Gliotactin

Gene name - Gliotactin

Synonyms -

Cytological map position - 35E1-2

Function - maintains blood-nerve barrier

Keywords - glial, junctions

Symbol - Gli

FlyBase ID:FBgn0001987

Genetic map position - 2-[51]

Classification - transmembrane protein

Cellular location - surface



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Khodaei, Z. S., Barmchi, M. P., Gilbert, M. M., Samarasekera, G., Fulga, T. A., Van Vactor, D. and Auld, V. J. (2016). The tricellular junction protein Gliotactin auto-regulates mRNA levels via BMP signaling induction of miR-184. J Cell Sci [Epub ahead of print]. PubMed ID: 26906422
Summary:
Epithelial bicellular and tricellular junctions are essential for establishing and maintaining permeability barriers. Tricellular junctions are formed by the convergence of three bicellular junctions at the corners of neighbouring epithelia. Gliotactin, a member of the Neuroligin family, is located to the Drosophila tricellular junction and is critical for the formation of tricellular and septate junctions and permeability barrier function. Gliotactin protein levels are tightly controlled by tyrosine phosphorylation and endocytosis. Blocking endocytosis or overexpression of Gliotactin triggers spread away from the tricellular junction, resulting in apoptosis, delamination and migration of epithelial cells. This study shows that Gliotactin levels are also regulated at the mRNA level by microRNA-mediated degradation targeted to a short region in the 3'UTR that includes a conserved miR-184 target site. miR-184 also targets a suite of septate junction proteins including Neurexin-IV, coracle and Mcr. miR-184 expression is triggered when Gliotactin is overexpressed leading to activation of the BMP signaling pathway. Gliotactin specifically interferes with Dad, an inhibitory SMAD, leading to activation of the Tkv type-I receptor, and Mad to elevate the biogenesis and expression of miR-184.
Resnik-Docampo, M., Koehler, C. L., Clark, R. I., Schinaman, J. M., Sauer, V., Wong, D. M., Lewis, S., D'Alterio, C., Walker, D. W. and Jones, D. L. (2017). Tricellular junctions regulate intestinal stem cell behaviour to maintain homeostasis. Nat Cell Biol 19(1): 52-59. PubMed ID: 27992405
Summary:
Ageing results in loss of tissue homeostasis across taxa. In the intestine of Drosophila melanogaster, ageing is correlated with an increase in intestinal stem cell (ISC) proliferation, a block in terminal differentiation of progenitor cells, activation of inflammatory pathways, and increased intestinal permeability. However, causal relationships between these phenotypes remain unclear. This study demonstrates that ageing results in altered localization and expression of septate junction proteins in the posterior midgut, which is quite pronounced in differentiated enterocytes (ECs) at tricellular junctions (TCJs). Acute loss of the TCJ protein Gliotactin (Gli) in ECs results in increased ISC proliferation and a block in differentiation in intestines from young flies, demonstrating that compromised TCJ function is sufficient to alter ISC behaviour in a non-autonomous manner. Blocking the Jun N-terminal kinase signalling pathway is sufficient to suppress changes in ISC behaviour, but has no effect on loss of intestinal barrier function, as a consequence of Gli depletion. This work demonstrates a pivotal link between TCJs, stem cell behaviour, and intestinal homeostasis and provides insights into causes of age-onset and gastrointestinal diseases.
Samarasekera, G. and Auld, V. J. (2017). C-terminal Src kinase (Csk) regulates the tricellular junction protein Gliotactin independent of Src. Mol Biol Cell 29(2):123-136. PubMed ID: 29167383
Summary:
Tricellular junctions (TCJs) are uniquely placed permeability barriers formed at the corners of polarized epithelia where tight junctions (TJ) in vertebrates or septate junctions (SJ) in invertebrates from three cells converge. Gliotactin is a Drosophila TCJ protein and loss of Gliotactin results in SJ and TCJ breakdown, and permeability barrier loss. When overexpressed, Gliotactin spreads away from the TCJs resulting in disrupted epithelial architecture including over-proliferation, cell delamination and migration. Gliotactin levels are tightly controlled at the mRNA level and at the protein level through endocytosis and degradation triggered by tyrosine phosphorylation. This study identified C-terminal Src kinase (Csk) as a tyrosine kinase responsible for regulating Gliotactin endocytosis. Increased Csk suppresses the Gliotactin overexpression phenotypes by increasing endocytosis. Loss of Csk causes Gliotactin to spread away from the TCJ. Although Csk is known as a negative regulator of Src kinases, the effects of Csk on Gliotactin are independent of Src, and likely occurs through an adherens junction (AJ) associated complex. Overall, this study identified a new Src-independent role for Csk in the control of Gliotactin, a key tricellular junction protein.
Resnik-Docampo, M., Sauer, V., Schinaman, J. M., Clark, R. I., Walker, D. W. and Jones, D. L. (2018). Keeping it tight: The relationship between bacterial dysbiosis, septate junctions, and the intestinal barrier in Drosophila. Fly (Austin): 1-7. PubMed ID: 29455581
Summary:
Maladaptive changes in the intestinal flora, typically referred to as bacterial dysbiosis, have been linked to intestinal aging phenotypes, including an increase in intestinal stem cell (ISC) proliferation, activation of inflammatory pathways, and increased intestinal permeability. However, the causal relationships between these phenotypes are only beginning to be unravelled. Age-related changes have been characterized that occur to septate junctions (SJ) between adjacent, absorptive enterocytes (EC) in the fly intestine. Changes could be observed in the overall level of SJ proteins, as well as the localization of a subset of SJ proteins. Such age-related changes were particularly noticeable at tricellular junctions (TCJ). Acute loss of the Drosophila TCJ protein Gliotactin (Gli) in ECs led to rapid activation of stress signalling in stem cells and an increase in ISC proliferation, even under axenic conditions; a gradual disruption of the intestinal barrier was also observed. The uncoupling of changes in bacteria from alterations in ISC behaviour and loss of barrier integrity has allowed exploration of the interrelationship of these intestinal aging phenotypes in more detail and has shed light on the importance of the proteins that contribute to maintenance of the intestinal barrier.
Wang, Z., Bosveld, F. and Bellaiche, Y. (2018). Tricellular junction proteins promote disentanglement of daughter and neighbour cells during epithelial cytokinesis. J Cell Sci. Pubmed ID: 29739875
Summary:
In epithelial tissue, new cell-cell junctions are formed upon cytokinesis. To understand junction formation during cytokinesis, this study explored in Drosophila epithelium, de novo formation of tricellular septate junctions (TCJs). Upon midbody formation, the membranes of the two daughter cells and of the neighbouring cells located below the adherens junction (AJ) remain entangled in a 4-cell structure apposed to the midbody. The septate junction protein Discs-Large and components of the TCJ, Gliotactin and Anakonda accumulate in this 4-cell structure. Subsequently, a basal movement of the midbody parallels the detachment of the neighbouring cell membranes from the midbody, the disengagement of the daughter cells from their neighbours and the reorganisation of TCJs between the two daughter cells and their neighbouring cells. While the movement of midbody is independent of the Alix and Shrub abscission regulators, the loss of Gliotactin or Anakonda function impedes both the resolution of the connection between the daughter-neighbour cells and midbody movement. TCJ proteins therefore control an additional step of cytokinesis necessary for the disentanglement of the daughter cells and their neighbours during cytokinesis.
BIOLOGICAL OVERVIEW

Gliotactin is a transmembrane protein expressed on glial cells. Its function results in proper protection of neurons from the high concentration of K+ in the hemolymph, thus ensuring a secure blood-brain barrier. Lack of proper insulation between nerves and hemolymph can cause a failure of synaptic transmission. Gliotactin mutant embryos are partially paralyzed and show no coordinated parastaltic movement. This is a consequence of the failure of a glial maintained peripheral blood-nerve barrier, resulting from a defect in the envelopment of neural cells by glia during development (Auld, 1995).

