InteractiveFly: GeneBrief

varicose: Biological Overview | References


Gene name - varicose

Synonyms -

Cytological map position - 38E1-39A3

Function - scaffolding protein

Keywords - cell adhesion, septate junction protein, tracheal development, PNS, brain, wings, eye

Symbol - vari

FlyBase ID: FBgn0250785

Genetic map position - 2L: 20,788,659..20,797,040 [-]

Classification - PDZ_signaling

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Jeong, S., Yang, D. S., Hong, Y. G., Mitchell, S. P., Brown, M. P. and Kolodkin, A. L. (2017). Varicose and cheerio collaborate with pebble to mediate semaphorin-1a reverse signaling in Drosophila. Proc Natl Acad Sci U S A 114(39): E8254-e8263. PubMed ID: 28894005
Summary:
The transmembrane semaphorin Sema-1a acts as both a ligand and a receptor to regulate axon-axon repulsion during neural development. Pebble (Pbl), a Rho guanine nucleotide exchange factor, mediates Sema-1a reverse signaling through association with the N-terminal region of the Sema-1a intracellular domain (ICD), resulting in cytoskeletal reorganization. This study uncover two additional Sema-1a interacting proteins, varicose (Vari) and cheerio (Cher), each with neuronal functions required for motor axon pathfinding. Vari is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins, members of which can serve as scaffolds to organize signaling complexes. Cher is related to actin filament cross-linking proteins that regulate actin cytoskeleton dynamics. The PDZ domain binding motif found in the most C-terminal region of the Sema-1a ICD is necessary for interaction with Vari, but not Cher, indicative of distinct binding modalities. Pbl/Sema-1a-mediated repulsive guidance is potentiated by both vari and cher Genetic analyses further suggest that scaffolding functions of Vari and Cher play an important role in Pbl-mediated Sema-1a reverse signaling. These results define intracellular components critical for signal transduction from the Sema-1a receptor to the cytoskeleton and provide insight into mechanisms underlying semaphorin-induced localized changes in cytoskeletal organization.
BIOLOGICAL OVERVIEW

Epithelial tubes are the functional units of many organs, but little is known about how tube sizes are established. Using the Drosophila tracheal system as a model, it has been shown that mutations in varicose (vari) cause tubes to become elongated without increasing cell number. This study shows how vari is required for accumulation of the tracheal size-control proteins Vermiform and Serpentine in the tracheal lumen. vari is an essential septate junction (SJ) gene encoding a membrane associated guanylate kinase (MAGUK). In vivo analyses of domains important for MAGUK scaffolding functions demonstrate that while the Vari HOOK domain is essential, the L27 domain is dispensable. Phylogenetic analyses reveal that Vari helps define a new MAGUK subgroup that includes mammalian PALS2. Importantly, both Vari and PALS2 are basolateral, and the interaction of Vari with the cell-adhesion protein Neurexin IV parallels the interaction of PALS2 and another cell-adhesion protein, Necl-2. Vari therefore bolsters the similarity between Drosophila and vertebrate epithelial basolateral regions, which had previously been limited to the common basolateral localization of Scrib, Dlg and Lgl, proteins required for epithelial polarization at the beginning of embryogenesis. However, by contrast to Scrib, Dlg and Lgl, Vari is not required for cell polarity but rather is part of a cell-adhesion complex. Thus, Vari fundamentally extends the similarity of Drosophila and vertebrate basolateral regions from sharing only polarity complexes to sharing both polarity and cell-adhesion complexes (Wu, 2007).

