmetrically locate cell fate determinants and properly orientate the mitotic spindle. Therefore, Cno seems to act through different mechanisms depending on the cell type. This study describes a novel function of Cno during neural differentiation. In the MG, Cno, through Shg, contributes to the tight adhesion between the MG and the commissural axons and perhaps even to the regulation of some intracellular signaling within the MG. Indeed, Cno has been shown to regulate different signaling cascades during development. No AJs or septate junctions (SJs) have been described at the MG-commissural axon interface. This suggests that the function of Cno in the midline is independent of AJs. In fact, the partner of Cno at this location, the Drosophila Nectin ortholog Echinoid, is not detected at the midline. In this context, it is worth pointing out that Shg is an epithelial cadherin key at AJs. This study has shown that Shg can also be found in non-epithelial tissues with an important function independent of AJs. A similar situation occurs with Nrx-IV. Despite Nrx-IV being a very well established component of SJs, no SJs are formed in the midline and no other known components of SJs are expressed there. Thus, different modes of Cno action, either as an AJ protein or as a signaling pathway regulator, are possible and they are not mutually exclusive: it all depends on the cell type and context (Slováková, 2011).

DEVELOPMENTAL BIOLOGY

Embryonic

The gene is transcribed only zygotically , with its peak of expression between 6 and 18 hours after egg lay. NRX is first detected at late stage 11/early stage 12. NRX is localized to epithelial cells of ectodermal origin such as epidermis, the tracheal system, pharynx, esophagus, proventriculus, hindgut, salivary gland, and cells of the peripheral and central nervous systems. Staining in the PNS is localized to the scolopales of the lateral chordotonal orgtans and is restricted to glial cells. NRX positive cells in the CNS and along the peripheral nerves express glial-specific markers and not neuronal markers. Two types of epithelia do not express NRX: amnioserosa and Malpighian tubules. All tissues that express NRX in the embryo are characterized by the presence of pleated synaptic junctions.

In the chordotonal organs, NRX is expressed in cells surrounding the scolopales where synaptic junctions have been localized. In addition, NRX is distributed in a ring at the apical end of many cell types, as shown for synaptic junctions (Baumgartner, 1996).

Organization and function of the blood-brain barrier in Drosophila

The function of a complex nervous system depends on an intricate interplay between neuronal and glial cell types. One of the many functions of glial cells is to provide an efficient insulation of the nervous system and thereby allowing a fine tuned homeostasis of ions and other small molecules. This study presents a detailed cellular analysis of the glial cell complement constituting the blood-brain barrier in Drosophila. Using electron microscopic analysis and single cell-labeling experiments, different glial cell layers at the surface of the nervous system, the perineurial glial layer, the subperineurial glial layer, the wrapping glial cell layer, and a thick layer of extracellular matrix, the neural lamella, were characterized. To test the functional roles of these sheaths a series of dye penetration experiments were performed in the nervous systems of wild-type and mutant embryos. Comparing the kinetics of uptake of different sized fluorescently labeled dyes in different mutants led to the conclusion that most of the barrier function is mediated by the septate junctions formed by the subperineurial cells, whereas the perineurial glial cell layer and the neural lamella contribute to barrier selectivity against much larger particles (i.e., the size of proteins). The requirements of different septate junction components were compared for the integrity of the blood-brain barrier, and evidence is provided that two of the six Claudin-like proteins found in Drosophila are needed for normal blood-brain barrier function (Stork, 2008).

Fast neuronal conductance requires a tight electrical insulation of the axons and in the mammalian nervous system, myelin and saltatory conductance evolved. Arthropods have not evolved saltatory conductance, but they are nevertheless in need for fast electrical conductance. In this respect it is not surprising that in marine shrimps myelin-like structures have been described . Drosophila follows two different and seemingly independent strategies to ensure fast conductance. In some central neuronal networks large caliber axons develop, whereas in the peripheral nervous system axons are insulated by several glial sheaths to ensure insulation. Initially, at the beginning of larval life, the different sensory and motor axons are kept as separate fascicles within the segmental nerves, suggesting there might be some degree of electrical cross talk within the different modalities. As the larva matures, the inner wrapping glia starts to grow around single axons, which may allow more sophisticated movements of the wandering larvae (Stork, 2008).

Whereas the wrapping glia insulates individual axons, do perineurial and subperineurial glia insulate the entire nervous system and set up the blood-brain-barrier? Genetic experiments and ultrastructural studies have long indicated that septate junctions provide the most effective part of this barrier. Indeed, the subperineurial cell are formed early in development and these cells are connected by septate junctions from late embryonic stages onwards. Using Gal4 driver strains specific to the subperineurial cells as well as in vivo septate junction markers, this study confirms that during larval life the subperineurial cells do not divide but grow enormously large in size. Septate junctions formed by the subperineurial cells are mostly found in interdigitated zones of cell-cell contact. Cell division would likely require disintegration of septate junctions and thus result in a temporal opening of the blood-brain barrier, which would be deleterious for the animal. This is in agreement with previous findings that Gliotactin expressing cells, forming septate junctions, do not divide during larval live (Stork, 2008).

The outermost glial cell layer is formed by the perineurial cells. Although these cells have long been described, their origin is still a matter of debate. In EM micrographs of late embryonic staged peripheral nerves some perineurial cells can be detected apically to the subperineurial cells. These glial cells divide during larval life and generate a large number of fine cell protrusions that cover the subperineurial cells. One function of the perineurium might be to influence the development and/or the tightness of the subperineurial layer. A comparable cellular function has been attributed to the astrocytes in the mammalian nervous system. Alternatively, the perineurial glial cells might provide a cellular basis for the response to injury. Unfortunately, to date there is no specific driver strains that allow manipulation of this glial cell population. Interestingly, a reverse relationship between subperineurial and perineurial cells has been suggested previously as subperineurial expression of activated Ras or PI3K (phosphoinositide 3-kinase) resulted in an thickening of the perineurial sheath (Stork, 2008).

Additionally, the fray gene has been shown to be required for normal axonal ensheathment. Interestingly the mutant phenotype could be rescued by expressing fray using three different Gal4 drivers. After the analysis of the specificity of these drivers (Mz317, subperineurial glia and weak wrapping glia; Mz709, all glial cell types; gliotactinGal4, subperineurial glia), it is concluded that fray is expressed in subperineurial glia and controls axonal ensheathment of wrapping glia in a noncell autonomous manner (Stork, 2008).

Given the different cellular barriers described in this report, questions arise concerning the functional contributions of the different layers. Kinetic studies were performed that supported the importance of the septate junctions in particular for small components. Animals lacking septate junctions are as leaky to a 10 kDa dextran as animals lacking all glial cell layers. However, when it comes to larger molecules, the relevance of the other cell layers becomes obvious. Although a 500 kDa dextran can easily penetrate into the nervous system of a glial cells missing embryo, its leakage into the nervous system of a neurexinIV mutant lacking septate junctions is greatly reduced. Thus, the other layers contribute to the function of the blood-brain barrier. Because a continuous perineurium is not fully formed in first instar larvae, the barrier function has to be assigned to the neural lamella and the inner glial layer. There are several reports showing that the neural lamella can act as an efficient filter for heavy metal ions. Possibly, large molecules such as the 500 kDa dextran are also trapped in this ECM. Alternatively, large particles are stopped by the diffusion barrier established by the normal cell-cell contacts between subperineurial cells and inner glial cell types like wrapping glia in the peripheral nerves and cortex and neuropile glia in the CNS (Stork, 2008).

The diffusion barrier provided by glial cells or epithelial sheaths is generated by special junctional complexes that help to tightly associate the involved cells. Drosophila epithelia as well as glial cells are characterized by septate junctions. Quite similar structures are also found at the mammalian paranodal junctions, which provide the structural basis for the tight electrical insulation of the nerve. A core component of the mammalian axoglial septate junctions is the NeurexinIV homolog Caspr that together with its binding partners, Contactin and Neurofascin155, sets up a tripartite adhesion complex at the paranode (Stork, 2008).

The function of this complex appears conserved in Drosophila, although there are some notable differences. The Caspr homolog NeurexinIV is expressed by glial cells as are Contactin and the Neurofascin155 homolog Neuroglian. As a consequence, in the fly septate junctions are formed between glial cells, whereas they are formed between neuronal and glial membranes in the mammalian system. The Caspr/Contactin/Neurofascin155 complex seals the paranodal junction and a similar function has been attributed to this protein complex in the invertebrate blood-brain barrier. This study found a less pronounced function of Contactin compared with NeurexinIV for the blood-brain barrier establishment, corroborating findings made in embryonic epithelia (Stork, 2008).

Another prominent component of the junctional complexes are the Claudin proteins. In mammals, members of these four transmembrane domain proteins are associated with tight junctions that are often considered to be functionally equivalent to the invertebrate septate junctions. In Drosophila two Claudin-like proteins have been described to be required for formation of normal epithelial barrier formation. This study shows that both Sinuous and Megatrachea are also needed for the establishment of normal blood-brain barrier formation. Similarly, it was shown that mammalian claudin5 is a major component of tight junctions of brain endothelial cells. claudin5 mutant mice show no structural or ultrastructural deficits, but have an impaired blood-brain barrier. The association of Claudins integrated in opposing membranes is thought to provide pores that can control the paracellular diffusion of small molecules. Although Drosophila Sinuous and Megatrachea clearly contribute to the barrier function, it is inconceivable that fly Claudins traverse the 20 nm wide septate gap to form a Claudin pore as it is discussed for the vertebrate Claudins. It has also been suggested that invertebrate Claudins might have lost their pore-like functions and exert only signaling function to establish the barrier (Stork, 2008).

Such a signaling function may control the size selectivity of the barrier and indeed sinuous mutants show only a weak barrier phenotype comparable with moody mutants, correlating with reduced septate junctions. This study demonstrates that a loss of septate junctions associated with neurexinIV mutants results in breakdown of the blood-brain barrier comparable with what is observed in animals lacking all glial cells. However, additional mechanisms are in place to control the paracellular diffusion of larger particles. A 500 kDa dextran can easily penetrate the nervous system of a glial cells missing embryo but cannot enter a nerve cord only lacking septate junctions (Stork, 2008).

Larval

Postembryonically, in third-instar larvae, staining can also be detected in areas where synaptic junctions have been repeated: the subperineural sheath of the larval CNS, imaginal disc cells, and salivary glands (Baumgartner, 1996).

Occluding junctions maintain stem cell niche homeostasis in the fly testes

Stem cells can be controlled by their local microenvironment, known as the stem cell niche. The Drosophila testes contain a morphologically distinct niche called the hub, composed of a cluster of between 8 and 20 cells known as hub cells, which contact and regulate germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Both hub cells and CySCs originate from somatic gonadal precursor cells during embryogenesis, but whereas hub cells, once specified, cease all mitotic activity, CySCs remain mitotic into adulthood. Cyst cells, derived from the CySCs, first encapsulate the germline and then, using occluding junctions, form an isolating permeability barrier. This barrier promotes germline differentiation by excluding niche-derived stem cell maintenance factors. This study shows that the somatic permeability barrier is also required to regulate stem cell niche homeostasis. Loss of occluding junction components in the somatic cells results in hub overgrowth. Enlarged hubs are active and recruit more GSCs and CySCs to the niche. Surprisingly, hub growth results from depletion of occluding junction components in cyst cells, not from depletion in the hub cells themselves. Moreover, hub growth is caused by incorporation of cells that previously express markers for cyst cells and not by hub cell proliferation. Importantly, depletion of occluding junctions disrupts Notch and mitogen-activated protein kinase (MAPK) signaling, and hub overgrowth defects are partially rescued by modulation of either signaling pathway. Overall, these data show that occluding junctions shape the signaling environment between the soma and the germline in order to maintain niche homeostasis (Fairchild, 2016).

The hub regulates stem cell behavior in multiple ways. First, the hub physically anchors the stem cells by forming an adhesive contact with both germline stem cells (GSCs) and cyst stem cells (CySCs). The hub thus provides a physical cue that orients centrosomes such that stem cells predominantly divide asymmetrically, perpendicular to the hub. Following asymmetric stem cell division, one daughter cell remains attached to the hub and retains stem cell identity while the other is displaced from the hub and differentiates. Second, hub cells produce signals, including the STAT ligand Unpaired-1 (Upd), Hedgehog (Hh), and the BMP ligands Decapentaplegic (Dpp) and Glass-bottomed boat (Gbb), that signal to the adjacent stem cells to maintain their identity. As germ cells leave the stem cell niche, two somatic cyst cells surround and encapsulate them to form a spermatocyst. As spermatocysts move from the apical to the basal end of the testis, both somatic cyst cells and germ cells undergo a coordinated program of differentiation. Previous studies have shown that differentiation of encapsulated germ cells requires their isolation behind a somatic occluding junction-based permeability barrier. Specifically, a role was identified for septate junctions, which are functionally equivalent to vertebrate tight junctions, in establishing and maintaining a permeability barrier for each individual spermatocyst (Fairchild, 2016).

During analysis of septate junction protein localization, it was observed that some, notably Coracle, were expressed in both the hub and the differentiating cyst cells. Moreover, knockdown of septate junction components in the somatic cells of the gonad resulted in enlarged hubs. Based on these results, the role of septate junction components in regulating the number of hub cells was explored in detail. To this end, RNAi was used to knock down the expression of the core septate junction components Neurexin-IV (Nrx-IV) and Coracle (Cora) in both the hub and cyst cell populations and the number of hub cells counted in testes from newly eclosed and 7-day-old adults. RNAi was expressed using three tissue-specific drivers: upd-Gal4, expressed in hub cells; tj-Gal4, expressed weakly in hub cells and strongly in both CySCs and early differentiating cyst cells; and eyaA3-Gal4, expressed strongly in all differentiating cyst cells, weakly in CySCs, and at negligible levels in the hub. To visualize hub cells, multiple established hub markers, including upd-Gal4, upd-lacZ, Fasciclin-III (FasIII), and DN-cadherin (DNcad) were used. Surprisingly, it was found that knockdown of Nrx-IV or cora driven by upd-Gal4 gave rise to normal hubs. In comparison, knockdown of Nrx-IV or cora using tj-Gal4 or eyaA3-Gal4 led to large increases in the number of the hub cells. Hub growth was not uniform and varied between testes, but median hub cells numbers in Nrx-IV and cora knockdown testes grew by 30% and 55%, respectively, between 1 and 7 days post-eclosion (DPEs). However, in extreme cases, hubs contained up to five times the number of cells found in age-matched control testes. This result was confirmed using a series of controls that discounted the possibility that hub overgrowth was due to temperature or leaky expression of the RNAi lines. These results suggested that hub growth occurred as a result of knockdown of septate junction proteins in cyst cells rather than the hub. This was further supported using another somatic driver that is not thought to be expressed in the hub, c587-Gal4. However, analysis of c587-Gal4 was complicated by the fact this driver severely impacted fly viability when combined with Nrx-IV or cora RNAi lines (Fairchild, 2016).

Intriguingly, hub growth largely occurred after adult flies eclosed and not in earlier developmental stages. For example, when the driver eyaA3-Gal4 was used to knock down Nrx-IV or cora, hubs from 1-day-old adults were not larger than controls, but hubs from 7-day-old adults were significantly larger. Moreover, overgrowth phenotypes were recapitulated when temperature-sensitive Gal80 was used to delay induction of eyaA3-Gal4-mediated Nrx-IV knockdown until after eclosion. Hub growth manifested both in a higher mean number of hub cells per testis and by a shift in the distribution of hub cells per testis upward, toward larger hubs sizes. This distribution suggested a gradual, stochastic process of hub growth, resulting in a population of testes containing a range of hub sizes. These results reveal progressive hub growth in adults upon knockdown of septate junction components in cyst cells and suggest that this growth is not driven by events occurring in the hub itself but rather by events occurring in cyst cells (Fairchild, 2016).

Niche size has been shown in various tissues, including vertebrate hematopoietic stem cells and somatic stem cells in the fly ovary, to be an important factor in regulating the number of stem cells that the niche can support. In the fly testes, it has been shown that mutants having few hub cells could nonetheless maintain a large population of GSCs. To determine how a larger hub, containing more cells, affects niche function, the number of GSCs and CySCs was monitored after knockdown of septate junction components in cyst cells. Overall, the average number of germ cells contacting the hub grew substantially in Nrx-IV or cora knockdown testes between 1 and 7 DPEs. To confirm that the germ cells contacting the hub were indeed GSCs, spectrosome morphology was studied, and it was found to be to be consistent with that seen in wild-type GSCs. Moreover, in individual testes, there was a positive correlation between the number of hub cells and the number of GSCs. Similar growth was also observed in the number of CySCs, defined as cyst cells expressing Zfh1, but not the hub cell marker DNcad. Control testes (from tj-Gal4 x w¹¹¹⁸ progeny) had on average 34.3 CySCs whereas Nrx-IV and cora knockdown testes had 53.4 and 50.2 CySCs, respectively. These results show the importance of maintaining a stable stem cell niche size, as enlarged hubs were active and could support additional stem cells, which may result in the excess production of both germ cells and cyst cells (Fairchild, 2016).

Next, it was of interest to determine the mechanism driving hub growth in adult flies upon knockdown of septate junction components in cyst cells. One possible mechanism that can explain this growth is hub cell proliferation. However, a defining feature of hub cells is that they are not mitotically active. Consistent with this, a large number of testes were stained for the mitotic marker phospho-histone H3 (pH3), and cells were never observed where upd-LacZ and pH3 were detected simultaneously. These results argue that division of hub cells is unlikely to explain hub growth in the adult Nrx-IV and cora knockdown testes. To determine the origin of the extra hub cells, the lineage of eyaA3-expressing cells was traced using G-TRACE (Evans, 2009). eyaA3 was chosed as both the expression pattern of septate junctions, and Nrx-IV or cora knockdown results suggested that hub growth involved differentiating eyaA3-positive cyst cells. The eyaA3-Gal4 driver utilizes a promoter region of the eya gene, which is required for somatic cyst cell differentiation and is expressed at very low levels in CySCs and at high levels in differentiating cyst cells. Using G-TRACE allows identification of both cells that previously expressed eyaA3-Gal4 (marked with GFP) and cells currently expressing eyaA3-Gal4 (marked with a red fluorescent protein [RFP]); additionally, the hub was identified using expression of upd-LacZ and FasIII. In control experiments at both 1 and 7 DPEs, few GFP-positive cells were observed in the hub. Those few GFP-positive cells could be explained by the transient expression of eya in the embryonic somatic gonadal precursor cells that form both hub and cyst cell lineages or extremely low levels of expression in adult hub cells. When G-TRACE was combined with knockdown of Nrx-IV, the results were strikingly different. Initially, 1 DPE, hubs were only slightly larger than controls and few GFP-positive hub cells were observed. In comparison, 7-DPE hubs contained on average more than twice as many cells compared to controls. Importantly, hub growth in Nrx-IV knockdowns was largely attributable to the incorporation of GFP-positive cells. Moreover, a population of upd-LacZ-labeled cells that were also RFP-positive was observed consistent with ongoing or recent expression of eyaA3-Gal4 in hub cells. These results suggest that knockdown of Nrx-IV or cora leads cyst cells to adopt hallmarks of hub cell identity and express hub-cell-specific genes (Fairchild, 2016).