Septate junctions (SJs), similar to tight junctions, function as transepithelial permeability barriers. As a cholinesterase-like molecule that is necessary for blood-nerve barrier integrity, Gli may contribute to SJ development or function. To address this hypothesis, Gli expression and the Gli mutant phenotype in Drosophila epithelia were analyzed. In Gli mutants, localization of SJ markers Neurexin-IV, Discs large, and Coracle are disrupted. SJ barrier function is lost as determined by dye permeability assays. These data suggest that Gli is necessary for SJ formation. Surprisingly, Gli distribution only colocalizes with other SJ markers at tricellular junctions, suggesting that Gli has a unique function in SJ development. Ultrastructural analysis of Gli mutants supports this notion. In contrast to other SJ mutants in which septa are missing, septa are present in Gli mutants, but the junction has an immature morphology. A model is proposed, whereby Gli acts at tricellular junctions to bind, anchor, or compact SJ strands apically during SJ development (Schulte, 2003).

Permeability barriers have important roles in many tissues in both vertebrates and invertebrates. In insects, the high potassium concentration of the hemolymph can block action potentials in neurons, and, thus, cause paralysis, if the blood-brain barrier is disrupted. Epithelial permeability barriers are formed by tight junctions (TJs) in chordates and by septate junctions (SJs) in most invertebrates. TJs and SJs differ in their ultrastructure, position in epithelial cells, and molecular composition, yet they share certain organizational similarities that enables them to form effective permeability barriers (Schulte, 2003 and references therein).

SJs are located in the apical portion of the lateral membrane of invertebrate epithelial cells, immediately below adherens junctions (AJs). TJs in contrast, lie apical to AJs in vertebrate epithelial cells. SJs are characterized by a ladder-like array of cross-bridges or septa that span the 15-20-nm intermembrane space of cell-cell contacts. TJs, in contrast, appear as multiple 'kissing-points', in transmission electron micrographs where adjacent plasma membranes are in direct contact. Both SJs and TJs are composed of multiple strands with some variation in strand number depending on cell type. For example, 10 or more strands typically compose an SJ in a locust epithelial cell, whereas in mammals 4-7 strands are found in a TJ of kidney distal tubule epithelial cell. SJ strands are tightly arrayed parallel to each other, whereas TJ strands are less compact and are organized into overlapping or anastomizing networks. The multi-stranded composition of both SJs and TJs appears to be necessary to effectively block the paracellular flow of substances. In insects, permeability studies have shown that heavy metal tracer dyes are often able to penetrate deep into the stacked arrays of SJ strands before they are blocked from paracellular passage (Swales and Lane, 1985). Similarly, studies of 'tight' and 'leaky' TJs in vertebrate epithelia have shown a positive correlation between strand number and TJ permeability (Schulte, 2003 and references therein).

In Drosophila, two types of SJs, smooth (sSJs) and pleated SJs (pSJs), have been observed. Smooth SJs and pSJs vary morphologically and have different tissue distributions, but they are functionally equivalent. In freeze-fracture electron micrographs, pSJ strands appear to lie in membrane depressions or grooves that are absent in sSJs. pSJs are found in ectodermally derived tissue, such as the foregut, hindgut, tracheae, and glia, whereas sSJs are found in endodermally derived tissue, such as the midgut (Schulte, 2003 and references therein).

TJs and SJs have been described to encircle epithelial cells as a continuous belt, though this view has been challenged. EM studies of epithelial cells, in both vertebrates and insects, have shown that the continuity of TJ and SJ belts is interrupted at sites of tricellular contact by 'pores' or 'channels' that span the depth of the epithelium. It is unclear what the function of these specialized structures may be. In insects, it has been proposed that diaphragms associated with these channels may serve as anchors for SJ strands. Similarly, developmental studies on rat olfactory epithelium suggest that de novo synthesis of TJ strands may occur at sites of tricellular contact. Recent studies on human umbilical vein cultures have suggested that the localized disruption of TJs at endothelial tricellular corners is important during acute immune responses as this enables neutrophils to migrate across capillaries and reach sites of inflammation or infection (Schulte, 2003 and references therein).

To more thoroughly appreciate the similarities and differences between vertebrate TJs and Drosophila SJs, a molecular characterization of these junctions has been performed over the last decade. Of the Drosophila SJ-associated proteins, four have clear roles in SJ formation. Mutations in neurexin-IV (Nrx), discs large (Dlg), scribble (Scrib), or coracle (Cora) prevent the formation of septa. Nrx is a transmembrane protein and a member of the neurexin family of synapse-associated proteins. Dlg and Scrib are cytosolic, PSD-95/Dlg/ZO-1 (PDZ) domain-containing proteins that also have roles in establishing epithelial cell polarity before SJ development. Cora is a band 4.1-related protein that possesses a four point one/ezrin/vadixin/moesin domain and physically associates with Nrx. In the case of vertebrate TJs, at least 25 claudins have been identified that are believed to play critical roles in TJ development. Claudins can interact in a homophilic or heterophilic fashion, and their mixing ratio is believed to moderate the permeability of TJ transepithelial barriers. In addition, numerous other transmembrane or cytoplasmic factors have been found to be associated with TJs (Schulte, 2003 and references therein).

No significant similarities between the molecular composition of vertebrate TJs and Drosophila SJs have been noted so far. Moreover, vertebrate homologs of Drosophila Nrx and Cora have been identified that localize to mammalian paranodal junctions, at the interface of axons and glia. These junctions are morphologically very similar to Drosophila SJs. It is important to more thoroughly characterize SJs, TJs, and paranodal junctions, in order to establish a better understanding of the relationship between these junction types and to gain insight into the mechanism of permeability barrier formation (Schulte, 2003 and references therein).

Gliotactin (Gli) is a noncatalytically active cholinesterase-like molecule that is a member of a class of adhesion proteins termed the electrotactins (Auld, 1995; Botti, 1998). In the peripheral nervous system, Gli is necessary for glial ensheathment of axons, and for the formation of the glial-based blood-nerve barrier (BNB; Auld, 1995). Although pSJs between glial wraps constitute the molecular seal of the BNB, it has not been determined if the BNB defects seen in Gli mutants arises from defective pSJ development or from inadequate axonal ensheathment (Schulte, 2003 and references therein).

Gli shows a tissue distribution pattern similar to that of the pSJ proteins Cora and Nrx. However, the subcellular distribution of Gli varies from other known pSJ proteins in that Gli is restricted to tricellular junctions. Three lines of evidence suggest that Gli is necessary for pSJ formation: (1) the pSJ markers Dlg, Cora, and Nrx are mislocalized in Gli mutant epithelial cells; (2) Gli mutants do not form effective transepithelial barriers as determined through a salivary gland dye permeability assay; (3) ultrastructural analysis indicates that pSJs are malformed in Gli mutants (Schulte, 2003).

