InteractiveFly: GeneBrief

: Biological Overview | References


Gene name - nervana 1 and nervana 2

Synonyms -

Cytological map positions - 27B1-27B1 and 27B1-27B2

Function - receptor and channel

Keywords - β subunit of Na,K-ATPase, plays an ion-pump-independent role in junction formation and tracheal morphogenesis

Symbol - nrv1 and nrv2

FlyBase ID: FBgn0015776 and FBgn0015777

Genetic map position - 2L:6,778,554..6,786,856 [-] and 2L:6,789,761..6,798,864 [-]

Classification - Sodium / potassium ATPase β chain

Cellular location - surface transmembrane



NCBI links for Nrv2: Precomputed BLAST | EntrezGene

NCBI links for Nrv2: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

The heterodimeric Na,K-ATPase (see Na pump α subunit) has been implicated in vertebrate and invertebrate epithelial cell junctions, morphogenesis and oncogenesis, but the mechanisms involved are unclear. It has been shown that the Drosophila Na,K-ATPase is required for septate junction (SJ) formation and that of the three β-subunit loci, only Nrv2 isoforms support epithelial SJ barrier function and tracheal tube-size control. This study shows that Nrv1 is endogenously co-expressed with Nrv2 in the epidermis and tracheal system, but Nrv1 has a basolateral localization and appears to be excluded from the Nrv2-containing SJs. When the normally neuronal Nrv3 is expressed in epithelial cells, it does not associate with SJs. Thus, the β-subunit is a key determinant of Na,K-ATPase subcellular localization as well as function. However, localization of the Na,K-ATPase to SJs is not sufficient for junctional activity because although several Nrv2/Nrv3 chimeric β-subunits localize to SJs, only those containing the extracellular domain of Nrv2 have junctional activity. Junctional activity is also specific to different α-subunit isoforms, with only some isoforms from the major α-subunit locus being able to provide full barrier function and produce normal tracheal tubes. Importantly, mutations predicted to inactivate ATPα catalytic function do not compromise junctional activity, demonstrating that the Drosophila Na,K-ATPase has an ion-pump-independent role in junction formation and tracheal morphogenesis. These results define new functions for the intensively studied Na,K-ATPase. Strikingly, the rat α1 isoform has full junctional activity and can rescue Atpα-null mutants to viability, suggesting that the Na,K-ATPase has an evolutionarily conserved role in junction formation and function (Paul, 2007).

Cell junctions are multifunctional complexes that play many crucial roles in epithelial development by providing adhesion, diffusion barrier, polarity and signaling functions. These functions are generally evolutionarily conserved, but combinations of these functions in different junctions can be divergent. For example, although the adherens junctions (AJs) are very similar between vertebrates and Drosophila, the organization and function of junctions that create diffusion barriers show significant differences. In vertebrates, the barrier is provided by the claudin-based tight junctions, which are apical to the AJs and also contain apical polarity proteins such as Par-3, Crumbs and aPKC. In Drosophila, the barrier is provided by a claudin-containing junction termed the septate junction (SJ). However, SJs are basolateral and contain basal polarity proteins such as Scribble (also known as Scribbled), Discs Large and Lethal Giant Larvae, and thus have similarity to both vertebrate tight junctions and basolateral regions (Paul, 2007 and references therein).

The functions and composition of SJs are only partially defined, but in addition to barrier function, SJs are required for proper regulation of tracheal tube size in Drosophila (Paul, 2003). The tracheal system is a ramifying network of epithelial tubes that serves as a combined pulmonary/vascular system to deliver oxygen to the body. After the tracheal system forms, the tubes are enlarged by a process called 'tube expansion' that is coincident with SJs assembly. Without SJs, multicellular tubes such as the dorsal trunk (DT) become too long and can have diameter abnormalities, and some branches such as the ganglionic branches (GBs) lose staining of lumenal markers (Paul, 2003). These tracheal defects arise in SJ mutants because SJs mediate apical secretion of the lumenal matrix-associated protein Verm, which is required to control tracheal tube size. SJ-mediated secretion of Verm occurs via a specialized pathway, as SJs are not required for the secretion of other apical markers. The nature of this specialized pathway is unclear, but it is an important and assayable cellular function of the SJ that is distinct from its paracellular barrier function (Paul, 2007).