To learn more about the differentiation state of non-endogenous hub cells in Nrx-IV and cora knockdown testes, various markers were used to label the stem cell niche. This analysis showed normal expression of hub cell markers, such as Upd, FasIII, DNcad, as well as Hedgehog (hh-LacZ), Armadillo (Arm), and DE-Cadherin (DEcad). It was asked how cells that were previously, and in some instances were still, eyaA3 positive could express multiple hub-cell fate markers. To answer this question, the signaling mechanisms that determine hub fate were investigated in Nrx-IV and cora knockdown testes. Hub growth phenotypes similar to those produced by Nrx-IV and cora knockdown have been described previously, most notably in agametic testes that lack germ cells, suggesting that the germline regulates the formation of hub cells. One specific germline-derived signal shown to regulate hub fate is the epidermal growth factor (EGF) ligand Spitz. In embryonic testes, somatic cells express the EGF receptor (EGFR), which, when activated, represses hub formation. EGFR-induced mitogen-activated protein kinase (MAPK) signaling, visualized by staining for di-phosphorylated-ERK (dpERK), was active in CySCs and spermatogonial-stage cyst cells. Quantifying dpERK-staining intensity in cyst cell nuclei showed that MAPK activity was lower in CySCs following knockdown of Nrx-IV or cora, suggesting reduced EGFR signaling. Moreover, the effect of Nrx-IV or cora knockdown on MAPK signaling was not restricted to CySCs, as lower dpERK staining was observed at a distance from the hub. To see whether disruption of EGFR signaling could underlie hub defects in Nrx-IV and cora knockdown testes, attempts were made to rescue these phenotypes by increasing EGF signaling. When a constitutively activated EGF receptor (EGFR-CA) was co-expressed in cyst cells along with Nrx-IV RNAi, hub growth was attenuated, resulting in a reduction in the average number of hub cells compared to expressing only Nrx-IV RNAi. Similar results were also observed in the growth of the GSC population, suggesting that reduced EGFR activation in cyst cells contributes to the overall growth of the stem cell niche caused by the knockdown of Nrx-IV or cora. Surprisingly, analysis of testes with loss-of-function mutations in the EGFR/MAPK pathway reveals different phenotypes than those observed: encapsulation is disrupted and CySCs are lost, but hub size is largely unaffected. This result shows that the partial reduction in EGFR/MAPK signaling seen in Nrx-IV and cora knockdown testes results in distinct phenotypes and highlights the complexity of EGFR signaling in the fly testis (Fairchild, 2016).

Another pathway that is documented to regulate hub cell fate is Notch signaling. Notch plays important roles in hub specification in embryos. The Notch ligand Delta is produced by the embryonic endoderm and acts to promote hub cell specification in the anterior-most somatic gonadal precursor cells. Whereas it has been suggested that Notch acts in the adult to regulate hub fate, such a role has not been clearly demonstrated. A reporter for the Notch ligand Delta (Dl-lacZ) was observed in hub cells of both control and Nrx-IV knockdown testes. Intriguingly, reducing Notch signaling efficiently rescued the hub overgrowth seen in adult Nrx-IV knockdown testes. When a dominant-negative Notch (Notch-DN) was co-expressed in the somatic cells, along with Nrx-IV RNAi, the growth of the hub was reduced compared to the expression of Nrx-IV RNAi alone. Growth in the GSC population was not significantly reduced by co-expression of Notch-DN, suggesting that the Notch pathways may modulate hub growth through a different mechanism compared to the EGFR pathway. Because Notch is well established to regulate hub growth in the embryo, temperature-sensitive Gal80 was used to delay expression of Notch-DN and confirm that the reduction in hub cells was due to disruption of post-embryonic Notch signaling. These results suggest that Notch signaling in cyst cells may contribute to the hub overgrowth phenotypes caused by septate junction knockdown in the adult testes (Fairchild, 2016).

In addition to Notch and EGFR, other signaling pathways that regulate hub size may contribute to the hub growth seen upon somatic knockdown of septate junction components. For example, it has been previously shown that the range of BMP signaling is expanded following Nrx-IV or cora knockdown in cyst cells. Constitutive activation of BMP signaling in the germline was shown to increase the size of the hub and the number of GSCs. Additionally, the relative expression levels of the genes drm, lines, and bowl regulate hub size in the adult. In particular, it is known that lines maintains a “steady state” in the testes by repressing expression of a subset of hub genes in the cyst cell population. Unlike lines mutants, Nrx-IV or cora knockdowns generally lack ectopic hubs. This may reflect the more gradual hub growth seen in septate junction knockdowns or, alternatively, highlight key mechanistic differences in how hub growth is achieved in each respective genetic background. The current work is consistent with the model whereby occluding junctions are required for proper soma-germline signaling in the fly testes. This signaling maintains stem cell niche homeostasis by preventing somatic cyst cells from adopting hub cell fate, which would lead to niche overgrowth. It is well established that, in embryonic testes, hub fate is both positively and negatively regulated by signals from the germline and the endoderm.The results, and recent findings about the genes lines and traffic jam, argue that, in the adult testes, hub fate is actively repressed in the cyst cell lineage. Failure to repress hub fate allows cyst cells to exhibit features of hub cells and act as a functional stem cell niche. However, these cyst-cell-derived hub cells are distinct from the true endogenous hub cells in that they show non-hub-cell features, including expression of the differentiating cyst cell markers eyaA3-Gal4 and β3-tubulin. The data suggest that, following disruption of septate junctions proteins, the signaling environment surrounding the somatic cells is altered such that cyst cells gradually begin expressing hub cell markers (Fairchild, 2016).

One major outstanding question is how eyaA3-Gal4-expressing cyst cells become incorporated into the endogenous hub. Previously, it was shown that a septate-junction-mediated permeability barrier forms by the four-cell spermatogonial-stage spermatocyst. The hub growth phenotypes induced by Nrx-IV and cora knockdowns may occur due to defects in cell-cell signaling, possibly involving EGFR and Notch, that manifest in these later spermatocysts. However, this model requires an explanation for how these cyst cells translocate back to and join the hub. Alternatively, signaling defects in these later spermatocysts are somehow instructing earlier cyst cells, such as CySCs, to join the hub. It is easier to envisage the latter model, as early cyst cells are spatially much closer to the hub, but the sequence of signaling events in such a case will be complex and require further elucidation. The ability of CySCs to convert into hub cells in wild-type testes is a controversial subject. However, the incorporation of CySCs into the hub does not necessitate complete conversion into hub cells but could rather involve simple de-repression or activation of genes that confer hub cell function, including regulators of the cell-cycle- and hub-cell-specific signaling ligands. Notably, the transition between CySC and hub cell fate is linked to the cell cycle (Fairchild, 2016).

Why would loss of the septate-junction-mediated somatic permeability barrier result in disruption of signaling between the soma and germline? There are many possible answers, but it is possible to speculate about two such mechanisms that explain hub overgrowth. One possibility is that germline differentiation, which is dependent on the permeability barrier, is required for the release of signals that maintain stem cell niche homeostasis. Another possibility is that the permeability barrier locally concentrates germline-derived signals that repress hub cell fate by trapping them in the luminal space between the encapsulating cyst cells and the germline. The latter scenario could explain the observation that activated EGFR signaling partially rescues hub overgrowth. In this model, septate junctions allow localized buildup of the EGF ligand Spitz, ensuring that sufficient signaling is available to repress hub fate. It is more difficult to draw strong conclusions about how Notch signaling is altered when septate junctions are disrupted, particularly as the Notch ligand Delta appears restricted to the hub. Overall, an unexpected role was found for an occluding-junction-based permeability barrier in mediating stem cell niche homeostasis. This work highlights how the architecture of the stem cell niche system in the fly testes, which is highly regular and contains a reproducible number of stem cells and niche cells, is in fact the result of an active and dynamic signaling environment (Fairchild, 2016).

EFFECTS OF MUTATION

Mutants produce no adult survivors, and most alleles are embryonic lethal. Rare adult escapers exhibit rough eyes, notched wings with vein broadening and duplicate legs with malformations (Baumgartner, 1996).

Nrx mutants exhibit behavioral defects. Mutants exhibit a severe reduction of coordinate muscle propagation waves. In addition, tracheae do not fill with air. These movement defects are similar to those observed in other mutants in which neurotransmitter release is abolished (Baumgartner, 1996).

Axons are seen to cross segmental boundaries in 10-20% of mutant embryos. Thus there is no absolute requirement for NRX in axonal pathfinding, but its absence results in a higher propensity for neuronal connectivity defects.

About 10% of the neuromuscular junctions examined fail to respond to nerve stimulation, indicating that a fraction of synapses fail to form in Nrx mutants. In the other 90% of the NMJs examined, the mean amplitude of evoked synaptic transmission is reduced by 40-45% in mutant embryos. Ionotophoresis of L-glutamate (the excitatory neurotransmitter) into mutant NMJs results in essentially wild-type responses, indicating that the postsynaptic receptor field develops normally. Thus NRX function appears to be required presynaptically to facilitate transmission. Bursts of activity at NMJs, which correlate with muscle contractions that permit first-instar larvae to hatch from the egg case, are reduced in mutants, suggesting a suppression of endogenous activity in motoneurons. Interesting, some muscle contractions are observed in Nrx mutant embryos upon dissection in low potassium ion recording solutions. A similar effect occurs in gliotactin mutant embryos in which the blood-nerve barrier is broken down. It is concluded that NRX expression in glial septate junctions is required for proper axonal insulation and blood-nerve barrier formation (Baumgartner, 1996).

coracle mutants display a cell-migration defect affecting dorsal closure (Fehon, 1994). Severe Nrx alleles also cause a defect in dorsal closure, resulting in a small than normal dorsal hole, consistent with abnormal function of D4.1/Coracle in Nrx mutant embryos. Hence, septate junctions may play a role in targeting cytoskeletal components and contributing to intercellular communication and cell migration during Dorsal closure (Baumgartner, 1996).

In Nrx mutants, smooth septate junctions remain unaffected. However, pleated septate junctions (pSJs) with a typical ladder-like structure are absent in ectodermal cells and in perineural glia-ensheathing peripheral axons. Apposing membranes in these tissues have a smooth and electron-dense appearance; unstructured cleft material is present. Thus pSJs in Nrx mutant embryos lose their characteristic ladder-like septae that distinguish them from smooth septate junctions. The affected pSJs retain some characteristics of septate junctions but lack the regularly arranged septae, the principle features distinguishing pSJs from smooth septate junctions. Obliquely sectioned septate junctions appear as honeycomb-like structures in ectodermal pSJs of wild type, and as regularly spaced parallel lines (normally seen in smooth septate junctions) in the ectoderm of mutant embryos (Baumgartner, 1996).

Myelinated fibers are organized into distinct domains that are necessary for saltatory conduction. These domains include the nodes of Ranvier and the flanking paranodal regions where glial cells closely appose and form specialized septate-like junctions with axons. These junctions contain a Drosophila Neurexin IV-related protein, Caspr/Paranodin (NCP1). NCP1 contains an open reading frame of 1385 amino acids (~153 kDa) with 93% identity to rat Caspr/Paranodin. NCP1 contains an amino-terminal discoidin domain, a laminin G domain, two Neurexin domains, and a PGY-enriched segment in the extracellular region. The cytoplasmic domain contains a band 4.1 binding motif and a proline-rich sequence with at least one consensus SH3 domain binding site. In contrast to Drosophila NRX IV and Caspr2, NCP1 lacks a PDZ binding domain consensus sequence at its carboxyl terminus. Mice that lack NCP1 exhibit tremor, ataxia, and significant motor paresis. In the absence of NCP1, normal paranodal junctions fail to form, and the organization of the paranodal loops is disrupted. Contactin is undetectable in the paranodes, and K+ channels are displaced from the juxtaparanodal into the paranodal domains. Loss of NCP1 also results in a severe decrease in peripheral nerve conduction velocity. These results show a critical role for NCP1 in the delineation of specific axonal domains and the axon-glia interactions required for normal saltatory conduction (Bhat, 2001).

Septate junctions form between the ensheathing glial cells that surround the nerve bundles in Drosophila and are essential for establishment of the blood-nerve barrier. The Drosophila NRX IV protein localizes to and is required for the formation of the ladder-like septae characteristic of these junctions. In nrx IV mutants, these septae are absent, and another component of these junctions, Coracle, a band 4.1 homolog, is mislocalized. The current studies demonstrate that the septate and paranodal junctions appear to serve conserved functions in maintaining the axonal milieu required for action potential propagation. However, the topology and localization of the proteins is clearly different. In Drosophila, NRX IV is expressed by and localized between glial cells. In mice and other vertebrates, NCP1 is expressed by neurons and localized between the axon and glial cells. Hence, even though there is functional conservation, significant changes in expression occurred during evolution (Bhat, 2001).

Kinesin heavy chain function in Drosophila glial cells controls neuronal activity

Kinesin heavy chain (Khc) is crucially required for axonal transport and khc mutants show axonal swellings and paralysis. This study demonstrates that in Drosophila khc is equally important in glial cells. Glial-specific downregulation of khc by RNA interference suppresses neuronal excitability and results in spastic flies. The specificity of the phenotype was verified by interspecies rescue experiments and further mutant analyses. Khc is mostly required in the subperineurial glia forming the blood-brain barrier. Following glial-specific knockdown, peripheral nerves are swollen with maldistributed mitochondria. To better understand khc function, Khc-dependent Rab proteins were determined in glia, and evidence is presented that Neurexin IV, a well known blood-brain barrier constituent, is one of the relevant cargo proteins. This work shows that the role of Khc for neuronal excitability must be considered in the light of its necessity for directed transport in glia (Schmidt, 2012).

It is well established that the modulation of neuronal functionality depends on the ability of glial cells to provide metabolic support, regulate neurotransmitter homeostasis and influence the electrical conductance. To decipher these processes a large-scale RNAi screen was performed and ~5000 genes were silenced specifically in glial cells. This study presents an unexpected glial function of Drosophila khc in regulating neuronal activity. The specificity of the phenotype was ascertained by interspecies rescue studies and cell type-specific rescue of the khc mutant. Furthermore, the fact that glial-specific knockdown of β3-tubulin results in identical neuronal phenotypes demonstrates the functional relevance of Khc-dependent transport along microtubules (Schmidt, 2012).

Drosophila Khc was identified more than 20 years ago. In khc mutant larvae, vesicles, lysosomes, and mitochondria accumulate in axons. This results in swollen axons and eventually in larval paralysis. This study has generated a number of UAS-driven khc constructs to examine cell type-specific requirements of Khc. Two different khc alleles, khc⁶ and khc⁸, were used in trans to a genomic deficiency to eliminate background effects. Surprisingly, ubiquitous expression of Khc is not able to rescue the hypomorphic allele khc⁶. In contrast the null mutation khc⁸ can be completely rescued by Khc expression. khc⁶ contains an alteration in coil 2 of the stalk domain, a region implemented to be important for Kinesin light chain and cargo linkage. Thus, khc⁶ may induce some antimorphic functions. But the introduction of a genomic rescue construct of khc (P-khc⁺) has been described as being able to rescue khc⁶-associated lethality, which points to very high levels of endogenous (Schmidt, 2012).

Using a null mutant background (khc⁸/Df(2R)Jp6), it was possible to perform cell type-specific rescue experiments. Following expression of khc in neurons using elavGal4 only 6% of the expected flies survived. These flies were very sluggish and died within 6 d. To analyze glial-specific requirements of Khc, the ubiquitous Gal4-dependent rescue was compare with and without a glial cell-specific Gal4 repressor (repoGal80). A significant difference was apparent, the repressor reduced viability down to ~60%, which further supports the crucial role of Khc in glial cells (Schmidt, 2012).

Glial-specific knockdown of khc revealed an adult phenotype, which resembled a hyperexcitation phenotype. khc knockdown flies have the ability to walk. However, to initiate flight, flies need to jump. Upon khc knockdown, flies fail to perform this jump but rather directly start with a wing beat. Such flies then turn on their backs or spin over the floor, which then results in the characteristic popcorn-like movements. Thus, khc knockdown delays or prevents the jumping response (Schmidt, 2012).

In larvae, glial knockdown of khc results in reduced and delayed evoked responses at the muscle. Concomitantly, swellings of the peripheral nerves were apparent. In the majority of the swellings (and only in the swellings) a mislocalization of the septate junction protein Neurexin IV was found. This indicates that knockdown of khc results in an opening of the blood-brain barrier. In turn, this results in influx of potassium into the nervous system. Such influx is known to induce changes in axonal physiology and conductance velocity. Recently the role of focal adhesion kinase 56 (fak56) in Drosophila nerves was reported. Focal adhesion kinase acts downstream of integrin receptors, which are also known to be expressed by Drosophila glial cells. Interestingly, loss of fak56 function in the subperineurial glial cells (which constitute the blood-brain barrier) lowers axonal conductance in larvae and also renders the larvae sluggish pointing toward the notion that adhesion is required for a full establishment of the blood-brain barrier (Schmidt, 2012).

The observed suppression of EJP could in principle also be due to presynaptic defects or defects in the glutamate receptors expressed by the muscle. However, in contrast to vertebrates, where terminal Schwann cells cover the NMJ, the NMJ of Drosophila larvae grows into the muscle cell, which forms a so called subsynaptic reticulum around the NMJ. In addition, only few (subperineurial) glial processes invade parts of the NMJ in a highly dynamic fashion and where they participate in the removal of presynaptic debris. To further discriminate between a possible synaptic or an axonal defect the latency of EJP suppression was compared by injecting the current in the nerve 200-300 μm distant from the NMJ or in the ventral nerve cord 1000 μm distant from the NMJ. In this setting the latency doubled suggesting that axonal conductance is impaired upon glial khc knockdown. Therefore, it is anticipated that the suppression of the EJPs caused by glial reduction of khc function is unlikely due to synaptic defects (Schmidt, 2012).

Moreover, if khc would modulate synaptic activities, it would be acting in adult flies. However, when khc function was silenced only during adult stages using the TARGET system, no abnormal locomotion was observed, demonstrating that Khc is needed during development and not during adult stages (Schmidt, 2012).

Several Rab proteins were identified as highly motile in glia and their motility was significantly reduced upon khc suppression. Rab30 has been identified as a downstream target of JNK signaling, which can control kinesin cargo linkage. In addition, JNK-interacting proteins (JIPs) link Kinesin with membrane vesicles and are involved in Khc activation and cargo release. For vertebrate Rab21 a possible role in targeted Integrin trafficking from and to the cleavage furrow during cell division and an involvement in the endocytic pathway have been described (Schmidt, 2012).

