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First published online December 12, 2006
doi: 10.1242/10.1242/dev.02710
1 Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern
University, Evanston, IL 60208, USA.
2 Laboratory of Genetics, University of Wisconsin, Madison, WI 53706, USA.
3 Department of Pharmacology and Pittsburgh Institute for Neurodegenerative
Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260,
USA.
* Author for correspondence (e-mail: beitel{at}northwestern.edu)
Accepted 23 October 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-subunit isoforms, with only some isoforms from the major
-subunit locus being able to provide full barrier function and produce
normal tracheal tubes. Importantly, mutations predicted to inactivate
ATP 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 1 isoform has full junctional activity and can
rescue Atp-null mutants to viability, suggesting that the
Na,K-ATPase has an evolutionarily conserved role in junction formation and
function.
Key words: Na, K-ATPase, Epithelial junctions, Drosophila, Septate junctions, Trachea
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Tepass et al.,
2001). 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
(Van Itallie and Anderson,
2006). In Drosophila, the barrier is provided by a
claudin-containing junction termed the septate junction (SJ)
(Behr et al., 2003;
Wu et al., 2004). 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
(reviewed by Tepass et al.,
2001; Wu and Beitel,
2004).
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 et al., 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 (reviewed by
Affolter et al., 2003;
Manning and Krasnow, 1993).
After the tracheal system forms, the tubes are enlarged by a process called
`tube expansion' that is coincident with SJs assembly
(Beitel and Krasnow, 2000).
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 et al., 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 (Luschnig et al.,
2006; Wang et al.,
2006). SJ-mediated secretion of Verm occurs via a specialized
pathway, as SJs are not required for the secretion of other apical markers
(Llimargas et al., 2004;
Paul et al., 2003;
Genova and Fehon, 2003;
Hemphala et al., 2003). 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.
In screens for tracheal tube-size or barrier junction mutants, we and
others previously found that the Na,K-ATPase localizes to and is required for
SJ formation (Genova and Fehon,
2003; Paul et al.,
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 /ß heterodimer (reviewed by
Kaplan, 2002). The
-subunit is a large, 
1000 amino acid (aa) tentransmembrane protein
that contains the Na+ and K+ antiporter function coupled
to ATPase activity. The ß-subunit is a small, 330 aa single-transmembrane
protein that is thought to chaperone the -subunit from the ER to the
plasma membrane and to modulate ion transport (reviewed by
Geering, 2001).
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 et al.,
2001a). Alternatively, there are multiple reports of
ion-transport-independent roles for the Na,K-ATPase. For example, the human
3 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 et al., 2006;
Xie and Askari, 2002). In this
report, we show that the junctional activity of the Drosophila
Na,K-ATPase is mediated by specific isoforms of the ATP-subunit
and by the extracellular domain of the Nrv2 ß-subunit, but is not
affected by mutations predicted to block ion-pump activity. Furthermore, we
show that expression of the rat 1-subunit in Drosophila
Atp-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 (see Discussion) (reviewed
by Cereijido et al., 2004).
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Breakpoints for the Nrv2/Nrv3 extracellular domain chimeras were as follows:
Nrv2 Region A, W70-D131; Region B, F132-A186; Region C, P187-R260; Region D,
G261-D322. Fulllength Atp clones were generated from RT-PCR
amplicons utilizing whole adult RNA and the TOPO T/A Cloning System
(Invitrogen). Clones were sequenced to identify inserts representing
previously described cDNA isoforms without missense or nonsense mutations. The
cloning primers contained EcoRI (5') and XhoI
(3') linkers that were used to shuttle the insert into the pUAST
transgenesis vector bearing the w[mc+] gene. The rat 1 clone
with an N-terminal tag has been shown to be functional
(Dada et al., 2003). Isoforms
predicted to be catalytically inactive were generated by sitedirected PCR
mutagenesis with oligonucleotide primers producing the D355N (short isoform)
or D394N (long isoform) mutation. The primers also introduced a silent GGC to
GGT codon change 3' to the mutation site, creating a unique
AgeI endonuclease site that was used to generate the
UASATP [D
N] constructs. All pUAST
construct inserts were resequenced and standard P-element-mediated
transgenesis techniques were used to generate transgenic flies.
The da-Gal4 on chromosome III
(Wodarz et al., 1995) was
crossed into the ATP[DTS2R3] mutant
(Palladino et al., 2003)
background to generate a driver line for the -subunit rescue
experiments. da-Gal4 was mobilized to chromosome II and crossed into
the nrv2[23B] (Genova and Fehon,
2003) background to generate a driver for the ß-subunit
rescues.