In screens for tracheal tube-size or barrier junction mutants, it has been found that the Na,K-ATPase localizes to and is required for SJ formation (Genova, 2003; Paul, 2003). This finding was unexpected because the Na,K-ATPase is expressed in essentially all animal cells and had not previously been reported to be part of a junctional complex. The Na,K-ATPase is a P-type ion transporter that is an α/β heterodimer (reviewed by Kaplan, 2002). The α-subunit is a large, ~1000 amino acid (aa) ten-transmembrane protein that contains the Na+ and K+ antiporter function coupled to ATPase activity. The β-subunit is a small, 330 aa single-transmembrane protein that is thought to chaperone the α-subunit from the ER to the plasma membrane and to modulate ion transport (reviewed by Geering, 2001; Paul, 2007 and references therein).

How could the ATPase function in SJ formation? One possibility is that Na,K-ATPase activity is required to keep intracellular Na+ concentration low to allow junction formation, which has been demonstrated for MDCK cells (Rajasekaran, 2001a). Alternatively, there are multiple reports of ion-transport-independent roles for the Na,K-ATPase. For example, the human α3 Na,K-ATPase serves as a neural receptor for the agrin protein and, through mechanisms that are gradually being defined, the Na,K-ATPase appears to transduce a reactive oxygen-mediated signal initiated by ouabain (Hilgenberg, 2006; Xie, 2002). This report shows that the junctional activity of the Drosophila Na,K-ATPase is mediated by specific isoforms of the ATPα α-subunit and by the extracellular domain of the Nrv2 β-subunit, but is not affected by mutations predicted to block ion-pump activity. Furthermore, expression of the rat α1-subunit in Drosophila Atpα-null mutants can completely restore junctional function and rescue the mutants to viability. This result suggests that the Na,K-ATPase could have an evolutionarily conserved role in cell junction formation and is consistent with evidence that the Na,K-ATPase can promote cell junction formation and cell polarity in vertebrate systems (reviewed by Cereijido, 2004; Paul, 2007).

The Na,K-ATPase has been intensively studied as an ion-transporter over the last 60 years (reviewed by Kaplan, 2002), and although there is increasing evidence that it has roles beyond this, in most cases the details of these ion-transport-independent functions are unclear. In particular, in vertebrate epithelia there are multiple reports that implicate a role for Na,K-ATPase in cell adhesion and/or polarity (Cereijido, 2004). For example, Shoshani (2005) provided evidence that the β1-subunit acts as a homophilic cell adhesion molecule, and Rajasekaran (2001b) showed that expression of the β1-subunit and E-cadherin, but not of either alone, could cause viral-transformed unpolarized MDCK cells to form adherens and tight junctions, and to polarize. However, to date the vertebrate Na,K-ATPase has not been shown to be a component of known cell adhesion or polarity complexes. In Drosophila, the Na,K-ATPase is part of the SJ which also contains the basolateral polarity proteins Scrib, Dlg and Lgl (Genova, 2003; Paul, 2003). This study shows that the junctional activity of the Na,K-ATPase is mediated by the extracellular domain of the Nrv2 β-subunit and that the junctional activity does not require ion pumping by the α-subunit (Paul, 2007).

Using chimeric β-subunits composed of domains from the Nrv2 isoform that has junctional activity, and from the Nrv3 isoform that lacks junctional activity, it was discovered that only chimeras containing the Nrv2 extracellular domain could properly target the chimera to the SJ and provide junctional activity. Although the extracellular domains of Na,K-ATPase β-subunits have previously been shown to mediate α-subunit ion-transport activity (Laughery, 2003; Noguchi, 1994) and cell-cell adhesion interactions (Contreras, 1999; Muller-Husmann, 1993), this is the first demonstration that the extracellular domain of the β-subunit organizes a junctional complex rather than simply acting as a cell adhesion molecule. Although the extracellular domain could simply serve to localize the Na,K-ATPase to the SJ, evidence that the Nrv2 extracellular domain has additional roles in junctional activity is provided by the observation that the Nrv2IT/3E chimera localized to the SJ but did not provide junctional activity. Thus, the Nrv2 extracellular domain is likely to interact with other extracellular SJ components to help organize SJ complexes. Whether these are cis interactions that organize the other transmembrane SJ components such as Neurexin, Neuroglian, or LachesinF, or trans interactions that organize septa between cells, or both, remains to be determined (Paul, 2007).