The function of organs such as the lung, kidney and vascular system depends on epithelial and endothelial tubes of specific sizes. However, the cell biological and molecular processes that control tube sizes are largely unknown. The Drosophila tracheal system is a network of ramifying epithelial tubes that serves as a combined pulmonary-vascular system to directly deliver oxygen to tissues. The comparative simplicity and genetic tractability of the tracheal system has made it one of the best models of tubular epithelial morphogenesis. The tracheal system develops from a series of sacs into a complex network of branches through a highly orchestrated series of cell migrations, cell shape changes and rearrangements of cell-cell junctions. An important element of these morphogenetic events is that changes in tube size occur reproducibly during specific developmental periods. Each tracheal branch has a specific size that results from the action of branch-specific signaling events that at least in some branches are known to act through transcription factors such as Spalt-Major (Spalt). At least one additional transcription factor, Grainyhead, is required to control tube length and apical cell surface in the major tracheal branches, but the transcriptional targets that more directly mediate these functions remain to be identified. Recent work by multiple groups has produced a basic molecular framework of the mechanisms that execute the size changes of 'tube expansion', a process that increases the diameter - but not the length - of the major tracheal tubes over a 2 hour period, and then gradually lengthens the tubes without changing their diameters. These tube size changes result from changes in cell shape and possibly cell size, but do not involve changes in cell number (Wu, 2007).

The tube expansion mechanism depends upon a fibrillar, chitin-based extracellular matrix that is assembled in the tracheal lumen at the beginning of the diameter dilation. As development progresses, chitin at the apical cell surface is organized into a highly patterned, multilayered cuticle. Lumenal chitin is eliminated before hatching. Defects in chitin synthesis or organization cause tracheal tube diameters to become either too large or too small, and tube lengths to become over-elongated. The exact role of the chitin-based matrix in controlling tracheal cell shape is unclear. Although the lumenal matrix and cuticle may serve as structural forms or 'mandrils' that mechanically shape the tracheal cells and tubes, an instructive or signaling role for the matrix is suggested by the observation that the organization of the βH-spectrin cytoskeleton is altered in chitin-synthetase mutants (Wu, 2007).

Beginning at stage 15, organization of the lumenal matrix requires the lumenal secretion of the putative chitin deacetylases, Vermiform (Verm) and Serpentine (Serp). In verm and serp mutants, the chitin-based matrix becomes disorganized and tracheal tubes become too long. Surprisingly, lumenal secretion of Verm requires a cell-cell junction termed the septate junction (SJ). Septate junctions are complex cell adhesion junctions that have at least 15 known components. These include transmembrane cell-adhesion proteins such as Neurexin IV (Nrx-IV; herein referred to as Nrx) and Neuroglian (Nrg), cytoplasmic proteins such as the FERM-domain protein Coracle (Cor; Cora - Flybase), the basal polarity proteins Scribbled (Scrib), Discs large (Dlg; Dlg1 -Flybase), and Lethal giant larvae (Lgl; L(2)gl - Flybase), and proteins with roles that remain to be determined, such as the Na+/K+-ATPase. Mutations in most known SJ components cause tracheal phenotypes indistinguishable from the verm mutant phenotype, consistent with the failure of Verm to be secreted into the tracheal lumen in the SJ mutants so far examined. Secretion of other apical lumenal markers appears normal in SJ mutants, indicating that Verm is secreted by a specialized pathway, the mechanism of which remains to be determined (Wu, 2007).

Although the role of SJs in lumenal (apical) secretion is not understood, other SJ functions are well defined. SJs have functional and molecular similarity to vertebrate tight junctions (TJs), in that both junctions require members of the claudin protein family to create the paracellular diffusion barriers between epithelial cells that are essential to the survival of multicellular animals. However, SJs are not simply the homologs of TJs, because there are significant ultrastructural, molecular and functional differences between SJ and TJs. For example, TJs are apical of adherens junctions (AJs) and contain conserved apical polarity complexes, while SJs are basal of AJs and contain the polarity proteins Scrib, Dlg and Lgl, which have vertebrate homologs that also localize basolaterally. Thus, in some respects SJs are more related to complexes found in the basolateral regions of vertebrate epithelial cells than to TJs (Wu, 2007).