The mutant phenotype of Gli, in many respects, is similar to that of Nrx and Cora, however, there are also clear differences between these mutants. In all mutants, SJ markers are mislocalized, but apical-basal polarity appears unaffected. All mutants have dorsal closure defects: whereas in Nrx and Cora mutants dorsal closure fails, and is accompanied with prominent cuticular defects, in Gli mutants, dorsal closure is slightly delayed at a low frequency but eventually completed. The most striking difference between Gli and Nrx/Cora mutants is that at the ultrastructural level they have distinct pSJ morphologies. In Nrx/Cora mutants, septa fail to form, whereas in Gli mutants, a normal number of pSJ strands form, but they are not tightly arrayed. This suggests that Nrx/Cora are necessary for pSJ strand synthesis, whereas Gli appears to be required for the maturation of the pSJ, which involves the compaction of SJ strands. pSJs in wild-type embryos, at stage 14 of development, have a morphology that resembles that of late stage Gli mutants. Evidently, compaction of pSJ septa is essential to form an effective transepithelial barrier because Gli mutants have leaky salivary glands. This result is in agreement with findings in locust that tracer dyes are often able to cross individual septa, as well as small groups, but that the wild-type distribution of multiple large groups of septa do form effective permeability barriers. It is also possible that the detachment of lateral membranes observed in Gli mutants may contribute to a compromised permeability barrier (Schulte, 2003).

Several ultrastructural studies of various insects, including Drosophila, have demonstrated specialized structures at the tricellular corners of epithelial cells that are linked to SJs. A detailed freeze-fracture EM analysis of epithelial tissue has been performed in crustaceans and cockroaches and channels have been identified at the tricellular corners of abutting epithelial cells. These channels span the length of the cells. In the region of the SJ domain, the channels are filled with what appears to be a series of diaphragms that are stacked on top of each other. The diaphragms make contact with the lateral membranes of all three epithelial cells comprising a channel. In the vicinity of the tricellular corners, SJ strands run parallel to the axis of the channel. This organization is different from that of SJ strands elsewhere in the cell. SJ strands typically run parallel to the axis of the apical membrane domain. It has been suggested that SJ strands anchor on the stacked arrays of diaphragms at tricellular corners. These structures have been referred to as tricellular plugs (TCPs), and it has been suggested that they serve as occlusive devices during transepithelial barrier formation in addition to acting as anchors for SJ strands (Schulte, 2003 and references therein).

The development of pSJs occurs in a step wise process. Early in pSJ development, intermembrane particles (IMPs), the building blocks of SJ strands, are homogeneously distributed throughout the lateral membrane. They polymerize at random sites in the lateral membrane (in small depressions) to form short pSJ strands. These, in turn, lengthen and 'stack' to form pSJ placodes, which eventually anchor on TCP diaphragms. TCPs are not observed early in pSJ development and, thus, they are believed to be mature features of pSJs. Observations that Gli is localized to the tricellular corners of epithelial cells, and that it is necessary for pSJ maturation are consistent with this TCP model. EM images, which have shown that TCP diaphragms are associated with pSJ strands in the apical half of tricellular channels, agree with the observations that Gli is restricted to the apical half of tricellular corners. These data suggest that Gli is an integral component of TCPs, a notion that could be confirmed by future immuno-EM experiments (Schulte, 2003 and references therein).

Combining the TCP model with EM observations, and the analysis of Gli, a model is proposed to suggest how Gli is involved in the formation of pSJ. During SJ development, Gli may serve as a linker between SJ strands and tricellular channel diaphragms (TCDs). Gli has the potential to associate with TCDs, through its extracellular domain and with SJ strands through its intracellular tail. Nrx and Cora (and possibly Dlg and Scrib) could be integral components of pSJ strands and may represent the pSJ intermembrane particles. Nrx and Cora behave like IMPs in that they are homogeneously distributed in the lateral membranes during stage 12 of embryogenesis, and are concentrated in the apical half of the lateral membrane at stage 15. It is possible that Gli could physically associate with Nrx and, thus, be linked to SJ strands, because Gli-like vertebrate neuroligins have been shown to bind to neurexin-1ß via their cholinesterase-like domains (Ichtchenko, 1995). However, Nrx is more similar to the alpha-neurexins than the ß-neurexins, and the latter is not known to bind neuroligins (Ichtchenko, 1995; Schulte, 2003 and references therein). Gli and Nrx also fail to interact in S2 cell aggregation assays. Alternatively, Gli may associate through its intracellular domain with SJ strands since Gli contains a COOH-terminal PDZ recognition sequence, and Dlg and Scrib are PDZ domain proteins. In vertebrates, it has been shown that PSD-95 (a Dlg-related protein) binds the intracellular tail of various neuroligins (Irie, 1997). Irrespective of the mechanism, the results reported in this study support the notion that Gli is physically associated with SJ strands, since Dlg, Nrx, and Cora fail to be confined to the pSJ domain within the lateral membrane, and pSJ strands are unorganized in Gli mutant epithelial cells (Schulte, 2003).

Many interesting questions remain to be addressed regarding the specific role of Gli in TCP and pSJ development. Is Gli only necessary to anchor pSJ strands to TCP diaphragms, or is it also directly involved in the formation of the TCP diaphragms? The molecular nature of the diaphragms is not known. One possibility is that the diaphragms are composed of secreted molecules that are linked via Gli to the pSJ strands. This linkage may be critical for the compaction of SJ strands (Schulte, 2003).

Tricellular channels are not features unique to the insect epithelium. The organization of TJs at tricellular corners is strikingly similar to that of SJs in insects. It will be interesting to determine if any of the vertebrate neuroligins are localized to the tricellular corners of epithelial cells, and if they have a role in TJ maturation similar to Gli's role in SJ development. Of particular interest are human Neuroligins 3 and 4, and rat neuroligins 2 and 3, which are not nervous system-specific and which have a broader tissue distribution than other vertebrate neuroligins. Given the structural and molecular similarities between Drosophila SJs and mammalian paranodal junctions, it will also be interesting to determine if neuroligins, or tricellular junctions are present at axon-glial contacts. However, at least two neuroligins, 1 and 3, are not found at paranodal junctions in mice or rat: rather, neuroligin 3 appears to be associated with nonmyelinating Schwann cells (Schulte, 2003 and references therein).


GENE STRUCTURE
Bases in 5' UTR - 178

Exons - three or more. The first intron interrupts the 3'UTR.


PROTEIN STRUCTURE

Amino Acids - 956

Structural Domains

The extracellular domain consists of 200 amino acids. There is a putative signal sequence ensuring transit of the extracellular part of the protein through the membrane during protein synthesis (Auld, 1995).

Evolutionary Homologs

The extracellular domain has sequence similarity to the serine esterase family of proteins. This sequence motif is shared by Glutactin and Neurotactin, and both human and fly acetylcholine esterase (Auld, 1995).