The swollen nerves are likely due to a defective blood-brain barrier. The integrity of septate junctions for normal locomotion has also been suggested by the analysis of mutants affecting several septate junction components. At the end of embryogenesis, lachesin, neurexin IV, and gliotactin mutant embryos are hyperactive before becoming immobile. Possibly, the influx of potassium and sodium ions into the nervous system affects conductivity. Interestingly, panglial downregulation of the Na-K-Cl cotransporter Ncc69 or the serine/threonine kinase Fray also results in locomotion defects characterized by jumping flies. Fray and Ncc69 act in the subperineurial glia but no affect on neuronal conductance had been determined in peripheral nerves (Leiserson, 2011a; Leiserson, 2011b). Thus, the disruption of septate junctions as seen following knockdown of khc likely affects ion homeostasis more dramatically (Schmidt, 2012).

Drosophila NrxIV is highly mislocalized upon glial khc suppression. Other examples of a role for kinesin in cell polarization have been described. KIF5B and KIF5C are involved in polarized transport of certain apically distributed proteins in cultured mammalian epithelial cells. Additionally, KIF13B has been shown to directly transport proteins like Dlg1 to myelinating sites in Schwann cells. Thus, a similar function might be performed by Drosophila Khc in subperineurial glial cells and Khc might be involved in the directed transport of transmembrane proteins to septate junctions (Schmidt, 2012).

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).

Epithelial polarity proteins regulate Drosophila tracheal tube size in parallel to the luminal matrix pathway

The integrity of polarized epithelia is critical for development and human health. Many questions remain concerning the full complement and the function of the proteins that regulate cell polarity. This study reports that the Drosophila FERM proteins Yurt (Yrt) and Coracle (Cora) and the membrane proteins Neurexin IV (Nrx-IV) and Na+,K+-ATPase are a new group of functionally cooperating epithelial polarity proteins. This 'Yrt/Cora group' promotes basolateral membrane stability and shows negative regulatory interactions with the apical determinant Crumbs (Crb). Genetic analyses indicate that Nrx-IV and Na+,K+-ATPase act together with Cora in one pathway, whereas Yrt acts in a second redundant pathway. Moreover, it was shown that the Yrt/Cora group is essential for epithelial polarity during organogenesis but not when epithelial polarity is first established or during terminal differentiation. This property of Yrt/Cora group proteins explains the recovery of polarity in embryos lacking the function of the Lethal giant larvae (Lgl) group of basolateral polarity proteins. It was also found that the mammalian Yrt orthologue EPB41L5 (also known as YMO1 and Limulus) is required for lateral membrane formation, indicating a conserved function of Yrt proteins in epithelial polarity (Laprise, 2009).

To clarify the mechanisms of epithelial polarization, the function was examined of the FERM-domain protein Yrt, which was shown to act as a negative regulatory component of the Crb complex in both Drosophila and vertebrates. Crb regulates epithelial apical basal polarity by promoting apical membrane and apical junctional complex formation. Crb also controls the growth of the apical membrane at late stages of epithelial differentiation. Yrt binds to the cytoplasmic tail of Crb and restricts Crb activity in apical membrane growth. However, Yrt is predominantly a basolateral protein that is recruited into the Crb complex only at late stages of epithelial differentiation. Embryos completely lacking maternal and zygotic Yrt (yrt^M/Z) displayed polarity defects before Yrt is recruited into the Crb complex at late organogenesis. This raises the question whether Yrt has a function in epithelial organization as a basolateral protein (Laprise, 2009).

yrt^M/Z mutants and double-mutant combinations of yrt and genes encoding basolateral proteins were examined for synergistic genetic interactions that could indicate functional cooperation. yrt^M/Z embryos showed clear polarity defects at post-gastrula stages of development, as indicated by the basolateral mislocalization of Crb. In contrast, zygotic yrt mutants demonstrated only minor polarity defects in the trunk ectoderm. This indicates that yrt is required for polarity, and that zygotic yrt mutants could provide a sensitized background to reveal genetic interactions. Within the basolateral membrane, Yrt is enriched at the septate junction together with the Na+,K+-ATPase and other septate junction components from stage 14 onwards. Marked genetic interactions were found between yrt and Atpα (which encodes the α-subunit of Na+,K+-ATPase) and between yrt and Nrx-IV, but not between yrt and six other genes that encode septate junction transmembrane proteins. In contrast to wild type and single mutants, apical markers were mislocalized in yrt Atpα and yrt Nrx-IV double mutants similar to yrt^M/Z embryos. Enhancement of the Nrx-IV null phenotype by yrt indicates that Nrx-IV and yrt have overlapping functions and do not operate in a linear pathway. The pathway relationship between yrt and Atpα remains uncertain as embryos completely devoid of Na+,K+-ATPase function cannot be analysed. These findings reveal previously unrecognized functions of the Na+,K+-ATPase and Nrx-IV in epithelial polarity (Laprise, 2009).

The developmental timing of the yrt^M/Z and the yrt Atpα and yrt Nrx-IV polarity phenotypes is notable. These mutants show no polarity defects during cellularization when epithelial cells first form or during gastrulation when the apical junctional belt is assembled. Polarity defects are first seen at stage 11 or 12, and are most prominent at embryonic stage 13. In contrast to wild type, Crb and other apical markers are found throughout the plasma membrane and colocalize with basolateral markers such as Discs large (Dlg, also known as Dlg1) and Fasciclin 3 in yrt^M/Z, yrt Atpα and yrt Nrx-IV mutants. During late embryogenesis, apical markers become restricted again to the apical membrane, which, however, remains abnormally extended and dome-shaped. Thus, Yrt, Na+,K+-ATPase and Nrx-IV cooperate and have critical functions in maintaining epithelial polarity during early organogenesis (stages 11-13), well before septate junction assembly is observed, and Yrt is recruited to the apical membrane at stage 14 (Laprise, 2009).

Na+,K+-ATPase and Nrx-IV are required for septate junction assembly. Because Yrt also accumulates at septate junctions, it was asked whether Yrt has a role in septate junction formation or function. Septate junctions are basal to the adherens junction and, like vertebrate tight junctions, prevent diffusion of solutes between cells. Dye injection assays show that paracellular barriers in epithelia of yrt^M/Z embryos are compromised. However, yrt^M/Z embryos showed a normal complement of septa when examined ultrastructurally whereas Atpα and Nrx-IV mutants lack septa. Immunoprecipitation experiments demonstrated FERM-domain-dependent interactions between Yrt and the septate junction transmembrane protein Neuroglian (Nrg), and the enrichment of Yrt at septate junctions was less pronounced in Nrg mutants. However, Nrg or Nrg yrt mutant embryos did not display overt defects in epithelial polarity. These findings indicate that Yrt is a bona fide septate junction component that has a function distinct from Nrx-IV and Na+,K+-ATPase because it is not required for septa formation but essential for barrier function (Laprise, 2009).

Next, the analysis extended to cytoplasmic adaptor proteins associated with septate junctions. yrt does not show genetic interactions with dlg or lgl, genes encoding conserved basolateral polarity proteins required for septate junction formation and apical/basal polarity. varicose (vari), which encodes a membrane-associated guanylate kinase, and cora, which encodes a FERM protein that is the Drosophila orthologue of mammalian erythrocyte protein band 4.1 and its paralogues, were examined. vari and cora null mutant embryos did not show defects in apical basal polarity. No functional interactions were seen between yrt and vari. In contrast, yrt cora double-mutant embryos displayed marked apicalization defects, with strongly expanded apical membranes and reduced basolateral membranes in all ectodermal epithelia including the epidermis. This phenotype is significantly stronger than the yrt^M/Z or cora null mutant phenotypes indicating that Yrt and Cora have redundant functions in promoting epithelial polarity (Laprise, 2009).

Similar to yrt^M/Z mutants, yrt cora double mutants did not show polarity defects during gastrulation. By stage 11, polarity defects were more prominent in yrt cora mutants than in yrt^M/Z embryos, which correlates with Cora being expressed from stage 11 onwards. In yrt cora mutants, the most severe polarity defects characterized by overlapping distributions of apical and basolateral markers occurred during stage 13. However, by late embryogenesis, apical and basolateral markers were segregated, although expanded apical membranes and severe defects in tissue organization persisted. Interestingly, the yrt cora apicalization phenotype is very similar to the phenotype that results from high-level overexpression of Crb in both timeline and severity. The segregation of apical and basolateral markers in late-stage yrt cora mutants is also observed in late-stage yrt^M/Z, yrt Atpα and yrt Nrx-IV embryos. Thus, polarization mechanisms are at play in late embryos that are not dependent on Yrt and Cora. To address the possibility that this repolarization is the result of redundancy between Yrt/Cora and Lgl-group proteins, which include Scribble (Scrib) as well as Lgl and Dlg, embryos lacking Yrt and Scrib (yrt^M/Z scrib^M/Z) were examined. Apical and basolateral markers segregated in yrt^M/Z scrib^M/Z embryos indicating that a basolateral polarity mechanism exists in late embryos that is independent of Yrt/Cora and Lgl-group proteins (Laprise, 2009).

Together, these findings suggest that Yrt, Cora, Nrx-IV and the Na+,K+-ATPase are a new functionally cooperating group of basolateral epithelial polarity proteins, which is refered to as the ‘Yrt/Cora group’. In contrast to the enhancement of the cora and Nrx-IV null phenotypes by yrt mutations, no phenotypic enhancement was seen in cora Nrx-IV or cora Atpα double mutants, indicating that the Yrt/Cora group is composed of two functionally overlapping pathways. Cora, Nrx-IV and the Na+,K+-ATPase belong to one pathway, which is consistent with previously documented biochemical and genetic interactions between these proteins, whereas Yrt defines a second redundant pathway (Laprise, 2009).

One critical aspect of polarity regulation is that apical and basolateral proteins act antagonistically to set up mutually exclusive membrane domains. In gastrulating Drosophila embryos, this interaction occurs between apical factors and basolateral Lgl-group proteins. Similar to lgl, dlg and scrib mutations, yrt mutations partially suppress the crb mutant phenotype. Because Yrt can bind Crb directly, it is suggested that Yrt negatively regulates Crb function as a component of the apical Crb complex in both apical basal polarity and the control of apical membrane size. The data reported in this study argue that Yrt opposes Crb function also as a basolateral polarity protein. To test this hypothesis further it was asked whether the loss of other Yrt/Cora-group genes could suppress the crb mutant phenotype. It was found that mutations in Atpα, Nrx-IV and cora ameliorate the epithelial defects of crb mutants to an extent similar to that of yrt mutations. Remarkably, yrt cora crb triple-mutant embryos not only showed a suppression of the crb mutant phenotype, which was much stronger than the one observed in yrt crb or cora crb double mutants, but also caused a strong suppression of the apicalization effect observed in yrt cora mutants. These findings further support the conclusion that Yrt and Cora act redundantly, and indicate that mutual competition of basolateral Yrt/Cora-group proteins and apical Crb organizes epithelial membrane domains (Laprise, 2009).

Epithelial differentiation during Drosophila embryogenesis can be subdivided into four phases that are characterized by distinct mechanisms governing epithelial polarity. (1) Initial cues for polarity are given before or during cellularization. (2) Fully polarized epithelial cells are established during gastrulation through the interplay of apical Par and Crb complexes and basolateral Lgl-group proteins. (3) The Yrt/Cora group acts during organogenesis to promote basolateral polarity and counteracts apical determinants, thereby functionally replacing the Lgl group. (4) The function of Yrt/Cora and Lgl groups does not account for the polarization of epithelial cells in late embryos, because normalization of polarity is observed in the absence of these factors. This implies the existence of a yet unknown mechanism that stabilizes basolateral polarity. Septate junctions form well after Lgl-group and Yrt/Cora-group proteins have contributed to epithelial polarity, indicating that polarity and septate junction formation are independent functions of these proteins. The identification of a novel group of polarity factors that acts in a discrete time window during development highlights the temporal complexity of the regulation of the epithelial phenotype and further explains why the loss of individual polarity proteins does not completely compromise polarity (Laprise, 2009).

The vertebrate Yrt orthologues Mosaic eyes (Moe, also known as Epb41l5) in zebrafish and EPB41L5 (also known as YMO1 and Limulus) and EPB41L4B (also known as EHM2) in mammals bind to Crb proteins and contribute to epithelial organization. However, as in Drosophila, vertebrate Yrt homologues are predominantly associated with the basolateral membrane and seem to be recruited to the apical membrane only at later stages of epithelial development. To test whether EPB41L5 is required for basolateral differentiation, RNA interference was used in MDCK cells. Transient depletion of EPB41L5 using shRNA resulted in a notable cell flattening and an expansion of the cell perimeter, indicating that lateral membranes were strongly reduced and apical and basal membranes were enlarged. Consistently, it was found that the lateral markers Na+,K+-ATPase, Scrib and E-cadherin were reduced or lost from the plasma membrane. The same effects were observed using siRNA oligonucleotides targeted to a distinct region of EPB41L5. MDCK cells were established that were stably transfected with EPB41L5 short hairpin RNA (shRNA), and lateral membrane formation was examined after Ca2+-switch. After re-addition of Ca2+, EPB41L5 shRNA cells displayed significant delays in lateral membrane formation and recruitment of E-cadherin to cell-cell contacts. At 8 h after Ca2+-switch, E-cadherin levels appeared similar in control and knockdown cells although experimental cells remained slightly flatter. Interestingly, by 24 h after Ca2+-switch, E-cadherin levels at the plasma membrane had decreased again and appeared lower than in control cells but similar to EPB41L5 shRNA cells before Ca2+-switch. EPB41L5 knockdown cells ultimately established normal cell shape. Whether the formation of a normal lateral membrane in EPB41L5 shRNA cells is due to residual EPB41L5 expression, or reflects a transient requirement of EPB41L5 for cell polarization as in Drosophila epithelia, remains unclear. It is concluded that the function of Yrt as a basolateral polarity protein is conserved in mammalian epithelial cells (Laprise, 2009).

The loss of basolateral membrane in Drosophila embryos that lack Yrt/Cora-group function and in MDCK cells depleted of EPB41L5 is reminiscent of the loss of basolateral membrane in MDCK cells depleted of the phosphoinositide PtdIns(3,4,5)P3 and of human bronchial epithelial cells depleted of β2-spectrin or ankyrin-G. Cora/band 4.1 and the Na+,K+-ATPase can associate with either spectrin or the spectrin adaptor ankyrin, and loss or ectopic expression of EPB41L5 can cause defects in basolateral actin. This raises the possibility that Yrt/Cora-group proteins act by stabilizing the lateral actin/spectrin membrane cytoskeleton. Analysis of vertebrate development in the absence of the function of the Yrt orthologues Moe in zebrafish and EPB41L5 in mice revealed defects in epithelial organization that reflect Crb-dependent and probably also Crb-independent functions of Yrt proteins. For example, the abnormal cell shape and multi-layering defects seen in the developing neuroepithelium of mutant mouse embryos could result from defects in basolateral polarity. Moreover, mouse embryos lacking EPB41L5 show defects in epithelial-mesenchymal transition of mesodermal cells during gastrulation. These surprising findings indicate that Yrt proteins are core regulators of animal tissue organization that can enhance epithelial or mesenchymal cell differentiation (Laprise, 2009).

Epithelial polarity proteins regulate Drosophila tracheal tube size in parallel to the luminal matrix pathway

Regulation of epithelial tube size is critical for organ function. However, the mechanisms of tube size control remain poorly understood. In the Drosophila trachea, tube dimensions are regulated by a luminal extracellular matrix (ECM). ECM organization requires apical (luminal) secretion of the protein Vermiform (Verm), which depends on the basolateral septate junction (SJ). This study shows that apical and basolateral epithelial polarity proteins interact to control tracheal tube size independently of the Verm pathway. Mutations in yurt (yrt) and scribble (scrib), which encode SJ-associated polarity proteins, cause an expansion of tracheal tubes but do not disrupt Verm secretion. Reducing activity of the apical polarity protein Crumbs (Crb) suppresses the length defects in yrt but not scrib mutants, suggesting that Yrt acts by negatively regulating Crb. Conversely, Crb overexpression increases tracheal tube dimensions. Reducing crb dosage also rescues tracheal size defects caused by mutations in coracle (cora), which encodes an SJ-associated polarity protein. In addition, crb mutations suppress cora length defects without restoring Verm secretion. Together, these data indicate that Yrt, Cora, Crb, and Scrib operate independently of the Verm pathway. These data support a model in which Cora and Yrt act through Crb to regulate epithelial tube size (Laprise, 2010).

The transmembrane protein Crumbs (Crb) acts as an apical determinant during establishment of epithelial apical-basal polarity. During later stages of epithelial differentiation, Crb promotes apical membrane growth independently of its role in apical-basal polarity. Crb activity is counteracted by different groups of basolateral polarity proteins including the Yrt/Cora group, which is composed of Yurt (Yrt), Coracle (Cora), Na⁺/K⁺-ATPase, and Neurexin IV (Nrx-IV). Loss of Yrt results in Crb-dependent apical membrane growth during late stages of epithelial cell maturation in Drosophila. Thus, the equilibrium between the activities of these polarity proteins is important to define the size of the apical domain. Precise control of the apical surface of tracheal cells is crucial to define epithelial tube size in the Drosophila respiratory system, suggesting a potential role for polarity proteins in tube morphogenesis. However, the contribution of polarity regulators to the regulation of epithelial tube shape and size is poorly understood. Controlling length and diameter of the lumen is important for organ function, as illustrated by the deleterious tubule enlargements that occur in polycystic kidney disease (Laprise, 2010).

To better understand the role of polarity regulators and apical membrane growth in epithelial tube morphogenesis, the role of Yrt was investigated in the formation of the Drosophila respiratory system, a network of interconnected tubules that delivers oxygen throughout the body. Yrt is mainly associated with the lateral membrane in tracheal cells and is enriched at septate junctions (SJs), as shown by its colocalization with the SJ marker Nrx-IV. Tracheal development was characterized in zygotic yrt mutants or yrt null embryos devoid of both maternal and zygotic yrt (yrtM/Z). Segmental tracheal placodes invaginated and established a normal branching pattern of tracheal tubes with intersegmental connections in both yrt and yrtM/Z mutants. yrtM/Z embryos display apical-basal polarity defects and irregularities in the tracheal epithelium at midembryogenesis. However, apical-basal polarity normalizes during terminal differentiation. In contrast, zygotic yrt mutants have only minor, if any, polarity defects in tracheal cells. The most apparent defect in yrt mutant trachea was the presence of excessively long and convoluted dorsal trunks compared to the straight dorsal trunks seen wild-type. The average dorsal trunk length was 417 ± 12 μm in wild-type embryos, whereas it was 476 ± 22 μm in yrt and 470 ± 23 μm in yrtM/Z mutant embryos. Similar but milder tube length defects were observed in other tracheal branches. In addition, the diameter of dorsal trunks in yrt and yrtM/Z mutants was uniform, but wider than in wild-type embryos. The average dorsal trunk diameter was 9.1 ± 0.6 μm in tracheal segment seven of wild-type embryos compared to 12.2 ± 0.9 μm in yrt and 12.7 ± 0.6 μm in yrtM/Z mutant embryos. In smaller branches, some diameter expansions were also apparent. In addition, the smaller tracheal branches in yrtM/Z embryos showed frequent interruptions indicating either breaks or a failure in the luminal accumulation of the 2A12 antigen. These findings indicate that Yrt regulates the size of tracheal tubes and supports the integrity of segmental tracheal branches. Remarkably, despite the prominent differences in the apical-basal polarity defects between yrt and yrtM/Z mutant embryos, both mutants exhibit similar dorsal trunk elongation and diameter defects. This finding suggests that the transient loss of apical-basal polarity in yrtM/Z embryos is not the cause of dorsal trunk size defects and that Yrt therefore has distinct functions during early and late stages of tracheal morphogenesis (Laprise, 2010).