One of the most surprising results from these studies is that the junctional and tube-size functions of the Na,K-ATPase apparently do not require ion pumping. This contrasts with the traditional view of the ATPase, as an ion pump required for ion homeostasis in many cellular functions and developmental events. For example, Rajasekaran (2001a) showed that the low intracellular Na+ concentration maintained by the Na,K-ATPase is required for MDCK junction formation, and Lowery (2005) showed that ATPase-mediated ion transport was required for zebrafish neural tube inflation. Shu (2003) has shown that ion-transport by the α1β1 isoform is required for zebrafish heart morphogenesis. Hilgenberg (2006) has shown that the neuronal α3 ATPase binds to and acts as a receptor for Agrin, but the signal is transduced via changes in ATPase ion transport activity. Thus, the apparent ion-pump-independent junctional activity of Nrv2 appears to be a novel activity for an Na,K-ATPase (Paul, 2007).

Although ion pumping by the α-subunit is not required for SJ formation, the α-subunit nonetheless appears to have an important role in organizing SJs. All isoforms with the Long N-terminus fully support junctional assembly, whereas isoforms with the Short N-terminus have only partial activity at the phenotypic level and do not significantly support junctional assembly at the cellular level. The alternatively spliced sixth exon also appears to contain some junctional activity because different sixth exon isoforms vary widely in their ability to provide tube-size and barrier function when the Long N-terminus is absent (Paul, 2007).

A model for the role of the ATPase in SJ assembly that is consistent with the combined results of the α- and β-subunit data is that the extracellular domain of the β-subunit interacts with multiple extracellular SJ components to assemble an extracellular complex, whereas the α-subunit interacts with cytosolic proteins or intracellular portions of transmembrane proteins to promote junction formation, paracellular barrier formation and tracheal tube-size control. An example of a protein that could interact with the α-subunit to organize junctions is the cytoskeletal protein Ankyrin, which has been shown to bind two distinct sites on the rat α-subunit (Jordan, 1995; Zhang, 1998), sites which are conserved in the Drosophila ATPα (Paul, 2007).

The ability of the rat α1 isoform to rescue all junctional defects of Drosophila Atpα-null mutants is consistent with the 77% identity between the Drosophila ATPα and the rat α1 proteins, and supports the hypothesis that the Na,K-ATPase has a conserved role in cell junction formation. Why would the Na,K-ATPase have evolved and maintained a role in epithelial cell junction formation and/or polarity? Possibly, as metazoans first became multicellular, their epithelial cells would have needed to establish cell-cell junctions and asymmetrically distributed ion pumps (i.e., primitive cell polarity) to enable polarized ion transport. In the first epithelial cells, the asymmetric localization of the Na,K-ATPase may have been achieved by anchoring the pump to asymmetrically localized adhesion proteins. As cell junctional and polarity mechanisms evolved, the Na,K-ATPase could have transitioned from being associated with adhesion proteins only to serving an integral scaffolding role in a larger junctional complex. Although ATPase-mediated ion transport would still be required for ion homeostasis, the scaffolding function could be ion-transport-independent, consistent with the findings that Na,K-ATPase catalytic activity is required for Drosophila viability, but that ATPα-subunits predicted to be catalytically inactive fully support SJ formation (Paul, 2007).

The role of the β1 subunit of the Na,K-ATPase and its glycosylation in cell-cell adhesion