REGULATION

Protein Interactions

One essential function of epithelia is to form a barrier between the apical and basolateral surfaces of the epithelium. In vertebrate epithelia, the tight junction is the primary barrier to paracellular flow across epithelia, whereas in invertebrate epithelia, the septate junction (SJ) provides this function. New proteins have been identified that are required for a functional paracellular barrier in Drosophila. In addition to the previously known components Coracle (Cora) and Neurexin (Nrx), four other proteins [Gliotactin, Neuroglian (Nrg), and both the alpha and ß subunits of the Na+/K+ ATPase] are required for formation of the paracellular barrier. In contrast to previous reports, it is demonstrated that the Na pump is not localized basolaterally in epithelial cells, but instead is concentrated at the SJ. Data from immunoprecipitation and somatic mosaic studies suggest that Cora, Nrx, Nrg, and the Na+/K+ ATPase form an interdependent complex. Furthermore, the observation that Nrg, a Drosophila homolog of vertebrate neurofascin, is an SJ component and is consistent with the notion that the invertebrate SJ is homologous to the vertebrate paranodal SJ. These findings have implications not only for invertebrate epithelia and barrier functions, but also for understanding of neuron-glial interactions in the mammalian nervous system (Genova, 2003).

The SJ has historically been thought of as an invertebrate-specific junction; however, recent studies of the vertebrate nervous system have identified a junction that is both molecularly and structurally homologous, the paranodal SJ (PSJ) (Einheber, 1997; Tepass, 2001). The PSJ occurs between neurons and the glial cells that myelinate them, the oligodendrocytes and Schwann cells. Each glial cell wraps around and contacts the neuron multiple times in a spiral pattern to form the paranodal loops. The PSJ forms between the paranodal loops and the neuron and keeps the node of Ranvier distinct from the internodal region by providing a seal between the neuron and glial cell. This seal provides a barrier within the neuronal membrane that separates Na+ channels at the node of Ranvier from K+ channels under the glial cells, and a paracellular diffusion barrier between the neuron and the ensheathing glial cell. Consistent with these structural and functional similarities, the invertebrate epithelial SJ and the vertebrate PSJ also display similarities at the molecular level. Caspr (contactin-associated protein; also known as paranodin), a mammalian homolog of Nrx, is located on the neuronal face of the PSJ (Einheber, 1997), where it interacts with protein 4.1 (Menegoz, 1997), which is homologous to Drosophila Cor (Genova, 2003).

To identify additional components of the Drosophila SJ, a collection of P element insertion mutations was screened for a phenotype attributable to a loss of the paracellular barrier. Two genes, Na pump alpha subunit (Atpalpha) and Nervana 2 (Nrv2), which encodes the ß subunit of the Na+/K+ ATPase) were identified as essential for the barrier function of the SJ. In addition, Neuroglian (Nrg), which is homologous to known components of the PSJ, and Gli, which is necessary for the blood-brain barrier, were tested and found to be necessary for the paracellular barrier. Direct immunostaining, epitope-tagged expression constructs, and GFP-tagged proteins indicate that Nrv2, ATPalpha, and Nrg localize to the SJ, and that they are interdependent for this localization. In keeping with this finding, the existence of a protein complex containing Cora, Nrx, Nrg, and Nrv is demonstrated. Taken together, these results suggest a novel complex involving the Na+/K+ ATPase that is necessary for establishing and maintaining the primary paracellular barrier in invertebrate epithelia, the SJs. Thus these studies provide new insights into the structure and function of SJs in both invertebrate epithelial cells and in the homologous PSJ of the vertebrate nervous system (Genova, 2003).

A novel approach has been used to identify components of the pleated SJ, which provides the barrier to paracellular diffusion in Drosophila epithelial cells. Three independent lines of evidence indicate that the proteins encoded by these genes are essential to the structure and function of epithelial SJs. (1) Mutations in all four identified loci, Nrg, Gli, Nrv2, and Atpalpha, disrupt the paracellular barrier of the salivary gland epithelium and alter the ultrastructure of epithelial SJs. (2) The proteins encoded by three of these genes localize to the region of the SJ as judged by antibody staining of fixed tissues and observation of GFP-tagged proteins expressed in living epithelial cells (reagents were unavailable for observations of the fourth protein, Gli). (3) Somatic mosaic studies and IP experiments indicate that these proteins form an interdependent complex at the SJ. This complex also includes two previously identified SJ components, Nrx, a transmembrane protein, and Cora, a membrane-associated cytoplasmic protein with a FERM domain (Genova, 2003).

One of the most intriguing results of this study is the identification of the Na+/K+ ATPase as a functional member of the SJ. Mutations in either the alpha subunit (ATPalpha) or ß subunit (NRV2) disrupt the paracellular barrier of the embryonic salivary gland and this functional loss corresponds to the structural loss of septae in the junction. Although the SJ is localized just basal to the adherens junction near the apical end of the cell, previous characterizations of the Na+ pump have described it as having a basolateral localization. The localization of the Na+/K+ ATPase was examined using immunofluorescence; both subunits are found highly concentrated at the SJ in imaginal epithelia. In embryonic epithelia, the results differed depending upon the fixation and staining method; methanol treatment resulted in staining that appeared basolateral whereas staining of embryos fixed without methanol was localized to the SJ. Observations of GFP-exon trap lines enabled the confirmation that both ATPase subunits localize to the SJ in live embryos and imaginal epithelia. These results are limited to the examination of ectodermally derived epithelia such as the embryonic epidermis, foregut, hindgut, and salivary glands. Interestingly, the midgut does not contain pleated SJs but rather smooth SJs, and so observed differences in subcellular localization of the ATPase may be cell type dependent (Genova, 2003).

The Nrv2 and Nrv1 genes encode ß subunits of the Na+/K+ ATPase that differ in their cytoplasmic tails. The P-element insertion (l(2)k13315) disrupts the Nrv2 gene product but appears to have no affect on the Nrv1 protein. In addition, both NRV2.1 and NRV2.2 are able to rescue the dye diffusion phenotype of l(2)k13315 whereas NRV1 is not. Together these results indicate that l(2)k13315 is a mutation in the Nrv2 locus, and that NRV2 normally functions in the SJ. Although both NRV2 and NRV1 were previously described as being nervous system specific, evidence from immunostaining and from a GFP gene trap inserted within the Nrv2 locus indicates that Nrv2 is highly expressed in epithelial cells. Because NRV1 expression is not affected by the l(2)k13315 mutation and l(2)k13315 homozygous mutant cells in the wing imaginal disc lack NRV staining, it is proposed that Nrv1 is nervous system specific and epidermal cells express only NRV2 (Genova, 2003).

The observation that an Nrv1 transgene cannot rescue the Nrv2 dye diffusion phenotype, even though it localizes to the SJ when ectopically expressed in epithelial cells, suggests that the proteins encoded by these genes, although quite similar in structure, are functionally distinct. Given the sequence diversity within the cytoplasmic tail, the observation that when expressed ectopically all three proteins localize to the SJ strongly suggests that this localization is mediated by the extracellular or transmembrane domain, rather than by the intracellular domain. This complex pattern of ß subunit expression and functional interactions suggests a surprising degree of functional regulation of the Na+/K+ ATPase in epithelial and neuronal cells (Genova, 2003).

The question still remains, What is the function of localizing the Na pump to such a specialized membrane domain, one of whose functions is to create a paracellular diffusion barrier? Several characteristics of the Na pump might be important in SJ function. Previous studies suggest that the Na+/K+ ATPase functions in cell adhesion, though whether its role is structural or regulatory is unclear. Other studies suggest that the Na pump could function as a scaffold on which proteins essential for the paracellular barrier are organized. For example, both subunits bind to a variety of proteins, from those involved in signal transduction to cytoskeletal elements. In addition, it is possible that the ion pumping activity of the Na pump actively participates in the formation or maintenance of the diffusion barrier. Studies in mammalian cells have demonstrated a requirement for the ATPase, and specifically the Na+ gradient it produces, in cell polarity, adhesion, and the formation of tight junctions. Because the tight junction is responsible for creating the paracellular barrier in vertebrate epithelial cells, the ATPase might perform a similar function in the paracellular barrier of the Drosophila SJ. Further experiments, using point mutations that specifically affect the pump function of the ATPase, could address these questions (Genova, 2003 and references therein).