Polyclonal peptide antibodies were generated against intracellular epitopes of Nrv1 MSKNNGKGAKGEFEFPQPAKKQTFSE, Nrv2.1 MSKPVPMSPSFVDEDLHNLRKPKPF and Nrv3 MADKKIGEYYAPPVKMGKWEGFKK. All peptides were synthesized by GenScript (Piscataway, NJ) and antibodies were generated by Harlan (Indianapolis, IN) in rabbits (Nrv2.1) or guinea pigs (Nrv1 and Nrv3). The pattern of anti-Nrv3 immunoreactivity precisely matched the expression of nrv3 RNA determined by in situ hybridization.
Immunohistochemistry and microscopy
The lumenal antibody 2A12, embryo fixation, staining and staging procedures
were as described in Samakovlis et al.
(Samakovlis et al., 1996).
Other antibodies used were anti-a5
(Lebovitz et al., 1989),
anti-Coracle (Fehon et al.,
1994), anti-GFP (Molecular Probes and Abcam) and anti-Verm
(Luschnig et al., 2006).
Anti-Nrv1, 2 and 3 sera required heat fixation
(Peifer et al., 1994) and were
used at 1:500 for anti-Nrv2.1 and 1:100 for anti-Nrv1 and anti-Nrv3. Confocal
images were captured using a Leica TCS SP2. To assess protein levels,
heterozygotes and homozygous mutants were imaged at the same settings on the
same slide in the same session. Apparent variations in epithelial cell heights
were artefacts resulting from optical sectioning of an irregular epithelial
layer and/or differences between the angles at which optical sections
intersected the epithelia. Post-processing image adjustments were applied
equally to matched images. Embryos were at stage 16.
Assays of SJ function
SJ tube-size function was assayed by examining the tracheal DT, which is
fairly straight in wild-type (WT) animals and tortuous in mutants, and GBs,
which are contiguous in WT animals but have lumenal gaps in mutants. The GB
gaps are a sensitive indicator of SJ function as they are more easily rescued
than the DT phenotype. SJ barrier function was tested using the assay of Lamb
et al. (Lamb et al., 1998) as
modified by Paul et al. (Paul et al.,
2003), which tests the ability of an epithelium to block
paracellular diffusion of a 10 kDa fluorescent dye. SJ assembly was assessed
by the localization of the canonical SJ marker Coracle to the normal position
of SJs in the columnar epithelium of the salivary gland, rather than the
trachea, because SJs occupy much of lateral cell surface in tracheal cells,
precluding reliable assessment of changes in SJ component subcellular
localization (Tepass and Hartenstein,
1994).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)]. This
finding was surprising because in vitro studies concluded that virtually any
Na,K-ATPase ß-subunit can associate with an -subunit to generate a
functional ion pump (reviewed by Blanco and
Mercer, 1998; Schmalzing et
al., 1991). To begin to understand the molecular basis of these
Nrv2-specific activities, we created isoform-specific reagents to determine
the endogenous subcellular localizations of these proteins (Figs
1,
2 and see Materials and
methods). Although we were unable to generate antisera specific for Nrv2.2, an
antibody specific for Nrv2.1 showed that Nrv2.1 is expressed in all
ectodermally-derived tissues, consistent with the expression pattern of both
Nrv2 isoforms determined using in situ hybridization (data not shown). Nrv2.1
is found at SJs (Fig.
1A1,B1,C1), where it co-localizes with the canonical pleated SJ
marker Coracle (Fehon et al.,
1994) (Fig. 2A4).
SJs are basolateral to adherens junctions and occupy the apical third or fifth
of columnar epithelia such as salivary glands
(Fig. 1B1) and surface ectoderm
(Fig. 1A1).
By contrast, Nrv3 is not expressed in epithelial tissues but was instead
found in the central nervous system and the chordotonal organs in the
peripheral nervous system (Fig.
1D1,D2; Fig. 2B2).
When Nrv3 was ectopically expressed in epithelial cells such as the salivary
glands using the Gal4/UAS system (Brand and
Perrimon, 1993), it was not efficiently transported to the cell
surface and either accumulated at low levels or appeared to become trapped
intracellularly (Fig. 2E5).