Although Scrib, Dlg and Lgl establish and currently define the similarity between SJ and vertebrate basolateral regions, it is notable that these proteins are not representative of most SJ components. Drosophila Scrib, Dlg and Lgl are maternally contributed and constitute a distinct subgroup of proteins required for initial epithelial cell polarization during embryonic stages 5-8. By contrast, most SJ components are not maternally expressed, are not required for cell polarity and only function relatively late in development when SJs begin forming during stage 13. Whether the Scrib, Dlg and Lgl proteins nucleate SJ assembly, or whether the nascent SJ recruits and incorporates Scrib, Dlg and Lgl has not been determined. It also has not yet been determined how Scrib, Dlg and Lgl are localized to the basolateral membrane in either Drosophila or vertebrate epithelia. Thus the similarity between Drosophila SJ and vertebrate basolateral regions has been limited to polarity complexes, and has not extended to cell adhesion complexes (Wu, 2007).

This report shows that vari encodes a previously uncharacterized, membrane-associated, guanylate kinase (MAGUK) scaffolding protein that is required for SJ organization and directly binds the cell adhesion protein Neurexin IV. Importantly, Vari helps define a new subgroup of MAGUKs that includes vertebrate PALS2. Both Vari and PALS2 localize basolaterally in epithelial cells and both interact through a PDZ domain with a basolateral adhesion protein. Thus, Vari is the first late-expressed SJ component to have a vertebrate homolog, and together Vari and PALS2 extend the similarity of Drosophila and vertebrate basolateral regions from polarity complexes to adhesion complexes (Wu, 2007).

Vari was originally identified as a gene required for regulating the size of epithelial tubes. In vari mutants, tracheal tubes become too long without changes in tracheal cell number (Beitel, 2000). This study shows that Vari encodes multiple isoforms of a MAGUK that helps define a new subgroup of MAGUKs. Vari functions in the assembly of the septate junctions and is required for the apical secretion of the protein Verm, which is thought to be responsible for modifying a chitin-based lumenal matrix (Luschnig, 2006; Wang, 2006). In vari and other SJ mutants, Verm is not secreted, the lumenal matrix becomes abnormal and tracheal tubes become elongated (Wu, 2007).

The protein-protein interaction domains present in Vari suggest it acts as a scaffolding protein that helps bring together different components of the SJ complex. This hypothesis is supported by GST-pull down assay results showing Vari's PDZ domain can directly bind the intracellular domain of Nrx, a transmembrane SJ adhesion protein. Binding of the Vari PDZ domain to Nrx would leave Vari's SH3, GUK and predicted C-terminal PDZ-binding motif available to anchor other SJ components to the membrane, or to bring together different transmembrane SJ components. One model is that Vari may help bring the Dlg-Scib complex to the membrane through interfolding of the Vari and Dlg SH3 domains, which is made possible by the unique HOOK domain insert in the MAGUK SH3 domains (McGee, 2001; Tavares, 2001). Whether or not Vari anchors the Dlg complex to the rest of the SJ, genetic evidence indicates that Vari has functions beyond simply bridging between transmembrane Nrx and intracellular SJ complexes, because vari mutations can strongly enhance the phenotypes caused by mutations in the Drosophila claudin sinuous, whereas nrx mutations do not enhance sinuous mutations (Wu, 2007).

By itself, the finding that Vari encodes a MAGUK was not unexpected, as many MAGUKs are associated with cell-cell junctions. However, it is significant that Vari helps define a new subgroup of MAGUKs that includes mammalian PALS2, because Vari and PALS2 both localize basolaterally and bind the C-termini of basolateral cell adhesion proteins. Thus, Vari and PALS2 bolster the similarity between Drosophila and vertebrate epithelial basolateral regions that was first evidenced by the common basolateral localization of the Scrib, Dlg and Lgl early polarity proteins. However, by contrast to the polarity proteins, Vari is not required for cell polarity but rather is expressed late in embryonic development and is part of a cell-adhesion complex. Thus, Vari fundamentally extends the similarity of Drosophila and vertebrate basolateral regions from containing only conserved polarity complexes to containing both conserved polarity and cell-adhesion complexes (Wu, 2007).