Cora has been shown to bind to the cytoplasmic tail of Nrx in the SJ. Studies of the PSJ have shown that the mammalian homologs of Nrx and Nrg interact via their extracellular domains. Together, these observations suggest the existence of a multiprotein complex at the SJ in which Cora binds to Nrx, which in turn binds to Nrg. The finding that Nrx and Nrg coimmunoprecipitate when either anti-Cora or anti-Nrg antibodies are used to immunoprecipitate is consistent with this model. Because Drosophila epithelial cells express all three proteins, it is not possible to rigorously distinguish whether this interaction occurs within the same cell or between adjacent cells. However, the observation that wild-type cells are unable to efficiently assemble Cora and Nrx at the boundary with cor- cells suggests that intercellular interaction with the same complex on adjacent cells is required for SJ assembly. In addition, Nrv is found to coimmunoprecipitates with both Cora and Nrx. Nrg has not been detected in this complex, suggesting that the interaction between NRV2 and the Cora-Nrx complex occurs independently of Nrg, perhaps on the cytoplasmic side of the membrane. Although these results imply the possibility of an interaction between Cora and the cytoplasmic tail of NRV2, this seems unlikely in light of observations that NRV1, 2.1, and 2.2 all localize to the SJ, despite having different cytoplasmic tails. Thus, it is more likely that the interaction between Cora and the ATPase occurs either through Nrx or the alpha subunit (Genova, 2003).

Somatic mosaic analysis has demonstrated that this complex of Cora, Nrx, Nrv, ATPalpha, and Nrg can be disrupted without affecting overall polarity, or other components of the SJ. No component essential for the paracellular barrier has been identified that is unaffected in mutant cells, suggesting that the substrate upon which this complex assembles has yet to be found. Previous studies have demonstrated that Ankyrin binds both the cytoplasmic domain of Nrg and, as has been described in mammalian cells, the alpha subunit of NA+/K+ ATPase. In addition, Ankyrin colocalizes with Nrg at points of Nrg-induced S2 cell adhesion complexes. Thus, one candidate for a substrate upon which this complex assembles is Ankyrin, a well-known member of the membrane skeleton (Genova, 2003).

Other candidate proteins for this scaffold are Scribble and Dlg. Both of these proteins are required early in Drosophila development for the establishment of epithelial cell polarity and growth control. If either is absent from epithelial cells, then the apical junctional complexes do not properly form and epithelial integrity is lost. Thus, Scribble and Dlg may be among the first constituents of the SJ upon which the subsequently expressed SJ proteins assemble (Genova, 2003).

Previous studies have suggested that the SJ may function in intercellular signaling, particularly in the regulation of cell proliferation. For example, dlg, which encodes a PDZ repeat-containing, membrane-associated guanylate kinase protein, has tumor suppressor functions. Loss of function dlg mutations are characterized by disruption of apical-basal polarity and an overproliferation of the larval imaginal discs. However, it is not known whether this overproliferation is due to a direct involvement of Dlg in a signal transduction cascade or to the disruption of apical-basal polarity within epithelial cells that could result in a disruption of apical signaling complexes. In addition to dlg, cor mutations were first isolated as dominant suppressors of a gain of function allele of the EGF receptor, EgfrElp (also known as EgfrE3), suggesting that Cora may function to positively regulate EGFR pathway function. Interestingly, a recent study of Nrg function in the developing Drosophila nervous system has proposed that it positively regulates EGF receptor function during axon guidance. The role of Nrg in regulating EGFR function in epithelial cells has not been investigated, but preliminary results indicate that Nrg mutations also dominantly suppress the rough eye caused by EgfrElp. This result may suggest that Nrg (or the entire complex) must be localized to the SJ in epithelial cells to regulate Egfr function. Alternatively, it is possible that the SJ complex is necessary to maintain polarized localization of the Egfr to the apical membrane, though no effect of cor mutations on Egfr localization has been observed (Genova, 2003).

The recent discovery of molecular, structural, and functional similarities between the invertebrate epithelial SJ and the vertebrate PSJ in the nervous system gives added significance to the identification of new SJ components in Drosophila. In addition to Cora/protein 4.1 and Nrx/paranodin, the SJ and PSJ have been shown to share neurofascin-155 and a Drosophila homolog, Nrg. This level of molecular homology strongly suggests that these two SJs are structurally and functionally homologous as well. It is therefore somewhat surprising that published reports indicate that the Na pump is uniformly distributed along the axonal membrane rather than being restricted to the PSJ (Genova, 2003 and references therein). One possible explanation is that only a subset of the several Na+/K+ ATPase isoforms found in the mammalian genome is localized to the PSJ, and that these isoforms have not yet been studied. Similarly, it is not known if the mammalian homologs of Drosophila Gli, the neuroligins, might localize to the PSJ, or if the Drosophila homolog of contactin, a protein that interacts with Nrx/paranodin, localizes to the SJ. Although it is possible that the invertebrate epithelial SJ and vertebrate PSJ are fundamentally different in some respects, this is unlikely given the remarkable degree of similarity between these two junctions. In any case, it is clear that the genetic and genomic tools available in Drosophila can provide important insights into both the SJ and its vertebrate counterpart, the PSJ (Genova, 2003).

The triple-repeat protein Anakonda controls epithelial tricellular junction formation in Drosophila

In epithelia, specialized tricellular junctions (TCJs) mediate cell contacts at three-cell vertices. TCJs are fundamental to epithelial biology and disease, but only a few TCJ components are known, and how they assemble at tricellular vertices is not understood. This study describes a transmembrane protein, Anakonda (Aka; also known as Bark beetle), which localizes to TCJs and is essential for the formation of tricellular, but not bicellular, junctions in Drosophila. Loss of Aka caused epithelial barrier defects associated with irregular TCJ structure and geometry, suggesting that Aka organized cell corners. Aka is necessary and sufficient for accumulation of Gliotactin at TCJs, suggesting that Aka initiated TCJ assembly by recruiting other proteins to tricellular vertices. Aka's extracellular domain had an unusual tripartite repeat structure that might mediate self-assembly, directed by the geometry of tricellular vertices. Conversely, Aka's cytoplasmic tail is dispensable for TCJ localization. Thus, extracellular interactions, rather than TCJ-directed intracellular transport, appear to mediate TCJ assembly (Byri, 2015).

Epithelial cells are linked via intercellular junctions that provide paracellular diffusion barriers, maintenance of polarity, and cell-to-cell communication. Bicellular junctions (BCJs) make up the most abundant intercellular contacts in epithelia. They connect two neighboring plasma membranes and are organized into distinct complexes along the apical-basal axis, collectively forming the apical junctional belt. However, at certain positions along the cell perimeter, three cell corners meet and the bicellular junctional complex is disjointed. Here, specialized tricellular junctions (TCJs) connect epithelial cells. TCJs play important roles in epithelial barrier functions and cytoskeletal organization, and are preferential sites for trans-endothelial migration of neutrophils and metastatic cancer cells, as well as for the spreading of intracellular pathogens (Byri, 2015).