Based on recent data showing that overexpression of the Na,K-ATPase β1 subunit increased cell-cell adhesion of nonpolarized cells, it was hypothesized that the β1 subunit can also be involved in the formation of cell-cell contacts in highly polarized epithelial cells. In support of this hypothesis, in Madin-Darby canine kidney (MDCK) cells, the Na,K-ATPase alpha1 and β1 subunits were detected as precisely co-localized with adherens junctions in all stages of the monolayer formation starting from the initiation of cell-cell contact. The Na,K-ATPase and adherens junction protein, β-catenin, stayed partially co-localized even after their internalization upon disruption of intercellular contacts by Ca(2+) depletion of the medium. The Na,K-ATPase subunits remained co-localized with the adherens junctions after detergent treatment of the cells. In contrast, the heterodimer formed by expressed unglycosylated Na,K-ATPase β1 subunit and the endogenous alpha1 subunit was easily dissociated from the adherens junctions and cytoskeleton by the detergent extraction. The MDCK cell line in which half of the endogenous β1 subunits in the lateral membrane were substituted by unglycosylated β1 subunits displayed a decreased ability to form cell-to-cell contacts. Incubation of surface-attached MDCK cells with an antibody against the extracellular domain of the Na,K-ATPase β1 subunit specifically inhibited cell-cell contact formation. It is concluded that the Na,K-ATPase β1 subunit is involved in the process of intercellular adhesion and is necessary for association of the heterodimeric Na,K-ATPase with the adherens junctions. Further, normal glycosylation of the Na,K-ATPase β1 subunit is essential for the stable association of the pump with the adherens junctions and plays an important role in cell-cell contact formation (Vagin, 2006).

Inverse correlation between the extent of N-glycan branching and intercellular adhesion in epithelia. Contribution of the Na,K-ATPase β1 subunit

The majority of cell adhesion molecules are N-glycosylated, but the role of N-glycans in intercellular adhesion in epithelia remains ill-defined. Reducing N-glycan branching of cellular glycoproteins by swainsonine, the inhibitor of N-glycan processing, tightens and stabilizes cell-cell junctions as detected by a 3-fold decrease in the paracellular permeability and a 2-3-fold increase in the resistance of the adherens junction proteins to extraction by non-ionic detergent. In addition, exposure of cells to swainsonine inhibits motility of MDCK cells. Mutagenic removal of N-glycosylation sites from the Na,K-ATPase β(1) subunit impairs cell-cell adhesion and decreases the effect of swainsonine on the paracellular permeability of the cell monolayer and also on detergent resistance of adherens junction proteins, indicating that the extent of N-glycan branching of this subunit is important for intercellular adhesion. The N-glycans of the Na,K-ATPase β(1) subunit and E-cadherin are less complex in tight renal epithelia than in the leakier intestinal epithelium. The complexity of the N-glycans linked to these proteins gradually decreases upon the formation of a tight monolayer from dispersed MDCK cells. This correlates with a cell-cell adhesion-induced increase in expression of GnT-III (stops N-glycan branching) and a decrease in expression of GnTs IVC and V (promote N-glycan branching) as detected by real-time quantitative PCR. Consistent with these results, partial silencing of the gene encoding GnT-III increases branching of N-glycans linked to the Na,K-ATPase β(1) subunit and other glycoproteins and results in a 2-fold increase in the paracellular permeability of MDCK cell monolayers. These results suggest epithelial cells can regulate tightness of cell junctions via remodeling of N-glycans, including those linked to the Na,K-ATPase β(1)-subunit (Vagin, 2008).

The Na+/K+ ATPase is required for septate junction function and epithelial tube-size control in the Drosophila tracheal system

Although the correct architecture of epithelial tubes is crucial for the function of organs such as the lung, kidney and vascular system, little is known about the molecular mechanisms that control tube size. Mutations in the ATPα and nrv2 β subunits of the Na+/K+ ATPase cause Drosophila tracheal tubes to have increased lengths and expanded diameters. ATPα and nrv2 mutations also disrupt stable formation of septate junctions, structures with some functional and molecular similarities to vertebrate tight junctions. The Nrv2 β subunit isoforms have unique tube size and junctional functions because Nrv2, but not other Drosophila Na+/K+ ATPase β subunits, can rescue nrv2 mutant phenotypes. Mutations in known septate junctions genes cause the same tracheal tube-size defects as ATPα and nrv2 mutations, indicating that septate junctions have a previously unidentified role in epithelial tube-size control. Double mutant analyses suggest that tube-size control by septate junctions is mediated by at least two discernable pathways, although the paracellular diffusion barrier function does not appear to involved because tube-size control and diffusion barrier function are genetically separable. Together, these results demonstrate that specific isoforms of the Na+/K+ ATPase play a crucial role in septate junction function and that septate junctions have multiple distinct functions that regulate paracellular transport and epithelial tube size (Paul, 2003).