The finding of more extensive similarity between SJ and vertebrate basolateral regions suggests that continued study of Drosophila SJs will provide insight into vertebrate epithelial basolateral regions. Further, these results support the idea that during evolution there has been conservation of different junctional functions, such as forming paracellular barriers and anchoring of polarity complexes. However, the comparison of TJs and SJs also makes it clear that there has been limited conservation of which particular functions have assorted to different junctions. An attractive explanation for these somewhat contradictory observations is that junctional functions are modular, and that the disparate junctions in different species represent alternative combinations of functional modules. For example, Drosophila SJs could be considered a combination of the claudin-based paracellular-barrier function and the basolateral polarity proteins Dlg, Scrib and Lgl. Alternatively, vertebrate TJs could be considered a combination of the claudin-based paracellular-barrier function and the apical polarity complexes of Crbs-Baz and Sdt-aPKC-Par-6. Thus, when comparing junctions between species, it is likely to be more useful to compare specific junctional functions, such as molecular details of polarity or barrier functions, than to attempt to directly compare junctions in their entirety (Wu, 2007).

If complex junctions such as TJs and SJs are comprised of functional modules, one would expect that these junctions should contain distinct molecular subcomplexes that mediate distinct functions. Consistent with this proposal, extensive work by many labs has shown that the polarity proteins of Crb-Sdt and Baz-cdc42-aPKC form specific complexes. Claudin proteins appear to be part of a 'barrier complex' because claudins are required for and co-localize with the paracellular barrier in both Drosophila and vertebrates. Functional demonstration of the independence of the barrier and polarity complexes in both species is provided by the observations that cell polarity is not affected by selective disruption of the barrier complex in either mammals by knockdown of ZO-1 and ZO-2, or in Drosophila by mutations in claudin genes (Behr, 2003; Wu, 2004). The Vari/PALS2 proteins could play a pivotal role in allowing cytoplasmic subcomplexes to associate different adhesion-junctional complexes, either in different cell types or during evolution, because changing which adhesion complex Vari or PALS2 associate with could be as simple as changing the four amino acid PDZ-binding motifs of one or a few transmembrane proteins. It seems likely that evolving a few unstructured amino acids would be significantly easier than evolving three-dimensional binding surfaces. Thus, Vari and its homologs could provide crucial (but malleable) links between conserved intracellular complexes and the divergent transmembrane junctional complexes found across the animal kingdom (Wu, 2007).

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 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 Drosophila Discs 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 Drosophila embryo, 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 Drosophila embryo. 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).

Varicose: a MAGUK required for the maturation and function of Drosophila septate junctions

Scaffolding proteins belonging to the membrane associated guanylate kinase (MAGUK) superfamily function as adapters linking cytoplasmic and cell surface proteins to the cytoskeleton to regulate cell-cell adhesion, cell-cell communication and signal transduction. Varicose (Vari), the homologue of vertebrate scaffolding protein PALS2 localizes to pleated septate junctions (pSJs) of all embryonic, ectodermally-derived epithelia and peripheral glia. In vari mutants, essential SJ proteins NeurexinIV and FasciclinIII are mislocalized basally and epithelia develop a leaky paracellular seal. In addition, vari mutants display irregular tracheal tube diameters and have reduced lumenal protein accumulation, suggesting involvement in tracheal morphogenesis. Vari is distributed in the cytoplasm of the optic lobe neuroepithelium, as well as in a subset of CNS and differentiated neurons of the nervous system. vari function during the development of adult epithelia was reduced with a partial rescue, RNA interference and generation of genetically mosaic tissue. All three approaches demonstrate that vari is required for the patterning and morphogenesis of adult epithelial hairs and bristles. It is concluded that Varicose is involved in scaffold assembly at the SJ and has a role in patterning and morphogenesis of adult epithelia (Moyer, 2008).