Due to the geometry of three-cell vertices, the sealing of the epithelium at these sites requires a dedicated junctional organization. Ultrastructural analyses using freeze-fracture electron microscopy (EM) showed that the zonula occludens, tight junctions (TJs) in vertebrates and septate junctions (SJs) in arthropods, changes characteristics when approaching a three-cell vertex. Instead of continuing parallel to the epithelial plane around the cell perimeter, the junctional strands extend basally, forming a bicellular seal along the apical-basal axis. In vertebrates, these structures are termed central sealing elements, and three such parallel TJ extensions enclose a narrow (approximately 10 nm) central canal at each TCJ. Similarly, in invertebrates the SJ strands turn by 90 degrees when approaching a tricellular corner, forming three parallel limiting strands that surround the tricellular juncture space, resembling the central sealing element in vertebrates. Within and perpendicular to the vertical juncture space are a series of diaphragms, which appear linked not only to three limiting septa, but also to the three cell corner membranes, thereby forming true tricellular contacts (Byri, 2015).

Despite the fundamental biological importance of TCJs, only few of their components are known, and the mechanism of their localized assembly at three-cell vertices is not understood. In vertebrates, the Occludin family protein Tricellulin localizes to TCJs and is recruited there by lipolysis-stimulated lipoprotein receptor (LSR) and related proteins (ILDR1 and ILDR2). Additionally, the cytoplasmic PDZ-domain-containing protein Tjp2iso3 associates with Tricellulin in Sertoli cells. The Neuroligin-like transmembrane protein Gliotactin in Drosophila is the only TCJ protein characterized so far in invertebrates. It is not clear what kind of cues direct the accumulation of these proteins at tricellular vertices, and the features of known TCJ proteins do not explain the distinct structure of three-cell contacts observed by EM. This study describes a transmembrane protein, Anakonda (Aka), which accumulates at TCJs in Drosophila epithelia. Aka can initiate TCJ assembly and may do so through its large extracellular domain, which exhibits an unusual triple-repeat structure (Byri, 2015).

Ultrastructural analyses revealed a unique junctional architecture at points in epithelia where three cells meet. Yet, only few TCJ-specific proteins are known, and how they assemble into a tightly localized complex exclusively at tricellular vertices is not understood. This study describes transmembrane protein Anakonda, which is shown to play a critical role in TCJ assembly and epithelial barrier formation. First, Aka was shown to localize to TCJs in ectodermal and endodermal epithelia, suggesting that Aka is a core TCJ component. Second, Aka was demonstrated to be specifically required for the assembly and correct geometry of tricellular, but not of bicellular SJs, and Aka was shown to localize to TCJs independently of bicellular SJs. Third, Aka is required for recruiting or maintaining Gli at tricellular vertices. Conversely, Gli is not required for TCJ localization of Aka. Fourth, Aka mis-expression causes premature accumulation of Gli at TCJs, indicating that Aka acts upstream of Gli in initiating TCJ formation (Byri, 2015).

Aka protein contains a large extracellular domain with a conserved tripartite repeat structure. This unique structure distinguishes Aka from other known TCJ proteins. Tricellulin, a four-pass transmembrane protein in vertebrates, localizes to the central sealing elements, suggesting that it participates in the specialized bicellular contacts that surround each three-cell vertex. Tricellulin is recruited to TCJs by the immunoglobulin domain transmembrane protein LSR, but how LSR localizes to TCJs is still unclear. In Drosophila, Gli, a transmembrane protein with a cholinesterase domain lacking catalytic activity, accumulates at TCJs, but is also involved in bicellular SJ organization. Phosphorylation-dependent endocytic turnover and indirect association with Dlg are required for accumulation of Gli at TCJs. However, the mechanisms underlying TCJ-specific localization are not understood for Gli or any other TCJ protein (Byri, 2015).

The results suggest that Aka acts at an early step during TCJ assembly by recruiting other TCJ components, including Gli, to tricellular vertices. TCJ localization of Aka and Gli does not require Aka’s C terminus, including the PDZ-binding motif. Similarly, Gli was found to localize to TCJs independently of its PDZ-binding motif. These findings suggest that Aka and Gli might be targeted to TCJs through their extracellular domains, rather than through a cytoplasmic localization machinery. Because mislocalized Aka protein is not sufficient to recruit Gli to ectopic locations, Aka might not interact directly with Gli, or only do so in the context of TCJs. Consistent with this notion, we were not able to detect co-immunoprecipitation of Aka and Gli. Interestingly, overexpression of Gli or absence of Aka causes Gli to spread from TCJs to BCJs. Thus, Gli does not have an intrinsic propensity to localize to TCJs, but by default localizes to the apicolateral membrane domain occupied by SJs along the cell perimeter. TCJ accumulation of Gli therefore requires (1) association with the apicolateral membrane and (2) Aka-dependent recruitment of Gli specifically to TCJs. The findings show that unlike Gli, Aka does not depend on bicellular SJs for its localization to TCJs, and suggesting that Aka protein might have intrinsic properties that lead to its accumulation at TCJs (Byri, 2015).

Tricellular vertices display a unique geometry where three plasma membranes are in close proximity and at fixed angles. The exceptional curvature of the plasma membrane at these sites implies distinct physical properties compared to bicellular contacts. Intriguingly, it was found that TCJ assembly depends on Aka expression in all three cells adjoining a vertex, suggesting that extracellular interactions between Aka molecules from different cells are essential for TCJ formation. Considering the size of Aka's extracellular domain (303 kDa), it is likely that a single Aka protein spans the entire TCJ canal (25–30 nm), although shorter Aka isoforms may constitute different structures. Shorter Aka species might carry out functions that may or may not be related to the function of full-length Aka at TCJs. Alternatively, Aka fragments may reflect turnover of TCJ complexes, e.g., during junctional remodeling (Byri, 2015).

It is tempting to speculate that the three repeat regions in full-length Aka protein, with apparent similar domain organization, could make equal contacts with the three cell corners in the plane perpendicular to the central TCJ canal. Possibly, interaction with membrane components in the tricellular region occurs via the three SR domains, since such domains are found in receptors that recognize a wide range of molecular patterns, including surface proteins, carbohydrates, lipids, lipopolysaccharides, and peptidoglycans associated with pathogens or apoptotic cells. Because such three-way contacts may, for steric reasons, only be possible at vertices, they might selectively stabilize Aka complexes at these sites. Computer simulations suggest that the enrichment of Aka at three-cell vertices could theoretically be enhanced by the reduction of dimensionality in the tricellular region, which may promote stacking interactions between Aka molecules within the central TCJ canal. This idea is consistent with the regular structure of the TCJ diaphragms and the equal dihedral angles near TCJs as observed with electron microscopy. Stacking might occur with Aka molecules rotated by 120 or 240 degrees within the stack, perhaps in a helical array, depending on the contributing cell. Notably, such a scenario would explain the finding that TCJ formation requires Aka protein production by all three cells adjoining a vertex. A high priority of future studies will be to investigate the arrangement of Aka proteins at TCJs. Together, the current results on Aka localization, the geometry of three-cell vertices, and the triple-repeat structure of Aka protein suggest a mechanism of TCJ formation, which is promoted by self-assembly of Aka at tricellular contacts. Such self-assembly might additionally involve interactions with other membrane-associated or extracellular components, and could cooperate with bicellular adhesion molecules that zip up bicellular contacts. It will be interesting to test whether the geometry of tricellular vertices and the specific properties of Aka protein are sufficient to direct its accumulation to TCJs. Conversely, perturbed TCJ geometry upon loss of Aka may have long-range effects, such as the loss of cell-cell adhesion that this study observed in late-stage aka embryos. Interestingly, depletion of Tricellulin from mammalian cells affects cell shape and the F-actin network, suggesting that TCJs may in fact have organizing activity on the entire cell (Byri, 2015).