Although in many systems, Na,K-ATPase ß-subunits must oligomerize with
-subunits in order to exit the ER
(Jaunin et al., 1993), it
seems unlikely that the general pool of -subunit is limiting in this
case because overexpression of Nrv2.1 or Nrv1 resulted in normal targeting
(Fig. 2C5,D5).
Like Nrv2, Nrv1 was found to be expressed in ectodermal epithelia such as the epidermis (at relatively high levels) and the trachea, but was expressed at very low levels in salivary glands and was absent from the hindgut (Fig. 1A1-C4 and data not shown). In contrast to Nrv2, Nrv1 was also expressed in some endodermal epithelia including the Malpighian tubules and midgut, which lack pleated SJs (data not shown). Importantly, in all epithelial tissues, the large majority of Nrv1 localized to the basolateral surface and did not significantly co-localize with Nrv2 or Coracle at SJs (Fig. 1 and data not shown). Further, even when overexpressed in a WT background, Nrv1 was largely excluded from the SJs (Fig. 2D5).
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Since all three Drosophila ß-subunits have different subcellular localizations when expressed in the same tissues, they are likely to have divergent functions and must contain different targeting signals. Furthermore, Nrv2 isoforms are the only isoforms that have an association with the SJ, suggesting that either targeting the Na,K-ATPase complex to SJs is important, or that Nrv2 isoforms might have a structural role in junctional assembly.
The extracellular domain of Nrv2 is required for septate junction function
We next investigated the ability of the different ß-subunits to
provide SJ function and to assemble SJ complexes. Expression of Nrv2.1 in a
nrv2-null mutant completely rescued the tracheal and barrier function
phenotypes and restored Coracle localization
(Fig. 2 and see Materials and
methods). Almost all Nrv2 protein localized to SJs. By contrast, expression of
Nrv1 and Nrv3 did not rescue tracheal or SJ barrier defects
(Fig. 2D1-D3,E1-E3) and did not
restore localization of Coracle to SJs
(Fig. 2D4,E4).
To define which regions of Nrv2 contained SJ function, we constructed a series of chimeras in which the intracellular (I), transmembrane (T) and extracellular (E) domains of Nrv2.1 were swapped with the corresponding domains of Nrv3. Because all ß-subunits share the same basic structure we reasoned that the chimeric proteins should fold properly. We found that both chimeras containing the extracellular domain - Nrv3I/2TE and Nrv3IT/2E - had full tracheal and SJ barrier function activity and were phenotypically indistinguishable from WT (Fig. 2F1-F3 and data not shown). Both chimeras could also restore strong Coracle localization to the SJs of nrv2 homozygotes. By contrast, although both chimeras with the Nrv3 extracellular domain appeared to be expressed at levels at least comparable to the UAS Nrv2 construct, these chimeras lacked detectable tracheal tube-size activity, SJ barrier function, or the ability to assemble SJ complexes as assayed by the ability to localize Coracle to SJs (Fig. 2G1-G4 and data not shown). Thus, the extracellular domain of the Nrv2 ß-subunit has a specific activity required for tube-size control, SJ barrier function and SJ complex assembly.
Importantly, although the Nrv2IT/3E chimera did not have rescue activity, it did have the ability to associate with SJs when expressed in nrv2/+ heterozygotes (Fig. 2G5), which argues that simply bringing the Na,K-ATPase to the SJ is insufficient for organizing the SJ and that the extracellular domain of the ß-subunit is involved in an extracellular complex required for SJ formation.
Multiple regions of the Nrv2 extracellular domain are required for junctional function
Having narrowed the essential functional region of Nrv2 to the
extracellular domain, we constructed an additional series of chimeras between
portions of the extracellular domains of Nrv2 and Nrv3 to further define the
regions of Nrv2 containing the junctional and tube-size control activities. We
divided the extracellular domain into four sections of roughly equal length,
using blocks of conserved regions as breakpoints for our chimera design given
that a high resolution crystal structure of a Na,K-ATPase ß-subunit is
not available (see Materials and methods). Unexpectedly, when expressed in the
nrv2 mutant background, none of these extracellular domain chimeras
rescued tracheal tube-size control or SJ barrier function
(Fig. 3B1-G4), suggesting that
the tube size and barrier junction function of the Nrv2 extracellular domain
is a three-dimensional epitope formed from multiple regions of linear sequence
or that full function of the extracellular domain requires several
interactions.