This study has characterised a MAGUK family member encoded by Drosophila CG9326, Varicose. Varicose localizes to pSJs of all embryonic ectodermally-derived epithelial tissues as well as the pSJs of the PNS. Vari expression was detected in the neuroepithelium of the developing optic lobe in a non-junction associated pattern, which is unique for a MAGUK member. Expression of Varicose in a subset of central brain neuroblasts and differentiated neurons of the adult nervous system emphasizes the importance and versatility of its function throughout development. Mutations in vari result in mislocalized SJ markers and disruption of the paracellular seal. Loss-of-function vari alleles display dilated and contorted tracheal tubes, implicating vari in tracheal morphogenesis. Furthermore, genetic mosaic and partial rescue phenotypes in the eye and wing suggests a role for vari during adult epithelial morphogenesis (Moyer, 2008).

Several lines of evidence demonstrate that Varicose is required for septate junction formation: (1) Varicose co-localizes with known SJ protein NrxIV in all embryonic pSJs, and NrxIV is mislocalized in the absence of varicose activity; (2) in vari null mutants, SJ do not mature to the point of septa formation; (3) the transepithelial barrier of vari mutants is 'leaky' to tracer dyes (Moyer, 2008).

Embryos mutant for varicose show mislocalization of SJ proteins like NrxIV, FasIII and the Na+K+ATPase basally along the plasma membrane of epithelia. The localization of SJ protein Dlg was not affected however, indicating Vari is not required to establish epithelial polarity. This is not a surprising result as the onset of Varicose expression appears midway through embryogenesis, a time when polarity has already been established and SJs begin to assemble. Proper localization of Vari requires Nrg. Varicose interacts with NrxIV and all three proteins share an mutually-dependent relationship necessary for proper subcellular localization. Vari reduces the lateral mobility of Nrg and NrxIV, suggesting that in the absence of vari, the assembly of key SJ proteins is interrupted, disrupting the architecture of the junctional region and triggering a cascade of mislocalized proteins (Moyer, 2008).

Ultrastructural analysis indicates that embryos null for vari die before intermembrane septa develop. Bachmann (2008) establish that septa do not develop in hypomorphs that develop further as embryos. nrxIV and cora mutants also lack septa, which are proposed to have a sealing function in the transepithelial barrier. An affinity approach has identified NrxIV as a potential Vari binding partner (Wu, 2007). These data are consistent with the failure of vari mutants to exclude dye in embryonic trachea. In contrast, SJ mutants, gliotactin and sinuous (sinu) show defects in septa array and septa number, respectively. Mutations in vari enhance the sinu phenotype (Wu, 2004). Together these results suggest Vari, like NrxIV and Cora, functions in assembling septa strands. However, the low levels of Vari in larvae suggest that Vari is not essential to maintain SJ (Moyer, 2008).

SJ integrity in Drosophila requires Megatrachea (Mega), a claudin that has a C-terminal PDZ binding domain (Behr, 2003). It has been suggested that a MAGUK member may act to tether Mega to the NrxIV/Cora complex to assemble the SJ. This paper proposes Vari as a candidate for this function (Moyer, 2008).

Expression of Vari in peripheral glia, but not the perineural sheath or midline glia of the embryonic nervous system is consistent with function in the establishment of ectodermally-derived pSJs. Neural expression was not detected in embryos. However, the distribution of Vari in the late larval and adult central nervous system suggests non-junctional roles for this MAGUK. In the optic lobe NE, which does express Dlg, Vari expression overlaps, and extends into the apical cytoplasm. Vari is not expressed in NBs of the embryo and medial optic lobe, yet is expressed in the cytoplasm of some central NBs of late third instar, and in low levels in the soma of differentiated neurons. This pattern of expression is not typical of other junctional or cell polarity markers like Bazooka, Glaikit or Miranda, or of MAGUKs in general and must be clarified by further study. This issue may be approached with nervous system specific RNAi knockdown of Vari, which was found to be pupal lethal (Moyer, 2008).