The enlargement of the tracheal tube lumen observed in yrt mutants could be caused by an increase in cell number or an increase in the dimension of the apical surface of tracheal cells that surround the lumen. To address this question, the number of dorsal trunk cells in yrt mutants and wild-type embryos was counted. No significant differences in cell numbers between wild-type and yrt mutant embryos were found, indicating that the enlargement of tracheal tubes observed in yrt and yrtM/Z mutants must be accompanied by an increase in the dimension of the apical surface of tracheal cells (Laprise, 2010).

Several other mutants display enlarged dorsal trunks similar to yrt. One group of genes required for limiting tube length encodes components of the SJ, including the Na⁺/K⁺-ATPase (α and β subunits), Cora, Nrx-IV, Scribble (Scrib), Lachesin (Lac), Sinuous (Sinu), Megatrachea (Mega), and Varicose (Vari). Among these SJ proteins, Na⁺/K⁺-ATPase, Cora, Nrx-IV, and Scrib also play a role as basolateral polarity proteins. In Drosophila and other invertebrates, SJs appear as a ladder-like group of septa basal to the cadherin-based adherens junctions. SJs have functions analogous to vertebrate tight junctions, because they provide a transepithelial diffusion barrier. Yrt is not required for normal septa formation or localization of SJ components such as Cora but is essential for the barrier function of SJs. Zygotic yrt mutants show only minor defects in paracellular barrier function, whereas barrier function is fully compromised in yrtM/Z embryos. This observation supports the notion that transepithelial barrier function and the regulation of tube dimension are independent functions of Yrt because yrt and yrtM/Z mutant embryos show similar tube size defects. This conclusion is consistent with previous findings suggesting that the regulation of tracheal tube elongation and the regulation of the paracellular diffusion barrier are distinct roles of SJ proteins (Laprise, 2010).

A second class of mutants exhibiting abnormally long tracheal tubes are defective in the genesvermiform (verm) and serpentine (serp), which encode enzymes predicted to modify the chitin-based luminal extracellular matrix (ECM), and mutants of which show structural defects in the luminal ECM. The chitin matrix filling the tracheal lumen is transiently present during lumen morphogenesis and is critical for determining lumen diameter and length. Interestingly, all of the mutations affecting SJ components tested so far are associated with a failure to secrete Verm into the tracheal lumen. This suggests that SJ proteins control tube size by regulating apical secretion and remodeling of the apical chitin matrix. Although Yrt is required for the barrier function of SJs, it was found that luminal secretion of Verm and Serp was normal in yrt and yrtM/Z mutants. In contrast, low but detectable levels of Verm were observed in cora mutants. This finding suggests that Yrt regulates tracheal tube length through a pathway that is independent of Verm and Serp (Laprise, 2010).

During late stages of epithelia maturation, Yrt is known to restrict apical membrane growth in epidermal and photoreceptor cells by limiting Crb activity. This interplay between Yrt and Crb governs apical membrane size in stage 14 and later embryos when tracheal tube size is defined. This raises the possibility that Crb-dependent apical membrane growth is responsible for dorsal trunk expansion in yrt mutant embryos. To test this hypothesis, crb dosage was reduced in yrt mutant embryos by introducing one copy of a crb null allele into a yrt mutant background. Loss of one copy of crb suppressed the dorsal trunk elongation defects seen in yrt mutants, because the dorsal trunks appeared similar to wild-type in yrt crb/yrt + mutants. In addition, moderate Crb overexpression increased dorsal trunk length and diameter without interfering with the integrity of the tracheal epithelium or the secretion of Verm or Serp. These results show that Crb is required for promoting the expansion of tracheal tubes at late stages of embryogenesis. It was previously suggested that Crb also acts during early tracheal branch outgrowth in addition to its role in apical-basal polarity. Therefore, Crb plays a critical role at several steps of tracheal development. Together, these findings indicate that the antagonistic interactions between Yrt and Crb determine tracheal tube size (Laprise, 2010).

Verm levels in yrt crb/yrt + and in yrt/yrt mutants were indistinguishable, indicating that the minor reduction in Verm levels sometimes seen in yrt mutants are not the cause of the tracheal elongation defects. Accordingly, reduction of crb dosage in a verm or serp mutant background did not suppress tube size defects. Similarly, loss of one copy of crb in sinu mutant embryos, which fail to secrete Verm, had no impact on the length of dorsal trunks that remained enlarged as in sinu single mutants. These findings suggest that the apical secretion of matrix-modifying enzymes such as Verm and the control of Crb activity by Yrt are two independent and nonredundant modes of tracheal tube size regulation. The data also establish that epithelial tube size control by SJ-associated proteins involves Verm-dependent and Verm-independent mechanisms (Laprise, 2010).

To further characterize the function of SJ-associated polarity proteins in the regulation of tracheal tube size, tube elongation, the integrity of SJs, and the secretion of Verm in scrib, lethal giant larvae (lgl), and discs large (dlg) mutant embryos were examined. Zygotic loss of scrib, lgl, or dlg resulted in excessively long dorsal trunks, indicating that these genes are critical for tube size control. Zygotic loss of lgl expression caused fully penetrant defects in SJ paracellular barrier function, whereas zygotic scrib or dlg mutants did not have compromised transepithelial barriers. Luminal Verm deposition was not detected in lgl mutants but appeared near normal in dlg and in scrib mutants. Thus, Scrib and Dlg act like Yrt by controlling tracheal tube size through mechanisms distinct from Verm secretion (Laprise, 2010).

Further analysis concentrated on Scrib and it was asked whether this protein, like Yrt, controls tracheal tube size by negatively regulating Crb activity. Scrib together with Lgl and Dlg shows antagonistic interactions with Crb to regulate apical-basal epithelial polarity in early Drosophila embryos. However, the tracheal tube defects were not ameliorated in scrib crb/scrib + embryos compared to scrib single mutants. Thus, in contrast to Yrt, Scrib does not seem to limit tube length by restricting Crb activity. Because Yrt and Scrib appear to control tracheal tube size through different mechanisms, tests were performed to see whether yrt scrib double homozygous mutant embryos had a more severe phenotype. The double mutants had Verm levels that were lower compared to yrtM/Z and scrib mutants. Moreover, the tracheal defects also appeared more severe in yrt scrib mutants than in yrt null mutant embryos, particularly in the smaller-diameter branches. The defects in small-diameter branches of the yrt scrib double mutants are not likely caused by the reduction in Verm secretion, because the complete loss of Verm has only a mild effect on smaller branches. The enhanced severity of the yrt scrib double-mutant tracheal defects compared to the defects seen in yrt null embryos and the differences in the genetic interactions of scrib and yrt with crb suggest that Scrib and Yrt act in separate pathways to regulate the size of tracheal tubes and that Scrib does not act by modulating Crb activity. It is possible that Scrib acts through other proteins, such as proteins of the Par complex, that promote apical domain formation. Therefore, SJ-associated polarity proteins use at least two Verm-independent mechanisms to restrict the dimension of tracheal tubes (Laprise, 2010).

Cora is an SJ-associated protein required for optimal secretion of Verm. This suggests that Cora may control tracheal tube length through a Verm-luminal matrix pathway. However, Cora is also a basolateral polarity protein restricting the activity of Crb, which promotes Verm-independent expansion of the dorsal trunk. This led to an investigation of the functional relationship between Cora and Crb in tracheal morphogenesis. In the epidermis, a striking redundancy between yrt and cora is observed in the regulation of apical-basal polarity. Similarly, it was found that tracheal cells in yrt cora double mutants show severe apicalization defects characterized by a broad expansion of the surface distribution of Crb. The antigen recognized by the monoclonal antibody 2A12 was found surrounding tracheal cells and was not confined to the luminal cavity. In addition, the 2A12 antigen was associated not only with tracheal cells but also with epidermal cells. These tracheal defects seen in cora yrt mutant embryos mimic defects that result from high levels of Crb overexpression. This observation argues that the tracheal defects observed in cora yrt double-mutant embryos result from strong Crb overactivation, which is associated with a loss of basolateral polarity and an expansion of apical membrane character. Because epidermal cells did not acquire expression of the tracheal cell marker Tango, it is unlikely that epidermal cells adopt a tracheal cell fate in cora yrt mutants. The association of 2A12 with epidermal cells is therefore presumably due to the apicalization of tracheal cells, which would consequently secrete the 2A12 antigen not only on the luminal side but all around their cell surface, allowing the 2A12 antigen to diffuse and bind to surrounding cells. Accordingly, cuticle deposition, taking place at the apical membrane, was seen at both luminal and abluminal sides of tracheal cells overexpressing Crb (Laprise, 2010).

The data indicate that Yrt and Cora cooperate to control apical-basal polarity of tracheal cells by limiting Crb to the apical cell pole, but they do not reveal whether Cora and Crb interact to control the length of tracheal tubes. To address this question, cora crb/cora + embryos were examined for a suppression of the tracheal size defects seen in cora single mutants. Reduction of crb dosage suppresses tube overelongation defects resulting from the loss of Cora. This restriction of dorsal trunk elongation does not result from the restoration of Verm secretion, because the level of Verm present in the dorsal trunk lumen was as low in cora crb/cora + embryos as in single cora mutants. Together, these data suggest that Crb overactivation is the primary cause of epithelial tube length defects observed in the absence of Cora. Thus, Cora and Yrt act independently from each other to counteract Crb activity and maintain the appropriate size of epithelial tubes. Because the reduction of crb dosage does not rescue the verm mutant phenotype, it is concluded that the residual amount of Verm found in cora mutants is sufficient to maintain Verm pathway activity (Laprise, 2010).

This analysis suggests that basolateral proteins that are enriched at SJs have several critical functions in determining the size of epithelial tubes in the Drosophila tracheal system. This study shows that the increase in tube size is not caused by an increase in cell number and therefore must be accompanied by an increase in the apical surface area of individual tracheal cells. Given that Crb is a well-known regulator of apical membrane size, these findings suggest that the interplay between Yrt, Cora, and Crb modulates the dimensions of the apical surface of tracheal cells to control tracheal tube size. Moreover, this mechanism acts independently of and in parallel to a previously proposed pathway depending on the apical secretion of the matrix-modifying enzymes Verm and Serp, which requires several SJ-associated proteins. Yet another mechanism is revealed by results demonstrating that scrib mutants also have long trachea with normal Verm levels but that, in contrast to cora and yrt, tracheal defects in scrib mutants are not suppressed by loss of one copy of crb. Together, these findings suggest that basolateral proteins utilize at least three distinct mechanisms to regulate tube size in the Drosophila tracheal system. Unexpectedly, these mechanisms involve functional interactions between polarity proteins that appear to be different from those involved in establishing apical-basal polarity at earlier stages of development. For example, in promoting apical-basal polarity, Yrt and Cora act redundantly so that cora mutants show polarity defects only in a yrt mutant background, and polarity defects in yrt mutants are strongly enhanced by removal of Cora. In contrast, both cora and yrt single mutants show similar strong tracheal size defects. Furthermore, Scrib and Crb display antagonistic functional interactions during establishment of apical-basal polarity, but not during tracheal elongation. An important challenge for future investigations will be to uncover the adaptations in the molecular pathways that allow polarity proteins to contribute to different aspects of epithelial development (Laprise, 2010).

EVOLUTIONARY HOMOLOGS

Neurexins: cloning and general biology

In the nervous system, glial cells greatly outnumber neurons but the full extent of their role in determining neural activity remains unknown. The axotactin (axo) gene of Drosophila is shown to encode a member of the neurexin protein superfamily secreted by glia and subsequently localized to axonal tracts. The axo gene was isolated in a screen for temperature-sensitive paralytic mutations. Null mutations of axo cause temperature-sensitive paralysis and a corresponding blockade of axonal conduction. At 37 degrees C., compound action potentials in mutant nerves are either lost or are reduced by more than 90%, in contrast with wild-type nerves, in which no failure is seen. Thus, the AXO protein appears to be a component of a glial-neuronal signaling mechanism that helps to determine the membrane electrical properties of target axons. It is hypothesized that an axo-dependent signal is required for the normal expression, localization, or clustering of some set of ion channels (Yuan, 1999).

The Axo protein contains several motifs, including an amino-terminal hydrophobic signal sequence followed by a consensus cleavage site, three cystein-rich epidermal growth factor repeats, five laminin G repeats, and a fibrinogen beta/gamma-like segment. Several features distinguish Axo from other members of the neurexin superfamily: a Kunitz-like domain; the presence of both a Laminin G domain (characteristic of neurexins) and a fibrinogen domain adjacent to the second EGF repeat; and the absence of a transmembrane domain, which suggests that Axo is secreted rather than membrane associated. Beginning at embryonic stage 13, axo transcripts are detected in the differentiating nervous system in a segmentally repeated pattern of discrete spots directly overlaying the longitudinal axonal tracts. This pattern corresponds with the location of a subset of glial cells, including longitudinal glia and segmental boundary cells. Axo protein distribution differs from that of the axo transcript. The protein coincides with axonal tracts in the ventral cord, brain and parts of the peripheral nervous system. Thus, Axo appears to be secreted from glia and subsequently localized to axonal tracts (Yuan, 1999).

In Caenorhabditis elegans, vulval induction is mediated by the let-23 receptor tyrosine kinase (RTK)/ Ras signaling pathway. The precise localization of let-23 RTK at epithelial junctions is essential for the vulval induction, and requires three genes, including lin-2, -7, and -10. The mammalian homolog of lin-2 has been identified as CASK, a protein interacting with neurexin, a neuronal adhesion molecule. CASK has recently been reported to interact with syndecans and an actin-binding protein, band 4.1, at epithelial and synaptic junctions, and to play central roles in the formation of cell-cell junctions. The product of C. elegans lin-7 directly interacts with let-23 RTK and localizes let-23 RTK at epithelial junctions. Three rat homologs of lin-7 are ubiquitously expressed in various tissues. These homologs accumulate at the junctional complex region in cultured Madin-Darby canine kidney cells, and are also localized at the synaptic junctions in neurons. The mammalian homologs of lin-7 may be implicated in the formation of cell-cell junctions (Irie, 1999).

Neurexins are highly variable transmembrane proteins hypothesized to be nerve terminal-specific cell adhesion molecules. As a test of the hypothesis that neurexin is restricted to the nerve terminal, neurexins were examined in the electric organ of the elasmobranch electric fish. Specific antibodies generated against the intracellular domain of electric fish neurexin were used in immunocytochemical and Western blot analyses of the electromotor neurons that innervate the electric organ. Neurexin is not expressed at electric organ nerve terminals, as would be expected by the neurexin hypothesis. Instead, neurexin is expressed by electromotor neurons and on myelinated axons. This neurexin has a molecular weight of 140 kDa, consistent with an alpha-neurexin. In addition, perineurial cells of the electromotor nerve also express a neurexin. These cells surround bundles of axons to form a diffusion barrier and are thought to be a special form of fibroblast. The results of the study argue against a universal role for neurexins as nerve terminal-specific proteins but suggest that neurexins are involved in axon-Schwann cell and perineurial cell interactions (Russell, 1997).

Neurexins, a family of cell surface proteins specific to brain, are transcribed from two promoters in three genes, resulting in three alpha- and three beta-neurexins. There are differential but overlapping distributions of neurexin isoforms in different classes of neurons. Alpha-neurexins are alternatively spliced at five canonical positions; beta-neurexins at two. Characterization of many independent bovine neurexin I alpha cDNAs suggests that different splice sites are used independently. This creates the potential to express more than 1000 distinct neurexin proteins in brain. The splicing pattern is conserved in rat and cow. Thus, in addition to somatic gene rearrangement (immunoglobulins and T cell receptors) and large gene families (odorant receptors), alternative splicing potentially represents a third mechanism for creating a large number of cell surface receptors that are expressed by specific subsets of cells (Ullrich, 1995).

Synaptotagmin I and neurexin I mRNAs, coding for proteins involved in neurotransmitter secretion, become detectable in primary sympathetic ganglia shortly after initial induction of the noradrenergic transmitter phenotype. To test whether the induction of these more general neuronal genes is mediated by signals known to initiate noradrenergic differentiation in a neuronal subpopulation, their expression was examined in noradrenergic neurons induced by ectopic overexpression of growth and transcription factors. Overexpression of BMP4 or Phox2a in vivo results in synaptotagmin I and neurexin I expression in ectopically located noradrenergic cells. In vitro, BMP4 initiates synaptotagmin I and neurexin I expression in addition to tyrosine hydroxylase induction. Thus, the induction of synaptotagmin I and neurexin I, which are expressed in a large number of different neuron populations, can be accomplished by growth and transcription factors available only to a subset of neurons. These findings suggest that the initial expression of proteins involved in neurotransmitter secretion is regulated by different signals in different neuron populations (Patzke, 2001).

Expression of the major isoforms of three neurexin genes was analyzed in the developing embryonic nervous system of mice. Transcripts of all three genes are detected as early as embryonic day 10 (E10) and increased with maturation of the nervous system. RNAs of the major neurexin isoforms (alpha and beta) are found throughout the central nervous system exclusively in postmitotic neurons and at least 1 d before synapses are formed. In contrast, in the PNS, the alpha- and beta-isoforms display differential expression patterns. Neurexin III mRNA shows a more restricted regional expression than neurexin I and II transcripts. These expression profiles are consistent with the hypothesis that the neurexins have a function in early neuronal differentiation and axogenesis (Puschel, 1995).