Previous showed that embryos homozygous for the l(2)k04223 strain had tracheal tube-size regulation defects (Beitel, 2000). Using inverse PCR, it was determined that the transposable element in this strain was inserted in an intron in the nervana2 (nrv2) locus, which encodes two alternatively spliced isoforms of a Na+/K+ ATPase β subunit (Sun, 1995a; Sun, 1995b). Two lines of evidence demonstrate that the tracheal phenotype of l(2)k04223 homozygotes results from disruption of the nrv2 locus. First, an independent transposable element insertion in the nrv2 locus, l(2)k13315, causes the same tracheal phenotypes as l(2)k04223 and fails to complement l(2)k04223 for tracheal phenotypes and viability. Second, in an EMS non-complementation screen isolated two additional mutations that fail to complement l(2)k04223. Both of these mutants contain a single nonsense base change in the exons common to both nrv2 isoforms, and both have the same tracheal phenotypes as l(2)k04223. Together, these results show that a Na+/K+ ATPaseβ subunit is required for tracheal tube-size control (Paul, 2003).

To better define the role of nrv2 in epithelial morphogenesis, a putative nrv2 null allele, nrv2nwu3, was generated that deletes the first three common nrv2 exons which encode transmembrane and extracellular domains. In addition, Genova (2003) provided a second putative null allele, nrv223B, that removes all nrv2 common exons. The phenotypes caused by nrv2nwu3 or nrv223B do not become more severe in trans to a chromosomal deficiency known to delete nrv2, providing genetic evidence that these are null alleles (Paul, 2003).

In nrv2-null embryos, beginning at the time of tracheal tube expansion, multicellular tracheal tubes become increasingly abnormal so that most tube lengths are significantly increased and all tube diameters are irregular with expansions and constrictions along their lengths. Defects are also present in regions of single cell tubes formed by autocellular junctions, particularly near the ends of the ganglionic branches where there are lumenal staining discontinuities (Paul, 2003).

Although the process of tracheal tube expansion is drastically disrupted in nrv2-null mutants, the earlier of phases of tracheal tube morphogenesis, including early tube-size regulation, are normal. Furthermore, overall embryonic morphogenesis of nrv2-null mutant embryos also appears to be grossly normal as evidenced by the correct outgrowth of all tracheal branches to their target tissues and the absence of major patterning or morphogeneic defects as assessed using DIC optics. One possible explanation for the apparent specific morphogenic requirement of nrv2 in tracheal tube expansion is that there is a maternal contribution of nrv2 that provides enough activity to support early morphogenic processes, but not enough to support tracheal tube expansion, which occurs late in embryonic development. However, in situ hybridization and microarray analysis did not reveal any significant maternal nrv2 transcript and embryos lacking both maternal and zygotic nrv2 are indistinguishable from nrv2 zygotic null embryos. Thus, nrv2 does not play a role in early epithelial formation or general embryonic morphogenesis, but instead appears to be specifically required for remodeling the length and diameter of tracheal tubes (Paul, 2003).

To test whether nrv2 functions as part of the Na+/K+ ATPase to control tracheal tube size, the embryonic phenotypes of mutations in the major Na+/K+ ATPase α subunit locus, ATPα (Lebovitz, 1989; Sun, 1998; Palladino, 2003) was examined. The transposable element insertions ATPαl(3)1278, ATPαl(3)04694 and ATPαl(3)07008 caused tracheal defects similar or identical to nrv2-null mutations, including tube length increases, diameter expansions and ganglionic branch discontinuities. The ATPα-null mutations ATPαDTS1R1 and ATPαDTS1R2 (Palladino, 2003) also caused nrv2-like length and ganglionic branch defects, but caused only mild diameter defects. Although one would normally expect the ATPα-null mutations to cause more severe phenotypes than partial loss-of-function mutations, the hypomorphic ATPα mutations might cause strong nrv2-like phenotypes by producing inactive α subunits that could unproductively interact with Nrv2 and deplete the pool of Nrv2 available for productive interactions with other binding partners, such as α subunits expressed from the secondary Na+/K+ ATPase α subunit locus CG17923. However, despite some differences between the phenotypic effects of different ATPα mutations, the observation that both null and partial loss-of-function mutations cause nrv2-like tracheal tube-size defects demonstrates that the ATPα locus is required for tracheal tube-size control and suggests that the nrv2 β and ATPα α subunits function together in this process (Paul, 2003).