The existence of aka homologs in invertebrates and cephalochordates correlates with the presence of SJs in these groups. However, the proposed self-assembly model for TCJ formation in Drosophila may apply also to vertebrates, although the corresponding proteins remain to be discovered. A better understanding of TCJ assembly will be a key step toward elucidating how these poorly characterized cellular structures provide epithelial barrier function, while at the same time allowing the passage of migrating lymphocytes, metastatic cells, and intracellular pathogens (Byri, 2015).


DEVELOPMENTAL BIOLOGY

Embryonic

Gliotactin is found on peripheral glia from stage 13. By stage 16 Gliotactin is found over the entire surface of peripheral glia. The glia protectively wrap motor nerves from just inside the CNS along much of the length of the axon as it extends peripherally, but wrapping does not extend to axon terminals. Gliotactin is not expressed on glia associated with sensory structures. Gliotactin is also found concentrated in small patches along tracheal trunks and at later stages is associated with the hindgut (Auld, 1995).

Larval

Little or no Gliotactin protein is found on larval peripheral glia. At this stage, glial wrapping extends out to the syntaptic terminals and fully enwraps the motor axons, but the protein is not found associated with these glia at the larval stage (Auld, 1995).

Effects of mutation or deletion

Mutant embryos appear morphologically normal, but show uncoordinated motion, and fail to hatch. Electrophysiological studies show normal axonic transmission in low K+, but failure of transmission in high K+. Electron microscopy reveals incomplete wrapping of peripheral axons by glia. Studies with ruthenium red, used to moniter the integrity of the blood-brain barrier, reveal that the barrier is leaky. Although glial wrappings appear normal in the mutants, there appears to be a failure to properly associate with neighboring glia to complete a glial sheath (Auld, 1995).

Transient apical polarization of Gliotactin and Coracle is required for parallel alignment of wing hairs

In Drosophila, wing hairs are aligned in a distally oriented, parallel array. The frizzled pathway determines proximal-distal cell polarity in the wing; however, in frizzled pathway mutants, wing hairs remain parallel. How wing hairs align has not been determined. A novel role for the septate junction proteins Gliotactin (Gli) and Coracle (Cora) in this process has been demonstrated. Prior to prehair extension, Gli and Cora are restricted to basolateral membranes. During pupal prehair development, Gli and Cora transiently form apical ribbons oriented from the distal wing tip to the proximal hinge. These ribbons are aligned beneath prehair bases and persist for several hours. During this time, Gli is lost entirely from the basolateral domain. A Gliotactin mutation alters the apical polarization Gli and Cora and induces defects in hair alignment in pupal and adult stages. Genetic and cell biological assays demonstrate that Gli and Cora function to align hairs independently of frizzled. Taken together, these results indicate that Gli and Cora function as the first-identified members of a long-predicted, frizzled-independent parallel alignment mechanism. A model is proposed whereby the apical polarization of Gli and Cora functions to stabilize and align prehairs relative to anterior-posterior cell boundaries during pupal wing development (Venema, 2004).

The Gliotactin null phenotype is paralysis and death due to an open blood-nerve barrier at the end of embryogenesis. No adult-viable mutant Gli genotypes have been reported. In order to extend the analysis of Gliotactin function postembryogenesis, a mutagenesis screen was performed and a number of novel, ethylmethane-sulfonate (EMS)-induced Gli alleles were isolated that failed to complement the null allele GliAE2Δ45. One novel allele, Glidv5, was homozygous viable. Sequencing of this allele revealed a single, nonconservative mutation in the extracellular serine esterase-like domain. Several of the novel and previously identified homozygous-lethal Gli alleles generated adult escapers in trans to Glidv5, resolving these alleles into an allelic series. Structural changes underlying this allelic series were determined by sequencing each allele. GliRAR77 was found to contain a nonsense mutation in the intracellular carboxyl-terminal region. This homozygous-lethal allele generated more adult escapers in trans to Glidv5 than Glidv5 itself, indicating that the extracellular and intracellular domains of Gli can function together when on separate proteins. The Glidv1 allele results from a premature stop codon within the serine esterase like domain; this allele escaped in trans to Glidv5 at a low frequency. Two alleles (GliP34 and Glidv3) rarely escaped in trans to Glidv5; both alleles are premature stop codons early in the Gli open reading frame (Venema, 2004).

The availability of Gli genotypes that survive to adult stages allowed an examination of adult epithelia for mutant phenotypes. Adult wings of Gli mutants were examined for defects. Gli mutants display an increasing severity of wing hair orientation defects that parallels the Gli allelic series. The primary Gli wing hair phenotype was disruption of parallel alignment between neighboring hairs such that adjacent hairs converge in a chevron pattern. This phenotype was present in Gli wings not mounted under a coverslip, as well as in Gli wings examined using scanning electron microscopy, and as such is not a mounting artifact. To quantify the Gli phenotype, veins were used to divide the wing into seven main regions, 20 wings of each genotype were scored, and the number of regions that contained patches of nonparallel hairs were counted. The Gli+ chromosome used for mutagenesis had the wild-type pattern of parallel, distally oriented hairs. In weak Gli genotypes, only small numbers of hairs are affected, resulting in small, infrequent mutant patches. Random samples of 20 Glidv5/GliRAR77 or Glidv5/Glidv5 wings ranged from wings with disrupted hairs in every wing region to completely wild-type wings. On average, Glidv5/GliRAR77 wings had 2.9 regions with altered alignment compared to 3.4 for Glidv5/Glidv5 wings. In stronger Gli genotypes, mutant patches were more frequent and larger. All 20 Glidv5/Glidv1 wings sampled had at least one region of disrupted alignment, with an average of 4.1 regions affected. In the severe Glidv5/Glidv3 genotype, mutant patches covered large portions of the wing and were consistently present. In a random sample of 20 Glidv5/Glidv3 wings, eight had an area of disrupted alignment in six wing regions; the remaining 12 wings had disrupted alignment in all seven intervein areas, giving an average of 6.7 disrupted regions per wing. Despite this consistent phenotype, hairs with correct alignment remained in even the most severely affected Glidv5/Glidv3 wings. In addition to nonparallel alignment, patches of deformed hairs were occasionally observed in all adult Gli genotypes, often at the anterior-posterior boundary. Deformed hairs were thinner than normal and were often bent. A third phenotype, blistering at the distal margin, was observed in the strongest Gli genotype, Glidv5/Glidv3. In a sample of 20 Glidv5/Glidv3 wings, 7 contained blisters. Such blisters were always in contact with the distal margin (Venema, 2004).