To explore the possibility that overproduction of neuronal acetylcholinesterase (AChE) confers changes in both cholinergic and morphogenic intercellular interactions, developmental responses to neuronal AChE overexpression were studied in motoneurons and neuromuscular junctions of AChE-transgenic mice. Perikarya of spinal cord motoneurons are consistently enlarged from embryonic through adult stages in AChE-transgenic mice. Atypical motoneuron development is accompanied by premature enhancement in the embryonic spinal cord expression of choline acetyltransferase mRNA, encoding the acetylcholine-synthesizing enzyme choline acetyltransferase. In contrast, the mRNA encoding for neurexin-Ibeta, the heterophilic ligand of the AChE-homologous neuronal cell surface protein neuroligin, is drastically lower in embryonic transgenic spinal cord than in controls. Postnatal cessation of these dual transcriptional responses is followed by late-onset deterioration in neuromotor performance that is associated with gross aberrations in neuromuscular ultrastructure and with pronounced amyotrophy. These findings demonstrate embryonic feedback mechanisms to neuronal AChE overexpression that are attributable to both cholinergic and cell-cell interaction pathways, suggesting that embryonic neurexin Ibeta expression is concerted in vivo with AChE levels and indicating that postnatal changes in neuronal AChE-associated proteins may be involved in late-onset neuromotor pathologies (Andres, 1997).

In yeast two-hybrid screens for intracellular molecules interacting with different neurexins, a single interacting protein called CASK has been identified. CASK is composed of an N-terminal Ca2+, calmodulin-dependent protein kinase sequence and a C-terminal region that is similar to the intercellular junction proteins dlg-A, PSD95/SAP90, SAP97, Z01, and Z02 (Drosophila homolog: Discs large). The C-terminal region also contains DHR-, SH3-, and guanylate kinase domains. CASK is enriched in the synaptic plasma membranes of the brain, but is also detectable at low levels in all tissues tested. The cytoplasmic domains of all three neurexins bind CASK in a salt-labile interaction. In neurexin I, this interaction is dependent on the C-terminal three residues. Thus, CASK is a membrane-associated protein that combines domains found in Ca(2+)-activated protein kinases and in proteins specific for intercellular junctions, suggesting that it may be a signaling molecule operating at the plasma membrane, possibly in conjunction with neurexins (Hata, 1996).

A novel multivalent PDZ domain protein, CIPP (for channel-interacting PDZ domain protein) is expressed exclusively in brain and kidney. Within the brain, the highest CIPP mRNA levels are found in neurons of the cerebellum, inferior colliculus, vestibular nucleus, facial nucleus, and thalamus. Furthermore, the inward rectifier K+ (Kir) channel, Kir4.1 (also called Kir1.2), has been identified as a cellular CIPP ligand. Among several other Kir channels tested, only the closely related Kir4.2 (or Kir1.3) also interacts with CIPP. In addition, specific PDZ domains within CIPP associate selectively with the C-termini of N-methyl-D-aspartate subtypes of glutamate receptors, as well as neurexins and neuroligins, cell surface molecules enriched in synaptic membranes. Thus, CIPP may serve as a scaffold that brings structurally diverse but functionally connected proteins into close proximity at the synapse. The functional consequences of CIPP expression on Kir4.1 channels were studied using whole-cell voltage clamp techniques in Kir4.1 transfected COS-7 cells. On average, Kir4.1 current densities are doubled by cotransfection with CIPP (Kurschner, 1998).

Synaptotagmin, a major intrinsic membrane protein of synaptic vesicles that binds Ca2+, was purified from bovine brain and immobilized onto Sepharose 4B. Affinity chromatography of brain membrane proteins on immobilized synaptotagmin reveals binding of alpha- and beta-neurexins to synaptotagmin in a Ca(2+)-independent manner. Synaptotagmin specifically interacts with the cytoplasmic domains of neurexins but not of control proteins. This interaction is dependent on a highly conserved, 40 amino acid sequence that makes up most of the cytoplasmic tails of the neurexins. These data suggest a direct interaction between the cytoplasmic domains of a plasma membrane protein (the neurexins) and a protein specific for a subcellular organelle (synaptotagmin). Such an interaction could have an important role in the docking and targeting of synaptic vesicles in the nerve terminal (Hata, 1993).

The interaction of the synaptic vesicle protein, synaptotagmin, and the presynaptic alpha-latrotoxin receptor, a neurexin, is thought to be involved in docking of synaptic vesicles at active sites or the modulation of neurotransmitter release. Pieces of synaptotagmin containing the carboxyl terminus are capable of purifying neurexins from solubilized brain homogenates. Pieces as small as a synthesized peptide corresponding to the COOH-terminal 34 amino acids are capable of enriching neurexins 100-fold. The binding of neurexins to synaptotagmin is calcium-independent and of moderate affinity. This COOH-terminal segment of synaptotagmin is conserved in all species characterized to date. Reflective of this, a synthetic peptide corresponding to the carboxyl terminus of Drosophila synaptotagmin is capable of purification of rat neurexins, suggesting the possibility that this interaction may also exist in Drosophila. It is proposed that the carboxyl terminus of synaptotagmin binds to the carboxyl terminus of the neurexins and that this interaction may mediate docking of synaptic vesicles or modulation of neurotransmitter release (Perin, 1994).

CASK Functions as a Mg2+-independent neurexin kinase

CASK is a unique MAGUK protein that contains an N-terminal CaM-kinase domain besides the typical MAGUK domains. The CASK CaM-kinase domain is presumed to be a catalytically inactive pseudokinase because it lacks the canonical DFG motif required for Mg2+ binding that is thought to be indispensable for kinase activity. This study shows, however, that CASK functions as an active protein kinase even without Mg2+ binding. High-resolution crystal structures reveal that the CASK CaM-kinase domain adopts a constitutively active conformation that binds ATP and catalyzes phosphotransfer without Mg2+. The CASK CaM-kinase domain phosphorylates itself and at least one physiological interactor, the synaptic protein neurexin-1, to which CASK is recruited via its PDZ domain. Thus, these data indicate that CASK combines the scaffolding activity of MAGUKs with an unusual kinase activity that phosphorylates substrates recuited by the scaffolding activity. Moreover, this study suggests that other pseudokinases (10% of the kinome) could also be catalytically active (Mukherjee, 2008).

Mg²⁺ acts as an obligate cofactor for ATP binding and phosphotransfer in all known kinases. This study demonstrates that the CASK CaM-kinase domain catalyzes phosphotransfer from ATP to proteins in the complete absence of Mg²⁺. CASK is the first kinase, indeed the first nucleotidase, known to catalyze phosphotransfers in the absence of Mg²⁺ (Mukherjee, 2008).

The structure of the CASK CaM-kinase domain, and comparison of its structure with those of other kinases, illustrates that the CaM-kinase domain of CASK adopts a constitutively active conformation. Biochemical and enzymatic assays demonstrated that CASK binds ATP and catalyzes autophosphorylation and neurexin-1 phosphorylation in the absence of Mg²⁺. Compared to other kinases, CASK contains noncanonical residues in the nucleotide-binding pocket that may account for its unusual catalytic mechanism. Both of the classical metal-coordinating residues in kinases are substituted in the CASK CaM-kinase domain. Moreover, Glu143 of the catalytic loop directly coordinates the metal ion in DAPK1, while in CASK, this Glu is altered to His (Glu145His). These changes likely contribute to the divalent cation-driven inhibition of the CASK CaM-kinase domain. Since the adenine base of ATP makes the most important contacts for the positioning of ATP in the nucleotide-binding pocket, an altered Mg²⁺-coordinating sequence does not exclude ATP binding and, as shown in this study, does not exclude catalysis. Importantly, similar to the CASK CaM-kinase domain, other pseudokinase domains with noncanonical Mg²⁺-binding motifs may coordinate ATP and phosphorylate physiological substrates as well (Mukherjee, 2008).

CASK also differs from other CaM-kinase family members in that its CaM-kinase domain exhibits a constitutively active conformation. In an archetypal CaM kinase, the catalytic domain is followed by an autoinhibitory domain that inhibits kinase activity and is disinhibited by Ca²⁺/calmodulin binding. The CASK CaM-kinase domain is followed by a sequence that is homologous to the autoinhibitory domain of CaM kinases and that also binds Ca²⁺/calmodulin. However, unlike typical CaM kinases, the autoinhibitory helix (αR1) of CASK does not engage in direct contacts with the ATP-binding cleft. No evidence was discerned for further C-terminal residues interacting with the ATP-binding cleft, as in CaMKI, and no stimulatory effect was detected of Ca²⁺ and/or calmodulin on the CASK kinase activity. Thus, the CASK CaM-kinase domain appears to retain a nonfunctional autoinhibitory domain as an evolutionary vestige of CaM kinases. CASK, therefore, differs from other, evolutionarily closely related CaM-kinase domains not only in its Mg²⁺ independence but also in its inherently “closed” active conformation that constitutively binds nucleotides (Mukherjee, 2008).

An almost essential consequence of the constitutively active conformation of the CASK CaM-kinase domain is that the domain exhibits a very low catalytic rate, as shown in autophosphorylation measurements and in measurements of neurexin-1 phosphorylation by the isolated CASK CaM-kinase domain lacking the neurexin-binding PDZ domain of CASK. Mechanistically, this low rate is likely due to the loss of Mg²⁺ coordination by the domain. The low catalytic rate of the CASK CaM-kinase domain presumably serves to ensure that the kinase does not phosphorylate potential substrates randomly. The phosphorylation rate of neurexin-1 is increased dramatically, however, when full-length CASK forms a complex with neurexin-1 via the CASK PDZ domain. This result suggests a general mechanism for CASK kinase activity, whereby CASK couples an intrinsically slow but constitutively active kinase domain to a PDZ domain that recruits the substrates to the kinase domain, thereby increasing the local substrate concentration by many orders of magnitude. According to the model, CASK unites two separate functions—the recruitment activity of MAGUKs and the kinase activity of the CaM-kinase domain—into a single unit whose objective is phosphorylation of specific interacting proteins (Mukherjee, 2008).

CASK phosphorylates neurexin-1 in vitro and in vivo in a reaction that depends on a catalytically active CaM-kinase domain. Neurexin, described in this study as a substrate of CASK, is a presynaptic cell-adhesion molecule. Its heterotypic binding to postsynaptic neuroligins may be involved in synaptic function and could induce synapse formation even on non-neuronal cocultured cells. The neurexin-neuroligin interaction is a candidate for synaptic specialization and pre-post-synapse communication. Both neurexin and neuroligin mutations have been linked to autism spectrum disorders. Deletion of CASK may be connected to X-linked optic atrophy and mental retardation. The evolutionary conservation of CASK and neurexins, and their central importance for survival and synaptic function in mice, indicate that neurexin phosphorylation by CASK may be crucial to neuronal function (Mukherjee, 2008).

In addition to the control of CASK kinase activity by the PDZ-domain-mediated substrate recruitment, whether it is regulated by synaptic activity-driven rises in Ca²⁺ and Mg²⁺ levels was examined. In neurons, synaptic activity triggers a surge in Mg²⁺ and Ca²⁺ levels that could regulate CASK kinase activity. Indeed, a strong increase was observed in neurexin phosphorylation upon silencing synapses in mature neurons, indicating that contrary to other kinases, CASK kinase is inhibited by neuronal activity. It was envisioned that CASK kinase activity is maximal during neuronal development and synaptogenesis and declines with the onset of synaptic function but is reactivated when neurons are silenced. This developmentally regulated activity is in line with the phenotypic defects in CASK knockout mice as well as the developmental nature of CASK- and neurexin-related pathologies (Mukherjee, 2008).

CASK is expressed ubiquitously at low levels. The non-neuronal functions of CASK are evident from developmental defects in CASK/Lin-2 null animals, such as cleft palate in mice and vulval dysgenesis in C. elegans. In non-neuronal cells, CASK-interacting adhesion molecules of the syndecan or JAM families could be substrates. These molecules share the PDZ-domain-mediated CASK association, and at least in the case of syndecan-2, serine residues in the cytoplasmic tail homologous to those of neurexins are phosphorylated in vivo (Mukherjee, 2008).

Finally, of the 518 known kinases in the human genome, 48 are predicted to be pseudokinases. In each of these pseudokinases, one or more of the invariant motifs are altered. Nine of the presumed pseudokinases, including CASK, lack a canonical DFG motif. Furthermore, this motif is altered along with other canonical motifs (HRD and/or VAIK) in 22 additional pseudokinases. These data on CASK suggest that other pseudokinases, especially those with atypical DFG motifs, could be active in physiologically relevant environments, indicating that the catalytically active kinome may be more diverse than originally envisioned (Mukherjee, 2008).

Neurexin functions at nodes

Ranvier nodes are flanked by paranodal regions where oligodendrocytes or Schwann cells interact closely with axons. Paranodes play a critical role in the physiological properties of myelinated nerve fibers. Paranodin, a prominent 180 kDa transmembrane neuronal glycoprotein, was purified and cloned from adult rat brain, and found to be highly concentrated in axonal membranes at their junction with myelinating glial cells, in paranodes of central and peripheral nerve fibers. The large extracellular domain of paranodin is related to neurexins, and its short intracellular tail binds protein 4.1, a cytoskeleton-anchoring protein. Paranodin may be a critical component of the macromolecular complex involved in the tight interactions between axons and myelinating glial cells characteristic of the paranodal region (Menegoz, 1997).

Mice incapable of synthesizing the abundant galactolipids of myelin exhibit disrupted paranodal axo-glial interactions in the central and peripheral nervous systems. Using these mutants, the role that axo-glial interactions play in the establishment of axonal protein distribution in the region of the node of Ranvier has been analyzed. Whereas the clustering of the nodal proteins, sodium channels, ankyrin(G), and neurofascin is only slightly affected, the distribution of potassium channels and paranodin, proteins that are normally concentrated in the regions juxtaposed to the node, is dramatically altered. The potassium channels, which are normally concentrated in the paranode/juxtaparanode, are not restricted to this region but are detected throughout the internode in the galactolipid-deficient mice. Paranodin/contactin-associated protein (Caspr), a paranodal protein that is a potential neuronal mediator of axon-myelin binding, is not concentrated in the paranodal regions but is diffusely distributed along the internodal regions. Collectively, these findings suggest that the myelin galactolipids are essential for the proper formation of axo-glial interactions and demonstrate that a disruption in these interactions results in profound abnormalities in the molecular organization of the paranodal axolemma (Dupree, 1999).

The axons of myelinated nerves in the adult nervous system are subdivided into several distinct structural and functional domains that each differ in their molecular composition. The generation of these specialized subcellular structures is essential for the efficient and rapid propagation of action potential via saltatory conduction. Several distinct subdomains can be found in the axonal membrane, the nodes of Ranvier, the paranodes, and the juxtaparanodes. The nodes of Ranvier are characterized by a high concentration of voltage-gated Na+ channels, which enable the regeneration of the action potential. Several other proteins are localized to the axolemma at the nodes, including Na+/K+ ATPases, the spectrin-binding protein ankyrin, and the cell adhesion proteins NrCAM and Neurofascin. Junctions that are formed between the axon and the myelinating cell (called the paranodal junctions) border the nodes of Ranvier. In this region, the compact myelin lamellae open up into a chain of cytoplasmic loops that form a series of septate-like junctions with the axon. The paranodal region is thought to serve several functions: to anchor the myelin to the axon; to form a partial barrier that isolates the periaxonal space under the myelin from the electrical activity at the nodes, and to physically demarcate boundaries that limit the lateral diffusion of membrane components, thereby confining them to the node. A third specialized region in myelinated axons is defined as the juxtaparanode. This region, often referred to as the paranodal main segment, is located in a short zone just beyond the innermost paranodal junctions, separating them from the internodes that lie beneath the compact myelin sheaths. In large fibers in the PNS, this region contributes to the axon-Schwann cell network, a structure of thin axonal processes that are enclosed by protrusions of the Schwann cell cytoplasmic layer. This complex network is implicated in axonal transport and in the lysosyme-mediated degradation of transported material. Although no prominent network is present in the CNS, some invasion of the axoplasm by the inner cytoplasmic loop occurs at this site. The juxtaparanodal region is characterized by the presence of heteromultimers of the Shaker-like K+ channel alpha subunits Kv1.1 and Kv1.2, and the cytoplasmic Kvbeta2 subunit. The precise localization of voltage-activated K+ channels to this region may stabilize conduction and help to maintain the internodal resting potential (Poliak, 1999 and references therein).

Contactin-associated protein (Caspr; also known as Paranodin) is a member of the neurexin superfamily, a group of transmembrane proteins that mediate cell-cell interactions in the nervous system. It was originally identified in a complex with contactin that binds to the receptor protein tyrosine phosphatase beta (RPTPbeta) and as a major lectin-binding protein from rat brain. Caspr is concentrated at the paranode of Ranvier in the septate-like junctions that are formed between axons and the terminal loops of oligodendrocytes and myelinating Schwann cells. Both the localization of Caspr and the development of the septate junctions occur with the maturation of the myelinated fiber, suggesting that Caspr may be an essential component of the paranodal junction. The notion that Caspr plays a role in the generation and integrity of the paranodal junctions is also supported by studies in Drosophila demonstrating that the Caspr-related Neurexin IV (Nrx-IV) is an essential component of the ectodermally derived pleated septate junctions (Baumgartner, 1996 ). Nrx-IV mutants are devoid of septate junctions between glial cells, resulting in the breakdown of the blood-nerve barrier. These mutants lack the transverse septate that is characteristic of these junctions, which are structurally similar to the paranodal junctions (Baumgartner, 1996 ). Although Caspr and the related Drosophila Nrx-IV have a similar primary structure, they differ in their cytoplasmic tail. Caspr contains a proline-rich sequence, while Drosophila Nrx-IV has a shorter cytoplasmic region containing a binding site for PDZ domains (Bellen, 1998). This structural difference suggests the existence of other members of this group in vertebrates. Caspr2, a new member of this family, is a component of the juxtaparanodal region in myelinated axons (Poliak, 1999).

Rapid conduction in myelinated axons depends on the generation of specialized subcellular domains to which different sets of ion channels are localized. Caspr2, a mammalian homolog of Drosophila Nrx-IV and the closely related molecule Caspr/Paranodin demarcate distinct subdomains in myelinated axons. An alignment of human Caspr proteins with the amino acid sequence of Drosophila Nrx-IV reveals that Caspr2 is more related (34% identity) to Nrx-IV than is Caspr (29% identity). Like Nrx-IV, Caspr2 lacks the proline-glycine-tyrosine repeats that are found near the transmembrane domain of Caspr. The major difference between Caspr2 and Caspr is found in the cytoplasmic domain. While both share a protein 4.1 binding sequence at the juxtamembrane region, they diverge thereafter. The intracellular region of Caspr2 is more similar to glycophorin C (65% identity) and Nrx-IV (37% identity) than to Caspr (29% identity). Caspr2, Nrx-IV, and all the neurexins each contain a short amino acid sequence at their C terminus, which serves as a binding site for type II PDZ domains. This consensus sequence is not found in Caspr. Altogether, these sequence similarities indicate that Caspr2 is most likely the mammalian ortholog of Drosophila Nrx-IV. While contactin-associated protein (Caspr) is present at the paranodal junctions, Caspr2 is precisely colocalized with Shaker-like K+ channels in the juxtaparanodal region. Caspr2 specifically associates with Kv1.1, Kv1.2, and their Kvbeta2 subunit. This association involves the C-terminal sequence of Caspr2, which contains the putative PDZ binding site. These results suggest a role for Caspr family members in the local differentiation of the axon into distinct functional subdomains (Poliak, 1999).