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Only some -subunit isoforms have full junctional activity
Given that only some of the Na,K-ATPase ß-subunit isoforms had
tube-size and barrier junction activities, we investigated whether all
-subunit isoforms would have junctional activities. In
Drosophila, most Na,K-ATPase -subunit isoforms are encoded by
one primary locus, Atp, that gives rise to numerous potential
protein products by alternative splicing. These isoforms differ at the extreme
N-terminus, where an additional 39 aa are found in the `Long' isoforms, and in
transmembrane domain six and the fourth intracellular domain, which together
are encoded by a mutually exclusive sixth exon cassette
(Fig. 4A,B)
(Palladino et al., 2003). To
date, the mutations in the Atp locus that cause defects in
tracheal tube-size control and SJ function
(Paul et al., 2003;
Genova and Fehon, 2003) affect
constitutive exons and disrupt all known ATP isoforms.
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isoforms were capable of organizing the SJ
and controlling tracheal tube size. To do this we generated UAS-expression
constructs, each containing a cDNA encoding one ATP isoform. When
expressed in an Atp-null mutant background under the
control of the da-Gal4 ubiquitous driver, isoforms Long A, B and C
were able to completely restore normal tracheal tube morphology, secretion of
Verm and paracellular barrier function
(Fig. 4F1-F3,F5 and
Table 1). At the cellular
level, the Long isoforms fully supported junctional assembly, with both the
Na,K-ATPase and Coracle localizing to SJs
(Fig. 4F4). By contrast, none
of the Short isoforms had full rescue ability despite being expressed at
levels comparable to or greater than those isoforms capable of full rescue
(e.g. Fig. 4, compare E4 with
F4 and H4); instead, the Short isoforms had a range of activity for tracheal
morphology and barrier function (Table
1). The Short C isoform had the least activity at the phenotypic
level, showing little rescue of DT morphology, Verm secretion or barrier
junction defects (Fig.
4E1-E3,E5). At the cellular level, Short C did not visibly support
junctional assembly, as there was no concentration of Coracle at the SJ, and
the Short C ATPase was uniformly distributed along the lateral membrane
(Fig. 4E4). Importantly, the
lateral distribution of the Short C isoform mimicked the lateral distribution
of SJ components in the absence of SJs, indicating that the Short C isoform
had little junctional organization activity. This result suggests that the
Long N-terminus is required for the -subunit to interact with
cytoplasmic proteins and/or cytoplasmic portions of membrane proteins to
support junctional assembly.
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-subunits predicted to
be catalytically inactive for tube-size control and barrier junction
activities. Across phylogeny, the Na,K-ATPase ion pump function is contained
within the -subunit and depends on an essential aspartic acid that
interacts with the terminal phosphate group of ATP to allow hydrolysis of the
high-energy bond (reviewed by Kaplan,
2002). Mutation of this invariant aspartic acid to asparagine
(D369N in vertebrates) has been shown in several systems to completely abolish
ATP hydrolysis and ion pumping (Ohtsubo et
al., 1990; Kuntzweiler et al.,
1995). Because regions of -subunits containing catalytic
function are nearly 100% identical between flies and vertebrates (for example,
the 60 residues flanking asparatic acid 369 are 98% identical between fly
ATP and the vertebrate -isoforms), it is highly likely that
mutation of the essential aspartic acid in fly -subunits completely
eliminates ion pump function. Strikingly, we found that several ATP
isoforms containing the fly equivalent of the D369N mutation rescued tube-size
and barrier junction functions as well as their WT counterparts
(Fig. 4 and
Table 1). For example,
expression of Long C in the Atp mutant background completely
restored tracheal tube-size control to wild type levels
(Fig. 4, compare C1 with F1).
In the absence of pump function, Long C D394N still retained the ability to
rescue tracheal morphology, Verm secretion and barrier function
(Fig. 4G1-G3) at the phenotypic
level, and SJ organization at the cellular level
(Fig. 4G4). Therefore,
Drosophila SJ formation requires an ion-pump-independent function of
the Na,K-ATPase. These results define a new function for a protein complex
that has been extensively studied for over 60 years.
Junctional activities are conserved between the Drosophila ATP and vertebrate 1 Na,K-ATPase subunits
The vertebrate Na,K-ATPase has been implicated in cell-cell adhesion and
polarity in a number of studies, although its exact role remains elusive.