During the current investigations of the role of the Na+/K+ ATPase in tube-size control, Genova and Fehon reported that Na+/K+ ATPase mutants had salivary gland septate junction defects. Therefore this study tested whether tracheal septate junction barrier function was defective in Na+/K+ ATPase mutants using a dye exclusion assay, which tests the ability of an epithelium to exclude a 10 kDa dye. In wild-type embryos, tracheal septate junction barriers become functional and excluded dye starting at late stage 15. However, the tracheal and salivary gland epithelia in nrv2 and ATPα mutants do not acquire the ability to regulate paracellular transport and cannot prevent the dye from inappropriately diffusing into the tracheal and salivary gland lumens (Paul, 2003).

To understand the nature of the septate junction defects in Na+/K+ ATPase mutants, the subcellular distribution of septate junction and cell polarity components were determined in stage 16 nrv2 and ATPα null mutants. Three ectodermally derived epithelia - trachea, epidermis and salivary glands - were examined and similar defects were found in all three tissues. The effects were most clearly seen in the large columnar cells of the salivary gland in which the septate junction occupies only a small region of the lateral surface of the cell. By contrast, septate junctions occupy most of the lateral surface of tracheal cells, making visualization of mislocalized septate junction components more difficult. In both nrv2 and ATPα mutants, the septate junction components Coracle, Neurexin and Discs Large mislocalize along the lateral and sometimes the basal cell surfaces, rather than being tightly localized to the apicolateral septate junction. In some cases, the levels of these proteins appeared to also be reduced. The septate junction component Fasciclin III was undetectable in nrv2 and ATPα mutant salivary glands and strongly reduced in trachea (Paul, 2003).

Although every septate junction marker tested is severely affected by the Na+/K+ ATPase mutations, the effects appear to be specific for septate junctions since the localizations and levels of the adherens junction components E-cadherin (Shotgun) and β-catenin (Armadillo), and the apical determinant Discs Lost were unaffected in nrv2 and ATPα null mutants. Together, these data demonstrate that Na+/K+ ATPase mutations specifically disrupt septate junctions (Paul, 2003).

A simple explanation for the abnormal sizes of tracheal tubes in mutants having septate junction defects would be that ionic or hydrostatic disequilibria across the tracheal epithelium disrupts tracheal cell morphogenesis. If so, then all mutants with similar paracellular barrier defects should have equivalent tracheal morphologies. However, it was found that barrier mutants had tracheal morphologies ranging from near wild type in the case of cor14* to diameter- or length-specific defects in cystic and megatrachea. These results support the conclusion that septate junction control of tube size is not dependent on regulation of paracellular diffusion (Paul, 2003).

The mutant phenotypes and genetic interactions among tracheal tube-expansion and septate junction mutants suggest there are at least two genetic pathways by which septate junctions regulate tracheal tube size. For example, nrv2 and coracle appear to act in the same genetic pathway, since nrv2 and coracle null mutants have the same tracheal phenotypes as each other and as nrv2 coracle double null mutants. This genetic evidence is supported by observations that the localization of Coracle to septate junctions is disrupted in nrv2 and ATPα mutants. By contrast, although nrv2-null and varicose mutants have the same tracheal phenotypes, nrv2 and varicose are unlikely to act in the same linear genetic pathway because nrv2 varicose double mutants have more severe tracheal phenotypes than nrv2-null mutants. This result suggests that either varicose and nrv2 function in separate pathways to control tube size, or there is redundancy between the functions of these genes (Paul, 2003).

Additional genetic pathways may also be revealed by the differential effects of tube-expansion mutations on septate junction barrier integrity. For example, in contrast to nrv2 and other mutations, the existing convoluted mutations do not affect septate junction barrier integrity and therefore may define a size-control pathway that acts in parallel to septate junction pathways. Consistent with this proposal, the double mutant combination of a nrv2 null mutation and a convoluted mutation result in more severe tracheal morphology defects than either nrv2-null or convoluted mutations alone. Alternatively, genes such as convoluted may function in a branch of a septate junction pathway to link septate junctions to tube size control. Although these models are necessarily incomplete, they offer testable predictions about gene interactions and subcellular localizations of uncharacterized gene products that should help define tube-size control and paracellular barrier pathways at the molecular level (Paul, 2003).