The localization of the Caspr proteins to distinct domains could be controlled by interactions with extracellular ligands, as well as with intracellular proteins within the axon. Candidate axonal proteins that may be involved in targeting and localization of Caspr2 include members of the protein 4.1 family and proteins containing PDZ domains. Caspr2 and Nrx-IV share in their cytoplasmic tail a high sequence homology with the erythrocyte transmembrane protein glycophorin C. This protein is found in a ternary complex also containing protein 4.1 and the membrane-associated guanylate kinase protein p55. The cytoplasmic domain of Caspr has been shown to interact with protein 4.1 from rat brain, and Nrx-IV associates directly with the N-terminal region of Coracle, a Drosophila protein 4.1 homolog. Genetic analyses in Drosophila indicate interdependence between protein 4.1/Coracle and Nrx-IV for correct localization. In Nrx-IV mutants, protein 4.1/Coracle is mislocalized and is not found in septate junctions. In coracle null flies, Nrx-IV reaches the lateral membrane but is not subsequently maintained at the septate junctions. Another protein that is required for the proper localization of Nrx-IV is Disc lost. This protein contains four PDZ domains, two of which interact with the C-terminal tail of Nrx-IV. By analogy with Nrx-IV and glycophorin C, it is likely that the localization of Caspr2 to the juxtaparanodal region may also involve a complex interaction with cytoplasmic proteins, such as members of the protein 4.1 family and PDZ domain-containing proteins (Poliak, 1999 and references therein).

Neurexins and synapses

Neurexins are a large family of proteins that act as neuronal cell-surface receptors. The function and localization of the various neurexins, however, have not yet been clarified. Beta-neurexins are candidate receptors for neuroligin-1, a postsynaptic membrane protein that can trigger synapse formation at axon contacts. Studies in mammalian cell culture reveal that neurexins are concentrated at synapses and purified neuroligin is sufficient to cluster neurexin and to induce presynaptic differentiation. Oligomerization of neuroligin is required for its function, and beta-neurexin clustering is found to be sufficient to trigger the recruitment of synaptic vesicles through interactions that require the cytoplasmic domain of neurexin. A two-step model is proposed in which postsynaptic neuroligin multimers initially cluster axonal neurexins. In response to this clustering, neurexins nucleate the assembly of a cytoplasmic scaffold to which the exocytotic apparatus is recruited (Dean, 2003).

Analysis of the molecular mechanism of neuroligin-induced synapse formation shows that that overexpression of neuroligin stimulates pre- and post-synaptic differentiation in cultured hippocampal neurons, suggesting that neuroligin is a limiting component of the postsynaptic machinery involved in synapse formation. Neuroligin activity depends on its interaction with neurexins. It was found that (1) endogenous neurexins are concentrated in synaptic terminals, (2) postsynaptic multimers of neuroligin-1 are sufficient to trigger the recruitment of neurexin to newly forming synaptic sites and (3) clustering of neurexin induces recruitment of synaptic vesicles (Dean, 2003).

The neurexin family of proteins was first identified as high-affinity receptors for the venom alpha-latrotoxin. Although an involvement of neurexins in the latrotoxin response of neuronal cells is now well documented, the subcellular localization and normal function of the neurexins are still unknown. Using a newly generated neurexin antibody that recognizes most neurexin isoforms, it has been shown that neurexins are concentrated at synapses. Neurexin immunoreactivity is not completely restricted to synapses, so it cannot be determined whether the additional non-synaptic neurexin pool consists of specific non-synaptic isoforms, or whether most neurexins are localized to both synapses and non-synaptic regions of the plasma membrane. In either case, the data show that at least a subset of neurexin family members is concentrated at CNS synapses.

Neuroligin was originally isolated as a splice-specific ligand of beta-neurexins by affinity chromatography. Using a functional in vitro assay, it has been demonstrated that neuroligins have a synaptogenic activity, but it remained unclear whether this activity of neuroligin is mediated through a neurexin. Strong evidence has now been provided that neurexin functions as a neuroligin receptor in synapse formation: overexpression of neuroligin in neurons induces recruitment of neurexins to newly forming terminals and promotes the formation of synaptic specializations. This activity of neuroligin-expressing cells can be mimicked by purified neuroligin presented to axons in a fluid lipid bilayer on a bead. Mutations in neuroligin that inhibit neurexin binding or adhesion with neurexin-expressing cells reduce the synapse-promoting activity of the protein (Dean, 2003).

Moreover, the results indicate that lateral clustering has a critical role in neuroligin-neurexin signaling. Clustering of beta-neurexin in axons with multimerized antibodies is sufficient to trigger the accumulation of synaptic vesicles, whereas crosslinking of neurexin with monomeric antibodies is ineffective. This suggests a highly cooperative process in which a minimum number of beta-neurexins (at least four) must be clustered to result in productive signaling. These findings support a striking mechanistic analogy between the induction of synaptic differentiation in CNS neurons and synapse formation in the immune system where receptor clustering represents a crucial event in T-cell activation (Dean, 2003).

How can the clustering of beta-neurexin trigger the assembly of presynaptic terminals? For beta-neurexin to be active it must have an intact cytoplasmic C-terminal tail, which contains a PDZ-binding motif. This domain is known to interact with the cytoplasmic scaffolding molecules CIPP, CASK/Lin-2 and Mint/Lin-10/X11alpha and may interact with other proteins that have not yet been identified. The clustering of beta-neurexin monomers in the axon by neuroligin tetramers may either recruit specific downstream signaling components to the beta-neurexin at the forming contacts or activate signaling through components that are 'pre-bound' to beta-neurexin. In either case, the small neurexin clusters would provide the minimal nucleation site for assembling a presynaptic protein scaffold and, subsequently, the secretory apparatus. Most of the cytoplasmic neurexin binding proteins are multivalent and can generate a scaffold with additional neurexin binding sites. Thus, the initial complexes could be enlarged by recruitment of more beta-neurexins, and, subsequently, additional neuroligins, forming an expanding cell-cell contact. Such a model might also account for the fact that clustering of ephrinB by EphB receptors induces the recruitment of adapters and signaling molecules resulting in a retrograde signal transmitted into the ephrinB-expressing cell (Dean, 2003).

The model predicts that a beta-neurexin is localized to the presynaptic terminal. With the immunological reagents currently available, direct evidence cannot be provided for a presynaptic localization of beta-neurexin. Several lines of evidence are provided, however, that support such a localization: (1) a pan-neurexin antibody that recognizes alpha- and beta-neurexins shows that neurexin isoforms are concentrated at synapses and in axonal growth cones; (2) clustering of endogenous neurexins with neuroligin-coated beads induces presynaptic specializations in the absence of postsynaptic elements and (3) clustering of epitope-tagged beta-neurexin in axons with antibodies triggers the recruitment of synaptic vesicles. It is possible that individual neurexin isoforms show differential distribution over pre- and postsynaptic plasma membrane domains. To address this question, beta-neurexin-specific antibodies are currently being generated, and ultrastructural studies with pan-neurexin antibodies are underway (Dean, 2003).

Whereas neuroligin-neurexin signaling is sufficient to promote synaptic differentiation, during development both molecules function in concert with several additional trans-synaptic signaling factors such as WNTs, cadherins, Ig-domain proteins and Eph receptors. One factor whose function at least partially overlaps with that of neuroligin is the Ig-domain protein SynCAM. Whereas neuroligin-neurexin interactions are heterophilic and therefore provide the synapse-induction process with directionality, SynCAM apparently functions through homophilic interactions. It is noteworthy that neurexin and SynCAM contain binding sites for the same cytoplasmic scaffolding proteins, indicating that they may converge on common downstream effectors. A common cytoplasmic scaffold may thus integrate adhesive and signaling cues from several extracellular pathways and allow for a stepwise assembly of trans-synaptic complexes. Future work should elucidate whether cross-talk between different adhesion and signaling system exists and, if so, how it contributes to the regulation of synapse formation and synaptic specificity in the CNS (Dean, 2003).

Synapses are specialized intercellular junctions in which cell adhesion molecules connect the presynaptic machinery for neurotransmitter release to the postsynaptic machinery for receptor signalling. Neurotransmitter release requires the presynaptic co-assembly of Ca²⁺ channels with the secretory apparatus, but little is known about how synaptic components are organized. alpha-Neurexins, a family of >1,000 presynaptic cell-surface proteins encoded by three genes, link the pre- and post-synaptic compartments of synapses by binding both extracellularly to postsynaptic cell adhesion molecules and intracellularly to presynaptic PDZ domain proteins. Using triple-knockout mice, it has been shown that alpha-neurexins are not required for synapse formation, but are essential for Ca²⁺-triggered neurotransmitter release. Neurotransmitter release is impaired because synaptic Ca²⁺ channel function is markedly reduced, although the number of cell-surface Ca²⁺ channels appear normal. These data suggest that alpha-neurexins organize presynaptic terminals by functionally coupling Ca²⁺ channels to the presynaptic machinery (Missler, 2003).

Marked advances have been made in the understanding of postsynaptic mechanisms, for example, the discovery of stargazins and the molecular description of glutamate receptor recycling. Furthermore, neuroligins and SynCAMs have been identified as postsynaptic molecules that can induce differentiation of presynaptic nerve terminals. However, little is known about the presynaptic mechanisms that assemble the secretory machinery for neurotransmitter release in nerve terminals precisely opposite to the postsynaptic density, and little is known about the mechanisms that control the activity and location of Ca²⁺ channels. In the present study, alpha-neurexins, presynaptic cell-adhesion molecules that are coupled to intracellular PDZ domain proteins, are shown to be required for normal neurotransmitter release, and deletion of alpha-neurexins impairs the function of synaptic Ca²⁺ channels. These results reveal a link between two previously unconnected processes -- synaptic cell adhesion and voltage-gated Ca²⁺-signalling -- and suggest the hypothesis that alpha-neurexins couple synaptic cell adhesion to presynaptic Ca²⁺-channels and other parts of the presynaptic secretory machinery (Missler, 2003).

Deletion of alpha-neurexins impairs both spontaneous and evoked neurotransmitter release as measured in excitatory and inhibitory synapses in the brainstem and neocortex. The decrease in mini frequency, the decrease in evoked responses, the increase in failure rates, and the lack of noticeable changes in postsynaptic receptor activity demonstrate a primary presynaptic impairment. This impairment is not due to developmental abnormalities. The only structural change found in alpha-neurexin KO mice is a selective decline in the number of inhibitory synapses that can not account for the much larger decrease in inhibitory synaptic transmission. This decline could be due to the special role of GABAergic synaptic transmission in driving early development, which may lead to enhanced activity-dependent elimination of inhibitory synapses in alpha-neurexin KO mice (Missler, 2003).

It is proposed that alpha-neurexins function as presynaptic cell adhesion and/or cell recognition molecules that couple extracellular synaptic interactions to the intracellular organization of the presynaptic secretory apparatus and Ca²⁺ channels. alpha-neurexins are clearly not subunits of Ca²⁺ channels because the function of Ca²⁺ channels is normal in many cells lacking alpha-neurexins (such as chromaffin or muscle cells). alpha-neurexins are not 'chaperones' for Ca²⁺ channels because at least N-type channels (which contributed predominantly to the Ca²⁺ current reduction) are transported to the cell-surface in alpha-neurexin KO mice and exhibit a normal function. One model to explain how alpha-neurexins determine the synaptic activity of Ca²⁺ channels is that Ca²⁺ channel activity is negatively regulated in mature neurons, and that synaptic alpha-neurexins function to selectively empower Ca²⁺ channels at the synapse. Such a mechanism would concentrate Ca²⁺ influx to synapses, and imply a neurexin-dependent pathway by which postsynaptic neurons could control presynaptic Ca²⁺ channel activity. According to this proposal, alpha-neurexins act as trans-synaptic cell-adhesion molecules that participate in a modular organization of presynaptic terminals by mediating the localized activation of Ca²⁺ channels (Missler, 2003).

Formation of synaptic connections requires alignment of neurotransmitter receptors on postsynaptic dendrites opposite matching transmitter release sites on presynaptic axons. ß-neurexins and neuroligins form a trans-synaptic link at glutamate synapses. Neurexin alone is sufficient to induce glutamate postsynaptic differentiation in contacting dendrites. Surprisingly, neurexin also induces GABA postsynaptic differentiation. Conversely, neuroligins induce presynaptic differentiation in both glutamate and GABA axons. Whereas neuroligins-1, -3, and -4 localize to glutamate postsynaptic sites, neuroligin-2 localizes primarily to GABA synapses. Direct aggregation of neuroligins reveals a linkage of neuroligin-2 to GABA and glutamate postsynaptic proteins, but the other neuroligins link only to glutamate postsynaptic proteins. Furthermore, mislocalized expression of neuroligin-2 disperses postsynaptic proteins and disrupts synaptic transmission. These findings indicate that the neurexin-neuroligin link is a core component mediating both GABAergic and glutamatergic synaptogenesis, and differences in isoform localization and binding affinities may contribute to appropriate differentiation and specificity (Graf, 2004).

Neurexins constitute a large family of highly variable cell-surface molecules that may function in synaptic transmission and/or synapse formation. Each of the three known neurexin genes encodes two major neurexin variants, alpha- and beta-neurexins, that are composed of distinct extracellular domains linked to identical intracellular sequences. Deletions of one, two, or all three alpha-neurexins in mice recently demonstrated their essential role at synapses. In multiple alpha-neurexin knock-outs, neurotransmitter release from excitatory and inhibitory synapses was severely reduced, primarily probably because voltage-dependent Ca2+ channels were impaired. It remained unclear, however, which neurexin variants actually influence exocytosis and Ca2+ channels, which domain of neurexins is required for this function, and which Ca2+-channel subtypes are regulated. This study shows by electrophysiological recordings that transgenic neurexin 1alpha rescues the release and Ca2+-current phenotypes, whereas transgenic neurexin 1beta has no effect, indicating the importance of the extracellular sequences for the function of neurexins. Because neurexin 1alpha rescued the knock-out phenotype independent of the alpha-neurexin gene deleted, these data are consistent with a redundant function among different alpha-neurexins. In both knock-out and transgenically rescued mice, alpha-neurexins selectively affected the component of neurotransmitter release that depended on activation of N- and P/Q-type Ca2+ channels, but left L-type Ca2+ channels unscathed. These findings indicate that alpha-neurexins represent organizer molecules in neurotransmission that regulate N- and P/Q-type Ca2+ channels, constituting an essential role at synapses that critically involves the extracellular domains of neurexins (Zhang, 2005).

The leucine-rich repeat transmembrane protein LRRTM2 has been identified as a key regulator of excitatory synapse development and function. LRRTM2 localizes to excitatory synapses in transfected hippocampal neurons, and shRNA-mediated knockdown of LRRTM2 leads to a decrease in excitatory synapses without affecting inhibitory synapses. LRRTM2 interacts with PSD-95 and regulates surface expression of AMPA receptors, and lentivirus-mediated knockdown of LRRTM2 in vivo decreases the strength of evoked excitatory synaptic currents. Structure-function studies indicate that LRRTM2 induces presynaptic differentiation via the extracellular LRR domain. Neurexin1 was identified as a receptor for LRRTM2 based on affinity chromatography. LRRTM2 binds to both Neurexin 1alpha and Neurexin 1beta, and shRNA-mediated knockdown of Neurexin1 abrogates LRRTM2-induced presynaptic differentiation. These observations indicate that an LRRTM2-Neurexin1 interaction plays a critical role in regulating excitatory synapse development (de Wit, 2009).

Alpha latrotoxin binds neurexin

alpha-Latrotoxin is a potent neurotoxin from black widow spider venom, stimulating neurotransmitter release. alpha-Latrotoxin is thought to act by binding to a high affinity receptor on presynaptic nerve terminals. High affinity alpha-latrotoxin binding proteins contain neurexin I alpha as a major component. Neurexin I alpha is a cell surface protein that exists in multiple differentially spliced isoforms, belonging to a large family of neuron-specific proteins. Using a series of neurexin I-IgG fusion proteins, it is shown that recombinant neurexin I alpha binds alpha-Latrotoxin directly with high affinity (Kd approximately 4 nM). Binding of alpha-latrotoxin to recombinant neurexin I alpha is dependent on Ca2+ (EC50 approximately 30 microM). These data suggest that neurexin I alpha is a Ca(2+)-dependent high affinity receptor for alpha-latrotoxin (Davletov, 1995).

Alpha-latrotoxin is a potent neurotoxin that triggers synaptic exocytosis. Two distinct neuronal receptors for alpha-latrotoxin have been described: CIRL/latrophilin 1 (CL1) and neurexin-1alpha. Alpha-latrotoxin is thought to trigger exocytosis by binding to CL1, while the role of neurexin 1alpha is uncertain. Using PC12 cells, neurexins are demonstrated to indeed function as alpha-latrotoxin receptors that are at least as potent as CL1. Both alpha- and beta-neurexins represent autonomous alpha-latrotoxin receptors that are regulated by alternative splicing. Similar to CL1, truncated neurexins without intracellular sequences are fully active; therefore, neurexins and CL1 recruit alpha-latrotoxin but are not themselves involved in exocytosis. Thus, alpha-latrotoxin is unique among neurotoxins, because it utilizes two unrelated receptors, probably to amplify recruitment of alpha-latrotoxin to active sites (Sugita, 1999).