In Drosophila, septate junctions are initially formed during embryogenesis: these septate junctions persist in epithelia derived from the epidermis, such as imaginal structures. In all previously examined tissues, septate junctions, once formed, appear static in the absence of cell division. In contrast, Gli and Cora are extensively remodeled during wing development at several time points in postmitotic cells. The initial Gli/Cora pattern in the wing before prehair extension is identical to that of the embryonic epidermis. Indeed, Gli and Cora remain stable until after prehair extension at approximately 32 h APF. Beginning at 35 h APF, Gli and Cora are apically polarized at the wing margins. Althought the initial stages of Cora polarization have been noted previously, previous studies did not assay Cora localization past 35 h APF or examine mutant cora genotypes. By 37 h APF, Gli and Cora colocalize to continuous apical ribbons across the pupal wing. These ribbons run along anterior-posterior cell boundaries and between proximal and distal vertices, passing just basal to prehair bases. At this stage, Gli is absent from basolateral membranes, yet Cora retains its basolateral pattern. This pattern remains stable until at least 40 h APF. By 47 h APF, Cora has lost its apical polarization and is present only around the basolateral cell periphery. In contrast, Gli is lost apically and basolaterally with only minimal Gli present on basolateral membranes, which is not predominantly tricellular at 47 h APF. It may be that the weak tricellular staining of Gli at this time point represents the start of relocalizing Gli to the tricellular septate junction; examination of Gli at stages after 47 h APF is underway to test this possibility (Venema, 2004).

The dynamic rearrangement of these junction components is without precedent in the literature. While septate junction components such as Discs-large, Coracle, Neurexin IV, Neuroglian, and the Na+/K+ ATPase are expressed in wing discs, these studies have not examined the localization of septate junction components past 30 h APF nor examined mutant genotypes for wing hair or adhesion phenotypes. Thus, it is an open question whether these septate junction proteins also are dynamic during later stages of wing development. It remains to be seen whether these components are recruited to the apical Gli/Cora ribbon and if their basolateral localization at septate junctions is modified during Gli and Cora polarization (Venema, 2004).

Loss of Dlg function in wing discs causes an overgrowth phenotype that includes loss of apical-basal polarity. In contrast to Dlg, cora null clones do not survive to adult stages. Additionally, clones for null alleles of Nrg and Nrv2 do not survive, consistent with the results that null Gli clones are cell lethal. Given the essential nature of the septate junction for both proliferation control and cell viability, it will not be possible to assess the adult wing phenotype for complete loss of septate-junction function. Attempts to induce null Gli clones after 30 h APF have not proved successful since cell division in the wing ceases around this stage. Null clones induced during the last round of cell division did not survive long enough to examine the start of Gli/Cora polarization at 35 h APF (Venema, 2004).

The polarization of Gli and Cora to apical ribbons is compromised in Gli mutant wings, which in turn leads to unstable prehairs later in development. The close proximity of the Gli ribbon to the prehair base, and the orientation of the ribbon along the axis the hairs will align to, is compelling evidence for a physical connection between prehairs and Gli/Cora ribbons that is required for hair alignment. In Gli mutant wings, the polarization of apical Gli and Cora is incomplete; additionally, it does not persist for the correct length of time. In wild-type wings, Gli and Cora remain polarized until at least 40 h APF; in contrast, in Gli mutant wings Gli is lost altogether by 40 h APF and Cora is no longer polarized. Thus, the Gli nonparallel phenotype seems to result from either a lack of apically polarized Gli/Cora or an insufficient time span of their polarization, or perhaps a combination of both. In either case, the nonparallel phenotype seems to arise from a failure to properly polarize apical Gli and Cora, which in turn leads to prehair instability later in development (Venema, 2004).

Though apical Gli and Cora define the distal-to-proximal axis in the wing, how they are connected to the prehair for its stabilization remains unresolved. The separation between the prehair base and the apical ribbon of Gli and Cora passing beneath it suggests an intermediate link between the two structures. A possible candidate to connect the Gli/Cora ribbon to the prehair pedestal is the cytoskeleton. Physical and genetic links between septate junction components and the cytoskeleton have previously been reported. Both subunits of the Na+/K+ ATPase are localized to the septate junction and physically interact with ankyrin, which acts as an adapter between transmembrane proteins and the spectrin/F-actin network. In Drosophila, the subcellular location of the Na+/K+ ATPase is disrupted in β spectrin mutants. Additionally, Neuroglian clustering assembles ankyrin at cell-cell contacts through physical association. Interactions through either component could physically connect the Gli/Cora ribbon to the F-actin prehair through spectrin; it remains to be seen, however, if the Na+/K+ ATPase and/or Neuroglian are apically polarized in conjunction with Gli and Cora. Previous studies in the wing have demonstrated that actin polymerization is necessary for prehair formation and cell viability. Disruption of F-actin regulation also causes wing hair phenotypes: perturbation of RhoA and its downstream effector Drok causes multiple hair phenotypes. Application of F-actin-disrupting drugs to pupal wings also causes a multiple hair phenotype. The Gli hair phenotype does not include multiple hairs per cell, suggesting that prehair F-actin is correctly regulated in Gli mutants (Venema, 2004).

The tubulin cytoskeleton is also a potential candidate for connecting the septate junction to the apical prehair. Microtubules form a web that contacts the cell periphery at the level of Cora staining at 30 h APF; additionally, microtubules are present inside the prehair after elongation. Experiments are underway to determine if the tubulin cytoskeleton is polarized to prehair bases in conjunction with Gli and Cora (Venema, 2004).

A relationship between septate junction components and the cytoskeleton may also explain the Gli/septate-junction marginal adhesion defects. Adhesion between wing layers is dependent on integrins at basal-basal contacts between wing layers and on the transalar cytoskeleton connecting them. Interestingly, this dependence does not extend to the wing margin: Clones lacking β-integrin adjacent to the wing margin do not cause blisters, even when the clone spans the wing margin, placing cells lacking β-integrin into basal-basal contact. Gli mutations are the first described that blister specifically at the wing margin. Adhesion in this area is thus dependent on Gli and independent of integrins. While the ultrastructure of the transalar cytoskeleton in this region has not been reported, the results suggest the possibility that the septate junction may be an alternative to integrin-based focal adhesions in this area of the wing (Venema, 2004).

When a mechanism functioning to align wing hairs in parallel was first suggested, it was predicted to be frizzled independent. The results have borne this prediction out. Gli and Cora are the first factors necessary for hair orientation shown to function independently of frizzled signaling: The localization and apical polarization of Gli and Cora does not require fz function; conversely, the fz pathway is unaffected by mutations in Gli. Gli and Cora are also the first factors shown to polarize in line with the distal-proximal axis in the pupal wing, the converse pattern of the fz pathway components. Thus, it appears that proximal-to-distal hair polarity is controlled by factors polarized in line with the anterior-posterior axis, while parallel alignment is controlled by factors polarized in line with the proximal-distal axis. Additionally, the Gli nonparallel phenotype appears to arise from unstable prehairs as a result of compromised, but partially functional, apically polarized Gli and Cora. The formation of the final wing hair pattern thus has two phases: the specification of the site of prehair emergence by the fz signaling pathway, and subsequent alignment and stabilization of extended prehairs through apically polarized septate junction components. Future experiments are necessary to determine the precise link between the apical Gli/Cora ribbon and the prehair pedestal; however, the demonstrated interactions between F-actin/ankyrin and the septate junction markers Neuroglian and the Na+/K+ ATPase are likely candidates for further investigation (Venema, 2004).


REFERENCES

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date revised:  20 June 2015
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