The Drosophila tracheal system is a well developed model for the dissection of pathways regulating tube formation. pSJ components are implicated in the regulation of tubule size. Genes regulating tube size fall into two phenotypic categories; those required to regulate tube length and those required for normal tube diameter. Several lines of evidence have suggested that pSJ components are involved in regulating tube length. Mutations in genes for SJ proteins like mega, sinu and the Na+K+ATPase β subunit, nrv2 have tortuous and elongated tracheal trunks, without affecting tube diameter. In contrast, mutations in vari do not appear to affect tube length. Tracheal tubes in vari mutants have irregular and enlarged tube diameters reminiscent of mutations affecting mmy/cystic and kkv, enzymes required for chitin synthesis. Epistatic analysis of vari and sinu reveals a tracheal phenotype in double mutants that is worse than either single mutant, suggesting these proteins function in different pathways (Wu, 2004; Moyer, 2008 and references therein).

The chitin matrix is secreted from the apical surface of tracheal cells and synthesis of the matrix has been linked to controlling tube diameter. During expansion of the dorsal trunk, the cylinder expands as the lumen dilates. It is suggested that formation of the chitin matrix is needed for the organized radial expansion of tracheal tubes. Tracheal enlargement in vari is similar to cystic and kkv mutants, and all three have reduced deposition of 2A12 antigen. Wu (2007) further showed that vari mutants fail to secrete apical protein Serpentine and secrete variable amounts of Vermiform. Unexpectedly, cuticle ultrastructure and taenidial ridges appear normal in vari mutants. This result is unlike tracheal mutants affecting tube length, such as sinu, where taenidial folds are irregular (Wu, 2004). The data suggests that lumenal protein secretion in vari mutants is sufficient to produce cuticle and regulate tube length, and that the SJ may also play a role in regulating tube diameter (Moyer, 2008).

Knowledge of the function of septate junctions during metamorphosis is limited. However a role for SJs in the adult ommatidium has been described. The SJ component NrxIV was shown to localize to junctional regions in the pupal and adult eye. Loss of nrxIV disrupts SJ function, which leads to structural disorganization resulting from a loss of adhesion between cells of the adult ommatidia. Although nrxIV- clones survive in the adult eye, vari- clones do not, indicating that vari has functions other than localizing NrxIV. Partial restoration of vari (by transgene rescue) during adult morphogenesis results in missing ommatidia and irregular bristle patterning. Vari may spatially organise adhesions during this developmental process, and reduced levels of Vari disrupts epithelial patterning. Vari levels are much lower subsequent to vari-RNAi treatment described by Bachmann (2008). The retinal and wing epithelia survive, but epithelial patterning is more disorganised. This study reports that mosaic null clones of vari generate duplicated bristles in the eye, and clumped sensory hairs on the thorax. The failure to establish SJs in these clones may result in delaminating mutant cells adopting a neurogenic fate, and thereby generate extra sensory structures (Moyer, 2008).

The involvement of SJs during wing imaginal disc to adult wing morphogenesis is also unclear. However, two known SJ components, Gli and Cora are required for survival of pre-hair cells during pupation. Similar to mutations in gli and cora, partial rescue of vari, and RNAi of vari resulted in patches of wing hairs that fail to point distally. Although reminiscent of a Frizzled (Fz) planar cell polarity phenotype, the mechanism regulating hair alignment acts independently of Fz. Patches of wing hairs, although not pointing distally, retain a parallel alignment with neighbouring hairs in Fzmutants. This is unlike the random alignment seen in vari, gli, and cora mutants where polarity of neighbouring wing hairs is different. During pupal development, the position and orientation of wing prehairs are determined and then stabilized during later stages. Vari and other SJ proteins play a role in prehair patterning (Moyer, 2008).