A central issue raised by these findings is the nature of the molecular functions(s) of the Na+/K+ ATPase and septate junctions in tube-size control. Although the Na+/K+ ATPase has been studied intensively for more than 40 years for its function as an ion pump (Chow, 1995; Blanco, 1998), the data indicate that the tracheal tube-size function of the Na+/K+ ATPase is intimately associated with its role in septate junction function. Furthermore the paracellular barrier and tube-size control functions of the septate junction are separable (Paul, 2003).

In one class of model that accounts for these observations, the role of the Na+/K+ ATPase is to organize septate junctions, which control tube size by an undetermined mechanism. The many functions of vertebrate tight junctions provide possible examples of non-barrier mechanisms by which septate junctions could control tube size. In particular, tight junctions organize polarized apical secretion mediated by the exocyst, bind cytoskeletal components such as ankyrin and fodrin, contain potential signaling molecules such as the tyrosine kinases Src, Yes and protein kinase C, and have recently been shown to regulate the activity of a Y-box transcription factor. In addition, both septate and tight junctions complexes contain proteins that organize epithelial cell apical/basal domains (Paul, 2003).

Of the tube-size control models that do not invoke ion-transport functions of the Na+/K+ ATPase, models involving apicobasal domain organization are particularly attractive. Apical surface regulation is a common theme in tubulogenesis, and has been shown to play an important role in tube-size control in the Drosophila salivary gland. Several observations support the possibility that septate junctions control tracheal tube size through the apical cell surface. First, the differential regulation of tracheal apical and basal cell surfaces suggests that tracheal tube size control is mediated at the apical cell surface. Second, the increased tracheal tube lengths and diameters present in tube-expansion mutants necessitate an increased apical cell surface area. Given that the Dlg/Scrib/Lgl complex normally present in septate junctions has an early embryonic function to negatively regulate the extent of the apical membrane domain, this complex could also act later to negatively regulate tracheal apical surface area (Paul, 2003).

In an alternative class of models that are not exclusive of the above possibilities, the ion-pump activity of the Na+/K+ ATPase may directly or indirectly mediate tube-size control. For example, pharmacologically blocking Na+/K+ ATPase ion-transport activity leads to increased intracellular Ca2+ levels in some cell types, and Ca2+ signaling abnormalities may be the molecular defect that causes the enlarged tubules of polycystic kidney disease (PKD). Another example is that the low intracellular Na+ level maintained by the Na+/K+ ATPase is required for formation of tight junctions and stress fibers in Madin-Darby canine kidney (MDCK) cells, an epithelial cell line that can form tubules in response to hepatocyte growth. Septate junction formation might also require low intracellular Na+ levels. Finally, disruption of the cellular Na+/K+ electrochemical gradient could impact secondary active transport of other solutes that may be important for proper tube-size regulation (Paul, 2003).

Although the exact biochemical roles of the Na+/K+ ATPase and septate junctions in tube-size control are unclear, identification of these complexes as parts of a tube-size control mechanism is an important step towards further understanding these mechanisms at the molecular level (Paul, 2003).

The Na+/K+ ATPase has been implicated in vertebrate tube-size control by the abnormal subcellular localization of the Na+/K+ ATPase in the inappropriately expanded tubules in individuals with PKD and in several animal models of cystic kidney diseases. However, it has not yet been determined whether this mislocalization contributes to the progression of cystic diseases or whether it is merely a secondary effect of other cellular defects. The finding that the Na+/K+ ATPase is required for normal tube-size control in the Drosophila tracheal system suggests that the vertebrate Na+/K+ ATPase may play an important role in maintaining the normal size of kidney and other epithelial and endothelial tubes. Ultimately, a molecular understanding of the tube-size control mechanisms should allow development of new strategies for preventing and treating PKD and other diseases resulting from epithelial and endothelial tube defects (Paul, 2003 and references therein).

Neuroglian, Gliotactin, and the Na+/K+ ATPase are essential for septate junction function

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). 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, where it interacts with protein 4.1, which is homologous to Drosophila Cor (Genova, 2003 and references therein).

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).