The potential role of contactin and contactin-associated protein (Caspr) has been examined in the axonal-glial interactions of myelination. In the nervous system, contactin is expressed by neurons, oligodendrocytes, and their progenitors, but not by Schwann cells. Expression of Caspr, a homolog of Neurexin IV, is restricted to neurons. Both contactin and Caspr are uniformly expressed at high levels on the surface of unensheathed neurites and are downregulated during myelination in vitro and in vivo. Contactin is downregulated along the entire myelinated nerve fiber. In contrast, Caspr expression initially remains elevated along segments of neurites associated with nascent myelin sheaths. With further maturation, Caspr is downregulated in the internode and becomes strikingly concentrated in the paranodal regions of the axon, suggesting that it redistributes from the internode to these sites. Caspr expression is similarly restricted to the paranodes of mature myelinated axons in the peripheral and central nervous systems; it is more diffusely and persistently expressed in gray matter and on unmyelinated axons. Immunoelectron microscopy has demonstrated that Caspr is localized to the septate-like junctions that form between axons and the paranodal loops of myelinating cells. Caspr is poorly extracted by nonionic detergents, suggesting that it is associated with the axon cytoskeleton at these junctions. These results indicate that contactin and Caspr function independently during myelination and that their expression is regulated by glial ensheathment. They strongly implicate Caspr as a major transmembrane component of the paranodal junctions, whose molecular composition has previously been unknown, and suggest its role in the reciprocal signaling between axons and glia (Einheber, 1997).

alpha-Latrotoxin stimulates neurotransmitter release probably by binding to two receptors, CIRL/latrophilin 1 (CL1) and neurexin Ialpha. Recombinant alpha-latrotoxin (LtxWT) has been produced that is as active as native alpha-latrotoxin in triggering synaptic release of glutamate, GABA and norepinephrine. Three alpha-latrotoxin mutants were generated with substitutions in conserved cysteine residues, and a fourth mutant with a four-residue insertion. All four alpha-latrotoxin mutants are unable to trigger release. Interestingly, the insertion mutant LtxN4C exhibits receptor-binding affinities identical to wild-type LtxWT; binds to CL1 and neurexin Ialpha as well as LtxWT, and similarly stimulates synaptic hydrolysis of phosphatidylinositolphosphates. Therefore, receptor binding by alpha-latrotoxin and stimulation of phospholipase C are insufficient to trigger exocytosis. This conclusion was confirmed in experiments with La3+ and Cd2+. La3+ blocks release triggered by LtxWT, whereas Cd2+ enhances it. Both cations, however, have no effect on the stimulation by LtxWT of phosphatidylinositolphosphate hydrolysis. These data show that receptor binding by alpha-latrotoxin and activation of phospholipase C do not by themselves trigger exocytosis. Thus receptors recruit alpha-latrotoxin to its point of action without activating exocytosis. Exocytosis probably requires an additional receptor-independent activity of alpha-latrotoxin that is selectively inhibited by the LtxN4C mutation and by La3+ (Ichtchenko, 1998).

alpha-Latrotoxin is a potent neurotoxin from black widow spider venom that binds to presynaptic receptors and causes massive neurotransmitter release. A surprising finding was the biochemical description of two distinct cell surface proteins that bind alpha-latrotoxin with nanomolar affinities: Neurexin I alpha binds alpha-latrotoxin in a Ca(2+)-dependent manner, and CIRL/latrophilin binds in a Ca(2+)-independent manner. Mice that lack neurexin I alpha were generated and analyzed to test the importance of neurexin I alpha in alpha-latrotoxin action. alpha-Latrotoxin binding to brain membranes from mutant mice is decreased by almost 50% compared with wild type membranes; the decrease is almost entirely due to a loss of Ca(2+)-dependent alpha-latrotoxin binding sites. In cultured hippocampal neurons, alpha-latrotoxin is still capable of activating neurotransmission in the absence of neurexin I alpha. Direct measurements of [3H]glutamate release from synaptosomes, however, shows a major decrease in the amount of release triggered by alpha-latrotoxin in the presence of Ca2+. Thus neurexin I alpha is not essential for alpha-latrotoxin action but contributes to alpha-latrotoxin action when Ca2+ is present. Viewed as a whole, these results show that mice contain two distinct types of alpha-latrotoxin receptors with similar affinities and abundance but different properties and functions. The action of alpha-latrotoxin may therefore be mediated by independent parallel pathways, of which the CIRL/latrophilin pathway is sufficient for neurotransmitter release, whereas the neurexin I alpha pathway contributes to the Ca(2+)-dependent action of alpha-latrotoxin (Geppert, 1998).

Neuroligins are ligands for neurexins

Neuroligin1 is a neuronal cell surface protein. It is enriched in synaptic plasma membranes and acts as a splice site-specific ligand for beta-neurexins. Neuroligin 1 binds only to beta-neurexins that lack an insert in the alternatively spliced sequence of the G domain; put another way, if the beta-neurexins have an insert, Neuroligin will not bind. The extracellular sequence of neuroligin 1 is composed of a catalytically inactive esterase domain homologous to acetylcholinesterase. Alternative splicing of neurexins at the site recognized by neuroligin 1 is highly regulated. A model proposed for this regulation suggests that the alternative splicing of neurexins creates a family of cell surface receptors that confer interactive specificity onto adjacent resident neurons (Ichtchenko, 1995).

Neuroligin 1 is a neuronal cell surface protein that binds to a subset of neurexins, polymorphic cell surface proteins that are also localized on neurons. Two novel neuroligins, neuroligins 2 and 3, are similar in structure and sequence to neuroligin 1. All neuroligins contain an N-terminal hydrophobic sequence with several characteristics in common: a cleaved signal peptide followed by a large esterase homology domain, a highly conserved single transmembrane region, and a short cytoplasmic domain. The three neuroligins are alternatively spliced at the same position and expressed at high levels only in the brain. Binding studies demonstrate that all three neuroligins bind to beta-neurexins both as native brain proteins and as recombinant proteins. Tight binding of the three neuroligins to beta-neurexins is observed only for beta-neurexins lacking an insert in splice site 4. Thus, neuroligins constitute a multigene family of brain-specific proteins with distinct isoforms that may have overlapping functions in mediating recognition processes between neurons (Ichtchenko, 1996).

PSD-95 is a component of postsynaptic densities in central synapses. It contains three PDZ domains that localize N-methyl-D-aspartate receptor subunit 2 (NMDA2 receptor) and K+ channels to synapses. In mouse forebrain, PSD-95 binds to the cytoplasmic COOH-termini of neuroligins, which are neuronal cell adhesion molecules that interact with beta-neurexins and form intercellular junctions. Neuroligins bind to the third PDZ domain of PSD-95, whereas NMDA2 receptors and K+ channels interact with the first and second PDZ domains. Thus different PDZ domains of PSD-95 are specialized for distinct functions. PSD-95 may recruit ion channels and neurotransmitter receptors to intercellular junctions formed between neurons by neuroligins and beta-neurexins (Irie, 1997).

beta-Neurexins and neuroligins are plasma membrane proteins that are displayed on the neuronal cell surface. The interaction of neurexin 1beta with neuroligin 1 was examined to evaluate their potential to function as heterophilic cell adhesion molecules. Using detergent-solubilized neuroligins and secreted neurexin 1beta-IgG fusion protein, the binding of these proteins to each other was observed only in the presence of Ca2+ and in no other divalent cation tested. Only neurexin 1beta, lacking an insert in splice site 4, binds neuroligins, whereas neurexin 1beta containing an insert is inactive. Half-maximal binding requires 1-3 microM free Ca2+, which probably acts by binding to neuroligin 1 but not to neurexin 1beta. To determine if neurexin 1beta and neuroligin 1 can also interact with each other when present in a native membrane environment on the cell surface, transfected cell lines expressing neuroligin 1 and neurexin 1beta were generated. Upon mixing different cell populations, cells are found to aggregate only if cells expressing neurexin 1beta are mixed with cells expressing neuroligin 1. Aggregation is dependent on Ca2+ and is inhibited by the addition of soluble neurexin 1beta lacking an insert in splice site 4 but not by the addition of neurexin 1beta containing an insert in splice site 4. It is concluded that neurexin 1beta and neuroligin 1 (and, by extension, other beta-neurexins and neuroligins) function as heterophilic cell adhesion molecules in a Ca2+-dependent reaction that is regulated by alternative splicing of beta-neurexins (Nguyen, 1997).

At the synapse, presynaptic membranes specialized for vesicular traffic are linked to postsynaptic membranes specialized for signal transduction. The mechanisms that connect pre- and postsynaptic membranes into synaptic junctions are unknown. Neuroligins and beta-neurexins are neuronal cell-surface proteins that bind to each other and form asymmetric intercellular junctions. To test whether the neuroligin/beta-neurexin junction is related to synapses, monoclonal antibodies to neuroligin 1 were generated and characterized. With these antibodies, neuroligin 1 has been shown to be synaptic. The neuronal localization, subcellular distribution, and developmental expression of neuroligin 1 are all similar to those of the postsynaptic marker proteins PSD-95 and NMDA-R1 receptor. Quantitative immunogold electron microscopy demonstrates that neuroligin 1 is clustered in synaptic clefts and postsynaptic densities. Double immunofluorescence labeling reveals that neuroligin 1 colocalizes with glutamatergic but not gamma-aminobutyric acid (GABA)ergic synapses. Thus neuroligin 1 is a synaptic cell-adhesion molecule that is enriched in postsynaptic densities where it may recruit receptors, channels, and signal-transduction molecules to synaptic sites of cell adhesion. In addition, the neuroligin/beta-neurexin junction may be involved in the specification of excitatory synapses (Song, 1999).

Accumulated evidence attributes one or more noncatalytic morphogenic activities to acetylcholinesterase (AChE). Despite sequence homologies, functional overlaps between AChE and catalytically inactive AChE-like cell surface adhesion proteins have been demonstrated only for the Drosophila protein Neurotactin. Furthermore, no mechanism had been proposed to enable signal transduction by AChE, an extracellular enzyme. Impaired neurite outgrowth and loss of neurexin Ialpha mRNA has been demonstrated under antisense suppression of AChE in PC12 cells (AS-ACHE cells). Neurite growth is partially rescued by the addition of recombinant AChE to the solid substrate or by transfection with various catalytically active and inactive AChE variants. Moreover, overexpression of the homologous neurexin I ligand, neuroligin-1, restores both neurite extension and expression of neurexin Ialpha. Differential PCR display reveals expression of a novel gene, nitzin, in AS-ACHE cells. Nitzin displays 42% homology to the band 4.1 protein superfamily capable of linking integral membrane proteins to the cytoskeleton. Nitzin mRNA is high throughout the developing nervous system; is partially colocalized with AChE, and increases in rescued AS-ACHE cells. These findings demonstrate redundant neurite growth-promoting activities for AChE and neuroligin and implicate interactions of AChE-like proteins and neurexins as potential mediators of cytoarchitectural changes supporting neuritogenesis (Grifman, 1998).

Most neurons form synapses exclusively with other neurons, but little is known about the molecular mechanisms mediating synaptogenesis in the central nervous system. Using an in vitro system, it has been demonstrated that neuroligin-1 and -2, both of which are postsynaptically localized proteins, can trigger the de novo formation of presynaptic structure. Nonneuronal cells engineered to express neuroligins induce morphological and functional presynaptic differentiation in contacting axons. This activity can be inhibited by addition of a soluble version of beta-neurexin, a receptor for neuroligin. Furthermore, addition of soluble beta-neurexin to a coculture of defined pre- and post-synaptic CNS neurons inhibits synaptic vesicle clustering in axons contacting target neurons. These results suggest that neuroligins are part of the machinery employed during the formation and remodeling of CNS synapses (Scheiffele, 2000).

Previous studies suggested that postsynaptic neuroligins form a trans-synaptic complex with presynaptic beta-neurexins, but not with presynaptic alpha-neurexins. Unexpectedly, neuroligins were also found to bind alpha-neurexins and that alpha- and beta-neurexin binding by neuroligin 1 is regulated by alternative splicing of neuroligin 1 (at splice site B) and of neurexins (at splice site 4). In neuroligin 1, splice site B is a master switch that determines alpha-neurexin binding but leaves beta-neurexin binding largely unaffected, whereas alternative splicing of neurexins modulates neuroligin binding. Moreover, neuroligin 1 splice variants with distinct neurexin binding properties differentially regulate synaptogenesis: neuroligin 1 that binds only beta-neurexins potently stimulates synapse formation, whereas neuroligin 1 that binds to both alpha- and beta-neurexins more effectively promotes synapse expansion. These findings suggest that neuroligin binding to alpha- and beta-neurexins mediates trans-synaptic cell adhesion but has distinct effects on synapse formation, indicating that expression of different neuroligin and neurexin isoforms specifies a trans-synaptic signaling code (Boucard, 2005).

Formation of synapses requires specific cellular interactions that organize pre- and postsynaptic compartments. The neuroligin-neurexin complex mediates heterophilic adhesion and can trigger assembly of glutamatergic and GABAergic synapses in cultured hippocampal neurons. Both neuroligins and neurexins are encoded by multiple genes. Alternative splicing generates large numbers of isoforms, which may engage in selective axo-dendritic interactions. This study explored whether alternative splicing of the postsynaptic neuroligins modifies their activity toward glutamatergic and GABAergic axons. It was found that small extracellular splice insertions restrict the function of neuroligin-1 and -2 to glutamatergic and GABAergic contacts and alter interaction with presynaptic neurexins. The neuroligin isoforms associated with GABAergic contacts bind to neurexin-1alpha and a subset of neurexin-1betas. In turn, these neurexin isoforms induce GABAergic but not glutamatergic postsynaptic differentiation. These findings suggest that alternative splicing plays a central role in regulating selective extracellular interactions through the neuroligin-neurexin complex at glutamatergic and GABAergic synapses (Chih, 2006).

Neuroligins enhance synapse formation in vitro, but surprisingly are not required for the generation of synapses in vivo. In cultured neurons, neuroligin-1 overexpression increases excitatory, but not inhibitory, synaptic responses and potentiates synaptic NMDAR/AMPAR ratios. In contrast, neuroligin-2 overexpression increases inhibitory, but not excitatory, synaptic responses. Accordingly, deletion of neuroligin-1 in knockout mice selectively decreases the NMDAR/AMPAR ratio, whereas deletion of neuroligin-2 selectively decreases inhibitory synaptic responses. Strikingly, chronic inhibition of NMDARs or CaM-Kinase II, which signals downstream of NMDARs, suppresses the synapse-boosting activity of neuroligin-1, whereas chronic inhibition of general synaptic activity suppresses the synapse-boosting activity of neuroligin-2. Taken together, these data indicate that neuroligins do not establish, but specify and validate, synapses via an activity-dependent mechanism, with different neuroligins acting on distinct types of synapses. This hypothesis reconciles the overexpression and knockout phenotypes and suggests that neuroligins contribute to the use-dependent formation of neural circuits (Chubykin, 2007).

The neuroligins are postsynaptic cell adhesion proteins whose associations with presynaptic neurexins participate in synaptogenesis. Mutations in the neuroligin and neurexin genes appear to be associated with autism and mental retardation. The crystal structure of a neuroligin reveals features not found in its catalytically active relatives, such as the fully hydrophobic interface forming the functional neuroligin dimer; the conformations of surface loops surrounding the vestigial active center; the location of determinants that are critical for folding and processing; and the absence of a macromolecular dipole and presence of an electronegative, hydrophilic surface for neurexin binding. The structure of a β-neurexin-neuroligin complex reveals the precise orientation of the bound neurexin and, despite a limited resolution, provides substantial information on the Ca2+-dependent interactions network involved in trans-synaptic neurexin-neuroligin association. These structures exemplify how an α/β-hydrolase fold varies in surface topography to confer adhesion properties and provide templates for analyzing abnormal processing or recognition events associated with autism (Fabrichny, 2007).

Neurexins and neuroligins provide trans-synaptic connectivity by the Ca²⁺-dependent interaction of their alternatively spliced extracellular domains. Neuroligins specify synapses in an activity-dependent manner, presumably by binding to neurexins. This study presents the crystal structures of neuroligin-1 in isolation and in complex with neurexin-1β. Neuroligin-1 forms a constitutive dimer, and two neurexin-1β monomers bind to two identical surfaces on the opposite faces of the neuroligin-1 dimer to form a heterotetramer. The neuroligin-1/neurexin-1β complex exhibits a nanomolar affinity and includes a large binding interface that contains bound Ca²⁺. Alternatively spliced sites in neurexin-1β and in neuroligin-1 are positioned nearby the binding interface, explaining how they regulate the interaction. Structure-based mutations of neuroligin-1 at the interface disrupt binding to neurexin-1β, but not the folding of neuroligin-1 and confirm the validity of the binding interface of the neuroligin-1/neurexin-1β complex. These results provide molecular insights for understanding the role of cell-adhesion proteins in synapse function (Araç, 2007).

Carbohydrates are ligands for Neurexins

Neurexins are expressed in hundreds of isoforms on the neuronal cell surface, where they may function as cell recognition molecules. Neurexins contain LNS domains, folding units found in many proteins like the G domain of laminin A, agrin, and slit. LNS domains are found in the G domain of Laminin A, Neurexins, and serum proteins, such as Sex hormone binding globulins. alpha-neurexins contain three EGF-like repeats and six LNS domains in their extracellular region, while beta-neurexins contain only a single LNS domain that is identical with the sixth LNS domain of alpha-neurexins. The crystal structure of neurexin Ibeta, a single LNS domain, reveals two seven-stranded beta sheets forming a jelly roll fold with unexpected structural similarity to lectins. The LNS domains of neurexin and agrin undergo alternative splicing that modulates their affinity for protein ligands in a neuron-specific manner. These splice sites are localized within loops at one edge of the jelly roll, suggesting a distinct protein interaction surface in LNS domains that is regulated by alternative splicing (Rudenko, 1999).

A large number of ligands for the LNS domains in the G domain of alpha-laminins has been reported, including heparin, sulfatides, integrins, dystroglycan, nidogen, and fibulin, but the specificity of most of these interactions is unknown. In neurexins, the functions of two LNS domains have been defined via their ligands neurexophilins, alpha-latrotoxin, and neuroligins, and binding is tightly regulated by alternative splicing. The structure of the LNS domain is very similar to lectin(-like) proteins, in particular, serum amyloid protein, S-lectin, and glucanase, all shown to bind sugars. In agrin and alpha-laminins, LNS domains bind to heparin and other glycosaminoglycan components of the extracellular matrix. The LNS domains in neurexins, however, are not known to bind carbohydrates. The structural homology between neurexin Ibeta and lectins raises the possibility that LNS domains may have a general function as carbohydrate-binding modules, and that protein:carbohydrate interactions might contribute to neurexin cell-adhesive properties at neuronal junctions. While it remains to be investigated whether LNS domains in neurexins do indeed bind sugars, the interactions of these domains with protein ligands are well characterized. There is no question that at least some of these domains are involved in high-affinity protein:protein interactions. For example, neurexin Ibeta binds alpha-latrotoxin with picomolar affinity. This interaction is not mediated by sugars because alpha-latrotoxin is not glycosylated (Rudenko, 1999).

Neurexophilins are secreted ligands for neurexins

A novel 29 kDa protein, neurexophilin, has been purified in a complex with neurexin l alpha. Cloning reveals that rat and bovine neurexophilins are composed of N-terminal signal peptides, nonconserved N-terminal domains (20% identity over 80 residues), and highly homologous C-terminal sequences (85% identity over 169 residues). There are two distinct neurexophilin genes in mice, one of which is more homologous to rat neurexophilin and the other to bovine neurexophilin. The first neurexophilin gene is expressed abundantly in adult rat and mouse brain, whereas no mRNA corresponding to the second gene is detected in rodents, despite its abundant expression in bovine brain, suggesting that rodents and cattle primarily express distinct neurexophilin genes. Neurexophilin is expressed in adult rat brain at high levels only in a scattered subpopulation of neurons that probably represent inhibitory interneurons; in contrast, neurexins are expressed in all neurons. Neurexophilin contains a signal sequence and is N-glycosylated at multiple sites, suggesting that it is secreted and binds to the extracellular domain of neurexin l alpha. This hypothesis has been confirmed by binding recombinant neurexophilin to the extracellular domains of neurexin l alpha. Together these data suggest that neurexophilin constitutes a secreted glycoprotein that is synthesized in a subclass of neurons and may be a ligand for neurexins (Petrenko, 1996).