The MAGUK protein PALS2 has been proposed to act in scaffold formation at the basolateral membrane of mammalian epithelia (Shingai, 2003). This study shows that a Drosophila homologue, Vari, is similarly distributed, and is required in ectodermally-derived epithelia to elaborate pSJs and establish a paracellular barrier. Embryos lacking vari function display mislocalization of essential pSJ membrane proteins, including NrxIV, Na+K+ATPase and FasIII, and are unable to control the permeability of the tracheal membrane. As a result, the trachea fail to fill with air, and the embryos die in early stage 17. The function of SJs in the morphogenesis of the wing and eye is less well characterised, yet imaginal epithelia lacking vari, cora or gli do not survive to the adult. The eye and wing phenotypes of reduced vari function overlaps with patterning defects of mutations in SJ genes nrxIV, gli and cora. Together, they indicate an uncharacterised role for SJs in establishing pattern in epithelial sheets. Vari is not expressed in the embryonic central nervous system, but is expressed apically in the neuroepithelium of the optic lobes and in neuronal cell bodies. These structures do not have pSJs, and indicate that there are uncharacterised functions of Vari, distinct from a role in the assembly of cell junctions (Moyer, 2008).

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).


REFERENCES

Search PubMed for articles about Drosophila Varicose

Bachmann, A., Draga, M., Grawe, F. and Knust, E. (2008). On the role of the MAGUK proteins encoded by Drosophila varicose during embryonic and postembryonic development. BMC Dev. Biol. 8: 55. PubMed ID: 18485238

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

Beitel, G. J. and Krasnow, M. A. (2000). Genetic control of epithelial tube size in the Drosophila tracheal system. Development 127: 3271-3282. PubMed ID: 10887083

Laval, M., Bel, C. and Faivre-Sarrailh, C. (2008). The lateral mobility of cell adhesion molecules is highly restricted at septate junctions in Drosophila. BMC Cell Biol. 9: 38. PubMed ID: 18638384

Luschnig, S., Batz, T., Armbruster, K. and Krasnow, M. A. (2006). serpentine and vermiform encode matrix proteins with chitin binding and deacetylation domains that limit tracheal tube length in Drosophila. Curr. Biol. 16(2): 186-94. PubMed ID: 16431371

McGee, A. W., et al. (2001). Structure of the SH3-guanylate kinase module from PSD-95 suggests a mechanism for regulated assembly of MAGUK scaffolding proteins. Mol. Cell 8: 1291-1301. PubMed ID: 11779504

Moyer, K. E. and Jacobs, J. R. (2008). Varicose: a MAGUK required for the maturation and function of Drosophila septate junctions. BMC Dev. Biol. 8: 99. PubMed ID: 18847477

Shingai, T., et al. (2003). Implications of nectin-like molecule-2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 in cell-cell adhesion and transmembrane protein localization in epithelial cells. J. Biol. Chem. 278: 35421-35427. PubMed ID: 12826663

Tavares, G. A., Panepucci, E. H. and Brunger, A. T. (2001). Structural characterization of the intramolecular interaction between the SH3 and guanylate kinase domains of PSD-95. Mol. Cell 8: 1313-1325. PubMed ID: 11779506

Tseng T.-C., et al. (2001). VAM-1: a new member of the MAGUK family binds to human Veli-1 through a conserved domain. Biochem. Biophys. Acta 1518: 249-259. PubMed ID: 11311936

Wang, S., et al. (2006). Septate-junction-dependent luminal deposition of chitin deacetylases restricts tube elongation in the Drosophila trachea. Curr. Biol. 16(2): 180-5. PubMed ID: 16431370

Wu, V. M., Schulte, J., Hirschi, A., Tepass, U. and Beitel, G. J. (2004). Sinuous is a Drosophila claudin required for septate junction organization and epithelial tube size control. J. Cell Biol. 164: 313-323. PubMed ID: 14734539

Wu, V. M., et al. (2007). Drosophila Varicose, a member of a new subgroup of basolateral MAGUKs, is required for septate junctions and tracheal morphogenesis. Development 134(5): 999-1009. PubMed ID: 17267446


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date revised: 25 October 2009

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