Given the high degree of conservation between -subunits in eukaryotes
(>70% identity between flies and vertebrates), and the fact that multiple
combinations of - and ß-subunits of the different species can form
functional complexes (Schmalzing et al.,
1991), we investigated whether a vertebrate -subunit could
function in the fly context. We selected rat 1 because, of the four
vertebrate -subunit isoforms, 1 is the only one expressed in all
epithelial tissues and therefore seemed the most likely to have a junctional
function (Blanco, 2005).
Strikingly, expression of this rat 1 with an N-terminal GFP tag in the
Drosophila Atp mutant rescued the tracheal mutant phenotypes
to wild type (Fig. 4H1,H2),
restored SJ barrier function (Fig.
4H3), rescued Coracle and Verm localization
(Fig. 4H4,H5), and was able to
rescue the lethality associated with null Atp alleles. Thus,
it appears that the functions of the Na,K-ATPase -subunit in SJ
formation are conserved between flies and vertebrates.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), 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 et al.,
2004). For example, Shoshani et al.
(Shoshani et al., 2005)
provided evidence that the ß1-subunit acts as a homophilic cell adhesion
molecule, and Rajasekaran et al.
(Rajasekaran et al., 2001b)
showed that expression of the ß1-subunit and E-cadherin, but not of
either alone, could cause viraltransformed 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 and Fehon, 2003;
Paul et al., 2003). Here we
show that the junctional activity of the Na,K-ATPase is mediated by the
extracellular domain of the Nrv2 ß-subunit and that the junctional
activity does not require ion pumping by the -subunit.
The extracellular domain of the ß-subunit mediates junction formation
Using chimeric ß-subunits composed of domains from the Nrv2 isoform
that has junctional activity, and from the Nrv3 isoform that lacks junctional
activity, we 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 ß-subunits
have previously been shown to mediate -subunit ion-transport activity
(Laughery et al., 2003;
Noguchi et al., 1994) and
cell-cell adhesion interactions (Contreras
et al., 1999; Muller-Husmann
et al., 1993), to our knowledge this is the first demonstration
that the extracellular domain of the ß-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 Lachesin (Genova and Fehon,
2003; Llimargas et al.,
2004), or trans interactions that organize septa between cells, or
both, remains to be determined.
Ion-pump-independent activity of the Na,K-ATPase in SJ function
One of the most surprising results from our 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 et al.
(Rajasekaran et al., 2001a)
showed that the low intracellular Na+ concentration maintained by
the Na,K-ATPase is required for MDCK junction formation, and Lowery and Sive
(Lowery and Sive, 2005) showed
that ATPase-mediated ion transport was required for zebrafish neural tube
inflation. Shu et al. (Shu et al.,
2003) have shown that ion-transport by the 1ß1 isoform
is required for zebrafish heart morphogenesis. Hilgenberg et al.
(Hilgenberg et al., 2006)
recently showed that the neuronal 3 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.
Although ion pumping by the -subunit is not required for SJ
formation, the -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.
A model for the role of the ATPase in SJ assembly that is consistent with
the combined results of the - and ß-subunit data is that the
extracellular domain of the ß-subunit interacts with multiple
extracellular SJ components to assemble an extracellular complex, whereas the
-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 -subunit to organize junctions is the cytoskeletal
protein Ankyrin, which has been shown to bind two distinct sites on the rat
-subunit (Jordan et al.,
1995; Zhang et al.,
1998), sites which are conserved in the Drosophila
ATP (data not shown).
An evolutionary conserved role for the Na,K-ATPase in cell junction and/or polarity formation?
The ability of the rat 1 isoform to rescue all junctional defects of
Drosophila Atp-null mutants is consistent with the 77%
identity between the Drosophila ATP and the rat 1
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 our findings that Na,K-ATPase
catalytic activity is required for Drosophila viability, but that
ATP-subunits predicted to be catalytically inactive fully support SJ
formation.
|
ACKNOWLEDGMENTS |
|---|
1 was a gift from J. I.
Sznajder. Support for M.J.P. was provided by NIH R01 AG025046 and NIH R01
NS15390. G.J.B. was a recipient of an NSF Career Award IBN-0133411 and NIH R01
GM069540. |
REFERENCES |
|---|
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Affolter, M., Bellusci, S., Itoh, N., Shilo, B., Thiery, J. P. and Werb, Z. (2003). Tube or not tube: remodeling epithelial tissues by branching morphogenesis. Dev. Cell 4, 11-18.[CrossRef][Medline]
Behr, M., Riedel, D. and Schuh, R. (2003). The claudin-like megatrachea is essential in septate junctions for the epithelial barrier function in Drosophila. Dev. Cell 5, 611-620.[CrossRef][Medline]
Beitel, G. and Krasnow, M. (2000). Genetic control of epithelial tube size in the Drosophila tracheal system. Development 127,3271 -3282.[Abstract]
Blanco, G. (2005). Na,K-ATPase subunit heterogeneity as a mechanism for tissuespecific ion regulation. Semin. Nephrol. 25,292 -303.[CrossRef][Medline]
Blanco, G. and Mercer, R. W. (1998). Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function. Am. J. Physiol. 275,F633 -F650.