Neurexophilin was discovered as a neuronal glycoprotein that is copurified with neurexin Ialpha during affinity chromatography on immobilized alpha-latrotoxin. An investigation has been carried out into how neurexophilin interacts with neurexins: whether neurexophilin is post-translationally processed by site-specific cleavage similar to neuropeptides, and whether related neuropeptide-like proteins are expressed in brain. Mammalian brains contain four genes for neurexophilins, whose products share a common structure composed of five domains: an N-terminal signal peptide, a variable N-terminal domain, a highly conserved central domain that is N-glycosylated, a short linker region, and a conserved C-terminal domain that is cysteine-rich. When expressed in pheochromocytoma (PC12) cells with a replication-deficient adenovirus, neurexophilin 1 is rapidly N-glycosylated and then slowly processed to a smaller mature form, probably by endoproteolytic cleavage. Similar expression experiments in other neuron-like cells and in fibroblastic cells revealed that N-glycosylation of neurexophilin 1 occurs in all cell types tested, whereas proteolytic processing is observed only in neuron-like cells. Finally, only recombinant neurexin Ialpha and IIIalpha but not neurexin Ibeta interact with neurexophilin 1 and are preferentially bound to the processed mature form of neurexophilin. Together these data demonstrate that neurexophilins form a family of related glycoproteins that are proteolytically processed after synthesis and bind to alpha-neurexins. The structure and characteristics of neurexophilins indicate that they function as neuropeptides that may signal via alpha-neurexins (Missler, 1998a).

alpha-Neurexins (Ialpha, IIalpha, and IIIalpha) are receptor-like proteins expressed in hundreds of isoforms on the neuronal cell surface. The extracellular domains of alpha-neurexins are composed of six LNS repeats, named after homologous sequences in the Laminin A G domain, Neurexins, and Sex hormone-binding globulin, with three interspersed epidermal growth factor-like domains. Purification of neurexin Ialpha reveals that it is tightly complexed to a secreted glycoprotein called neurexophilin 1. Neurexophilin 1 is a member of a family of at least four genes and resembles a neuropeptide, suggesting a function as an endogenous ligand for alpha-neurexins. Recombinant proteins and knockout mice have been used to investigate which isoforms and domains of different neurexins and neurexophilins interact with each other. Neurexophilins 1 and 3 but not 4 (neurexophilin 2 is not expressed in rodents) bind to a single individual LNS domain, the second overall LNS domain in all three alpha-neurexins. Although this domain is alternatively spliced, all splice variants bind, suggesting that alternative splicing does not regulate binding. Using homologous recombination to disrupt the neurexophilin 1 gene, mutant mice were generated that do not express detectable neurexophilin 1 mRNA. Mice lacking neurexophilin 1 are viable with no obvious morbidity or mortality. However, homozygous mutant mice exhibit male sterility, probably because homologous recombination resulted in the co-insertion into the neurexophilin gene of herpes simplex virus thymidine kinase, which is known to cause male sterility. In the neurexophilin 1 knockout mice, neurexin Ialpha is complexed with neurexophilin 3 but not neurexophilin 4, suggesting that neurexophilin 1 is redundant with neurexophilin 3 and that neurexophilins 1 and 3 but not 4 bind to neurexins. This hypothesis was confirmed using expression experiments. These data reveal that the six LNS and three epidermal growth factor domains of neurexins are independently folding ligand-binding domains that may interact with distinct targets. The results support the notion that neurexophilins represent a family of extracellular signaling molecules that interact with multiple receptors, including all three alpha-neurexins (Missler, 1998b).

LRRTM2 functions as a neurexin ligand in promoting excitatory synapse formation

Recently, leucine-rich repeat transmembrane proteins (LRRTMs) were found to be synaptic cell-adhesion molecules that, when expressed in nonneuronal cells, induce presynaptic differentiation in contacting axons. LRRTM2 induces only excitatory synapses, and it also acts to induce synapses in transfected neurons similarly to neuroligin-1. Using affinity chromatography, alpha- and beta-neurexins were identified as LRRTM2 ligands, again rendering LRRTM2 similar to neuroligin-1. However, whereas neuroligins bind neurexins containing or lacking an insert in splice site #4, LRRTM2 only binds neurexins lacking an insert in splice site #4. Binding of neurexins to LRRTM2 can produce cell-adhesion junctions, consistent with a trans-interaction regulated by neurexin alternative splicing, and recombinant neurexin-1beta blocks LRRTM2's ability to promote presynaptic differentiation. Thus, these data suggest that two unrelated postsynaptic cell-adhesion molecules, LRRTMs and neuroligins, unexpectedly bind to neurexins as the same presynaptic receptor, but that their binding is subject to distinct regulatory mechanisms (Ko, 2009).

Synaptic cell adhesion not only mediates the initial establishment of synapses, but also directs synapse specification, controls synapse maintenance, and regulates synapses during long-term synaptic plasticity. Moreover, the recent discovery of multiple synaptic cell-adhesion molecules as candidate genes for cognitive diseases such as autism, schizophrenia, and addiction has moved synaptic cell adhesion into the center of attention. In particular, neurexin-1 has been repeatedly linked to autism and schizophrenia (Kim, 2008: Kirov, 2009; Rujescu, 2009), LRRTM1 was linked to schizophrenia, and neurexin-3 has been associated with reward pathways and drug addiction (Bierut, 2007). Thus, the recent findings that LRRTMs are candidate synaptic cell-adhesion molecules that are potent effectors in the artificial synapse-formation assay were of great interest because they suggested that LRRTMs may define a novel trans-synaptic cell-adhesion pathway that may contribute to cognitive diseases. However, these results also raised important questions, namely whether the artificial synapse-formation activity of LRRTMs truly reflects a role in synapses formed between neurons, whether this role applies to all types or specific subtypes of synapses, and whether LRRTMs act as homophilic cell-adhesion molecules analogous to the leucine-rich repeat protein connectin, or function by binding to an as yet unidentified presynaptic ligand (Ko, 2009).

The present study provides initial answers to these questions by focusing on LRRTM2, the most abundant LRRTM isoform. LRRTM2 increases the abundance of excitatory, but not inhibitory, synapses, not only in the artificial synapse-formation assay, but also in transfected neurons. Moreover, LRRTM2 was shown not be a homophilic cell-adhesion molecule, but instead binds to presynaptic neurexins in a tight interaction that is regulated by alternative splicing of neurexins at splice-site #4. Thus, the data unexpectedly show that neurexins act by binding to two different downstream ligands, neuroligins and LRRTMs, in a mutually exclusive manner, and that their binding to these ligands is differentially regulated. These findings place neurexins at the core of two separate trans-synaptic cell-adhesion pathways, and indicate that the involvement of LRRTM1 and neurexin-1 in schizophrenia may delineate a common mechanism (Ko, 2009).

As synaptic cell-adhesion molecules, LRRTMs could be involved in one or several steps during synapse formation, from their initial establishment to their maturation and remodeling. The assays used in the present study -- the artificial synapse-formation and neuronal transfection assays -- do not allow conclusions about the steps in which a protein functions. For example, in the artificial synapse-formation assay, even control nonneuronal cells form transient synapses with cocultured neurons; thus, if a molecule stabilizes or validates synapses, it could in this assay stabilize synapses that are initially established by an independent mechanism. In vivo, neuroligins appear to be more important for synapse specification, function, and plasticity than for the initial establishment of synapses (Varoqueaux, 2006; Chubykin, 2007), and neurexins have a demonstrated role in synapse specification and function, but not synapse establishment. However, these findings do not necessarily mean that LRRTMs do not function in the establishment of initial synapses, and even neurexins might do so, because no complete neurexin deletion has yet been analyzed. Moreover, it is not known whether LRRTMs also bind to other presynaptic ligands, which is a distinct possibility because neuroligins also appear to interact with other presynaptic ligands besides neurexins. Again, these are issues that will have to be addressed in future studies (Ko, 2009).

Protein 4.1, the vertebrate homolog of Drosophila Coracle, interacts with Neurexin

Protein 4.1 (P4.1) is a multifunctional protein with heterogeneity in molecular weight, intracellular localization, tissue- and development-specific expression patterns. Mouse protein 4.1 gene, over 90 kilobases long, comprises at least 23 exons (13 constitutive exons, 10 alternative exons) interrupted by 22 introns. The donor and acceptor splice site sequences match the consensus sequences for the exon-intron boundaries of most eukaryotic genes. No significant sequence difference has been observed between splice junctions of alternative and constitutive exons. Apparently, most alternative exon-encoded peptides are located within particular functional domains of the P4.1 protein: two peptides encoded by alternative exons 4 and 5 are located near or within the glycophorin/calmodulin binding domain, whereas three other alternative exon-encoded peptides (19-amino acid encoded by exon 14, 14-amino acid encoded by exon 15, and 21-amino acid encoded by exon 16) are located near or within the spectrin-actin binding domain. Selective use of exon 2', which carries an upstream translation initiation codon (AUG), may produce an elongated P4.1 isoform (135 kDa) that is predominantly expressed in nonerythroid tissues. Combinatorial splicing of these exons may generate different isoforms that will exhibit complicated tissue-specific expression patterns (Huang, 1993).

Protein 4.1's interaction with the erythroid skeletal proteins spectrin and actin and its essential role in regulating membrane strength are both attributable to expression of an alternatively spliced 63-nucleotide exon. The corresponding 21-amino acid (21-aa) cassette is within the spectrin-actin binding domain of erythroid protein 4.1. This cassette is absent, however, in several isoforms that are generated by tissue- and development-specific RNA splicing. Four isoforms of the 10-kDa domain were constructed for comparative assessment of functions particularly relevant to red cells. In vitro translated isoforms containing the 21-aa cassette, bind spectrin, stabilize spectrin-actin complexes, and associate with red cell membrane. Isoforms replacing or lacking the 21-aa cassette do not function in these assays. The 21-aa sequence in protein 4.1 is critical to mechanical integrity of the red cell membrane. These results also allow the role of protein 4.1 in membrane mechanics to be interpreted primarily in terms of its spectrin-actin binding function. Alternatively expressed sequences within the 10-kDa domain of nonerythroid protein 4.1 suggest different, yet to be defined functions (Discher, 1993).

Mints are adaptors that directly bind to neurexins and recruit of munc18

Mint1 (X11/human Lin-10) and Mint2 are neuronal adaptor proteins that bind to Munc18-1 (n/rb-sec1), a protein essential for synaptic vesicle exocytosis. Mint1 has previously been characterized in a complex with CASK, another adaptor protein which in turn interacts with neurexins. Neurexins are neuron-specific cell surface proteins that act as receptors for the excitatory neurotoxin alpha-latrotoxin. Hence, one possible function for Mint1 is to mediate the recruitment of Munc18 to neurexins. In agreement with this hypothesis, it has been shown that the cytoplasmic tail of neurexins captures Munc18 via a multiprotein complex that involves Mint1. Furthermore, both Mint1 and Mint2 can directly bind to neurexins in a PDZ domain-mediated interaction. Various Mint and/or CASK-containing complexes can be assembled on neurexins, and Mint1 can bind to Munc18 and CASK simultaneously. These data support a model whereby one of the functions of Mints is to localize the vesicle fusion protein Munc18 to those sites at the plasma membrane that are defined by neurexins, presumably in the vicinity of points of exocytosis (Biederer, 2000).

A stoichiometric complex of neurexins and dystroglycan in brain

In nonneuronal cells, the cell surface protein dystroglycan links the intracellular cytoskeleton (via dystrophin or utrophin) to the extracellular matrix (via laminin, agrin, or perlecan). Impairment of this linkage is instrumental in the pathogenesis of muscular dystrophies. In brain, dystroglycan and dystrophin are expressed on neurons and astrocytes, and some muscular dystrophies cause cognitive dysfunction; however, no extracellular binding partner for neuronal dystroglycan is known. Regular components of the extracellular matrix, such as laminin, agrin, and perlecan, are not abundant in brain except in the perivascular space that is contacted by astrocytes but not by neurons, suggesting that other ligands for neuronal dystroglycan must exist. Alpha- and beta-neurexins, polymorphic neuron-specific cell surface proteins, have now been identified as neuronal dystroglycan receptors. The extracellular sequences of alpha- and beta-neurexins are largely composed of laminin-neurexin-sex hormone-binding globulin (LNS)/laminin G domains, which are also found in laminin, agrin, and perlecan, that are dystroglycan ligands. Dystroglycan binds specifically to a subset of the LNS domains of neurexins in a tight interaction that requires glycosylation of dystroglycan and is regulated by alternative splicing of neurexins. Neurexins are receptors for the excitatory neurotoxin alpha-latrotoxin; this toxin competes with dystroglycan for binding, suggesting overlapping binding sites on neurexins for dystroglycan and alpha-latrotoxin. These data indicate that dystroglycan is a physiological ligand for neurexins and that neurexins' tightly regulated interaction could mediate cell adhesion between brain cells (Sudhof, 2001).

Protein 4.1 interaction with Glycophorin, a protein that shares a domain with Neurexin

Protein 4.1 is the prototype of a family of proteins that include ezrin, talin, brain tumor suppressor merlin, and tyrosine phosphatases. All members of the protein 4.1 superfamily share a highly conserved N-terminal 30-kDa domain whose biological function is poorly understood. It is believed that the attachment of the cytoskeleton to the membrane may be mediated via this 30-kDa domain, a function that requires formation of multiprotein complexes at the plasma membrane. Synthetically tagged peptides and bacterially expressed proteins were used to map the protein 4.1 binding site on human erythroid glycophorin C, a transmembrane glycoprotein, and on human erythroid p55, a palmitoylated peripheral membrane phosphoprotein. The 30-kDa domain of protein 4.1 binds to a 12-amino acid segment within the cytoplasmic domain of glycophorin C and to a positively charged, 39-amino acid motif in p55. Sequences similar to this charged motif are conserved in other members of the p55 superfamily, including the Drosophila Discs-large tumor suppressor protein (Marfatia, 1995)

The major attachment site for protein 4.1 on the human erythrocyte is glycophorin (GP) C/D. Purified protein 4.1 can bind to two distinct sites on glycophorin C/D. One of these interactions is direct, involving residues 82-98 on glycophorin C (61-77 on glycophorin D), while the other interaction is mediated by p55. The binding site for p55 on glycophorin C is localized to residues 112-128 (glycophorin D91-107). Band 3 is an additional and minor binding site for Protein 4-1. The binding sites for band 3, glycophorin C/D, and p55 are all located within the 30-kDa domain of protein 4.1. The relative utilization of the three sites in normal membranes comprises 40% to p55, 40% to GPC/D, and 20% to band 3. The same region of protein 4.1 binds GPC/D and band 3, while the p55 binding site is distinct. The interactions involving protein 4.1 with p55 and p55 with GPC/D are of high affinity (nM), while those involving GPC/D and band 3 are 100-fold lower (microM). These results suggest that the most significant interactions between protein 4.1 and the membrane are those involving p55 (Hemming, 1995).

Contactin, a protein that interacts with Neurexins

Receptor protein tyrosine phosphatase beta (RPTPbeta) expressed on the surface of glial cells binds to the glycosylphosphatidylinositol (GPI)-anchored recognition molecule contactin on neuronal cells leading to neurite outgrowth. Contactin belongs to the Ig superfamily and is expressed on the cell surface of neurons. A novel contactin-associated transmembrane receptor (p190/Caspr) has been cloned. Caspr contains a mosaic of domains implicated in protein-protein interactions. The extracellular domain of Caspr contains a neurophilin/coagulation factor homology domain, as well as a region related to fibrinogen beta/gamma, epidermal growth factor-like repeats, neurexin motifs and unique PGY repeats found in a molluscan adhesive protein. The cytoplasmic domain of Caspr contains a proline-rich sequence capable of binding to a subclass of SH3 domains in signaling molecules. Caspr and contactin exist as a complex in rat brain and are bound to each other by means of lateral (cis) interactions in the plasma membrane. Caspr may function as a signaling component of contactin, enabling recruitment and activation of intracellular signaling pathways in neurons. The binding of RPTPbeta to the contactin-Caspr complex could provide a mechanism for cell-cell communication between glial cells and neurons during development. The sequence and domain structure of Caspr show a close similarity to Drosophila Neurexin IV; it is suggested that these two proteins could recruit PDZ or SH3 domains containing signaling molecules to specific regions of cell-cell contacts thereby regulating intracellular events in the nervous system and in other tissues. Indeed, the intracellular domain of Drosophila NrxIV has a binding site for PDZ domain-containing proteins and is required for the localization of Coracle protein to pleated septate junctions (Peles, 1997a and b).

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 (see Drosophila 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).

Carbohydrate binding function of Discoidin, homologous to the extracellular domain of Drosophila Neurexin

One of the common characteristics observed in different families of sugar-binding proteins is the presence of aromatic residues in proximity to the functional sugar-binding site. This general property has made these proteins a very appropriate subject for studies using intrinsic fluorescence assays. The galactose binding of the lectin discoidin I has been estimated, with an affinity constant of 1.8.10(-7) M-1 in the absence of calcium. In the presence of 1 mM Ca2+, the Kd of galactose binding is lowered to 2.7.10(-8) M-1. Calcium binding, by itself, seems to occur as two components with Kd values of 10(-7) and 10(-6) M-1. From these data, and sequence comparison of discoidin I with other lectins, a general model for ligand binding has been proposed in which a sequence from position 176 to 188, together with another region close to an apolar tryptophan residue, most probably Trp-50, would participate in the calcium- and sugar-binding site(s) of this protein (Valencia, 1989).

Disruption of axo-glial junctions causes cytoskeletal disorganization and degeneration of Purkinje neuron axons

Axo-glial junctions (AGJs) play a critical role in the organization and maintenance of molecular domains in myelinated axons. Neurexin IV/Caspr1/paranodin (NCP1) is an important player in the formation of AGJs because it recruits a paranodal complex implicated in the tethering of glial proteins to the axonal membrane and cytoskeleton. Mice deficient in either the axonal protein NCP1 or the glial ceramide galactosyltransferase (CGT) display disruptions in AGJs and severe ataxia. In this article, these two phenotypes were correlated and it was shown that both NCP1 and CGT mutants develop large swellings accompanied by cytoskeletal disorganization and degeneration in the axons of cerebellar Purkinje neurons. alphaII spectrin was found to be part of the paranodal complex and that, although not properly targeted, this complex is still formed in CGT mutants. Together, these findings establish a physiologically relevant link between AGJs and axonal cytoskeleton and raise the possibility that some neurodegenerative disorders arise from disruption of the AGJs (Garcia-Fresco, 2006; full text of article).

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