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).