In vivo modification of Na+,K+-ATPase activity in Drosophila

Transgenic Drosophila lines with modified Na+,K+-ATPase activity have been constructed and characterized. Using a temperature dependent promoter from the hsp70 gene to drive expression of wild-type α subunit cDNA, bang-sensitive paralysis and ouabain sensitivity can be conditionally rescue of a Drosophila Na+,K+-ATPase α subunit hypomorphic mutant, 2206. In contrast, a mutant α subunit (αD369N) leads to increased bang-sensitive paralysis and ouabain sensitivity. Temperature dependent phenotypes in wild-type Drosophila can be generated using the same hsp70 controlled α transgenes. Ouabain sensitivity was as expected, however, both bang sensitive paralysis or locomotor phenotypes became more severe regardless of the type of α subunit transgene. Using the Gal4-UAS system expression of α transgenes has been limited to cell types that normally express a particular Drosophila Na+,K+-ATPase beta (Nervana) subunit isoform (Nrv1 or 2). The Nrv1-Gal4 driver results in lethality while the Nrv2-Gal4 driver shows reduced viability, locomotor function and uncontrolled wing beating. These transgenic lines will be useful for disrupting function in a broad range of cell types (Sun, 2001).

Organization and transcriptional regulation of Drosophila Na+, K+-ATPase β subunit genes: Nrv1 and Nrv2

Drosophila melanogaster has two Na+,K+-ATPase β subunit genes (Nervana 1 and 2; Nrv), with tissue-specific expression patterns. Nrv1 produces a single β subunit isoform expressed primarily in muscle tissue, whereas Nrv2 codes for two different isoforms (2.1 and 2.2) expressed in the nervous system. This study determined the complete molecular genomic organization for both Nrv genes. Only 3kb of DNA separate the 3' end of Nrv2 from Nrv1. The cDNAs from all three forms of Nrv have been mapped onto the genomic structure and all intron-exon junctions have been confirmed by direct sequencing. The genomic DNA positioned in the 5' flanking region of each Nrv gene has also been tested for tissue-specific transcriptional regulatory activity. P-element transformation vectors were constructed, which contained either 7.7kb of Nrv2 or 3.5kb Nrv1 5' flanking DNA driving expression of a lacZ reporter gene. Multiple transgenic Drosophila lines were established for each construct and analyzed for their β-galactosidase expression pattern. The tissue-specific expression of each Nrv gene is independently regulated by the cis-element(s) present in the 5' flanking region. The Nrv2 5' flanking DNA directs expression exclusively to the nervous system, whereas Nrv1 5' flanking DNA directs expression primarily in muscle tissue (Xu, 1999).

Two Drosophila nervous system antigens, Nervana 1 and 2, are homologous to the β subunit of Na+,K+-ATPase

A nervous system-specific glycoprotein antigen from adult Drosophila heads, designated Nervana (Nrv), has been purified on the basis of reactivity of its carbohydrate epitope(s) with anti-horseradish peroxidase (HRP) antibodies that are specific markers for Drosophila neurons. Anti-Nrv monoclonal antibodies (mAbs), specific for the protein moiety of Nrv, were used to screen a Drosophila embryo cDNA expression library. Three cDNA clones (designated Nrv1, Nrv2.1, and Nrv2.2) were isolated that code for proteins recognized by anti-Nrv mAbs on Western blots. DNA sequencing and Southern blot analyses established that the cDNA clones are derived from two different genes. In situ hybridization to Drosophila polytene chromosomes showed that the cDNA clones map to the third chromosome near 92C-D. Nrv1 and Nrv2.1/2.2 have open reading frames of 309 and 322/323 amino acids, respectively, and they are 43.4% identical at the amino acid level. The proteins deduced from these clones exhibit significant homology in both primary sequence and predicted topology to the β subunit of Na+,K+-ATPase. Immunoaffinity-purified Nrv is associated with a protein (M(r) 100,000) recognized on Western blots by anti-ATPase alpha-subunit mAb. The results suggest that the Drosophila nervous system-specific antigens Nrv1 and -2 are neuronal forms of the β subunit of Na+,K+-ATPase (Sun, 1995a; Full text of article).


REFERENCES

Search PubMed for articles about Drosophila Nervana

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date revised: 10 May 2010

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