Neurexin IV
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-8G0397 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, Gαs 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).
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).
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).
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 vari03953b 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 vari38EFa2 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 variMD109, vari327 or variR979 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 variF033 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 vari38EFa2, 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 variMD109, 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).
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).
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-IVexon3 isoform is predominantly expressed in glial cells that can form septate junctions, the Nrx-IVexon4-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-IVexon4 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 IVexon3 is less pronounced compared with Neurexin IVexon4. 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).
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).
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