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Dev. Suppl. 118,401 -415.
Cereijido, M., Contreras, R. G. and Shoshani, L.
(2004). Cell adhesion, polarity, and epithelia in the dawn of
metazoans. Physiol. Rev.
84,1229
-1262.
Contreras, R. G., Shoshani, L., Flores-Maldonado, C., Lazaro, A. and Cereijido, M. (1999). Relationship between Na(+),K(+)-ATPase and cell attachment. J. Cell Sci. 112,4223 -4232.[Abstract]
Dada, L. A., Chandel, N. S., Ridge, K. M., Pedemonte, C., Bertorello, A. M. and Sznajder, J. I. (2003). Hypoxia-induced endocytosis of Na,K-ATPase in alveolar epithelial cells is mediated by mitochondrial reactive oxygen species and PKC-zeta. J. Clin. Invest. 111,1057 -1064.[CrossRef][Medline]
Fehon, R. G., Dawson, I. A. and Artavanis-Tsakonas, S. (1994). A Drosophila homologue of membrane-skeleton protein 4.1 is associated with septate junctions and is encoded by the coracle gene. Development 120,545 -557.[Abstract]
Geering, K. (2001). The functional role of beta subunits in oligomeric P-type ATPases. J. Bioenerg. Biomembr. 33,425 -438.[CrossRef][Medline]
Genova, J. L. and Fehon, R. G. (2003).
Neuroglian, Gliotactin, and the Na+/K+ ATPase are essential for septate
junction function in Drosophila. J. Cell Biol.
161,979
-989.
Hemphala, J., Uv, A., Cantera, R., Bray, S. and Samakovlis,
C. (2003). Grainy head controls apical membrane growth and
tube elongation in response to Branchless/FGF signalling.
Development 130,249
-258.
Hilgenberg, L. G., Su, H., Gu, H., O'Dowd, D. K. and Smith, M. A. (2006). Alpha3 Na+/K+-ATPase is a neuronal receptor for agrin. Cell 125,359 -369.[CrossRef][Medline]
Jaunin, P., Jaisser, F., Beggah, A. T., Takeyasu, K., Mangeat,
P., Rossier, B. C., Horisberger, J. D. and Geering, K.
(1993). Role of the transmembrane and extracytoplasmic domain of
beta subunits in subunit assembly, intracellular transport, and functional
expression of Na,K-pumps. J. Cell Biol.
123,1751
-1759.
Jordan, C., Puschel, B., Koob, R. and Drenckhahn, D.
(1995). Identification of a binding motif for ankyrin on the
alpha-subunit of Na+,K(+)-ATPase. J. Biol. Chem.
270,29971
-29975.
Kaplan, J. H. (2002). Biochemistry of Na,K-ATPase. Annu. Rev. Biochem. 71,511 -535.[CrossRef][Medline]
Knust, E. and Bossinger, O. (2002). Composition
and formation of intercellular junctions in epithelial cells.
Science 298,1955
-1959.
Kuntzweiler, T. A., Wallick, E. T., Johnson, C. L. and Lingrel,
J. B. (1995). Glutamic acid 327 in the sheep alpha1 isoform
of Na+,K+-ATPase stabilizes a K+-induced
conformational change. J. Biol. Chem.
270,2993
-3000.
Lamb, R. S., Ward, R. E., Schweizer, L. and Fehon, R. G.
(1998). Drosophila coracle, a member of the protein 4.1
superfamily, has essential structural functions in the septate junctions and
developmental functions in embryonic and adult epithelial cells.
Mol. Biol. Cell 9,3505
-3519.
Laughery, M. D., Todd, M. L. and Kaplan, J. H.
(2003). Mutational analysis of alpha-beta subunit interactions in
the delivery of Na,K-ATPase heterodimers to the plasma membrane. J.
Biol. Chem. 278,34794
-34803.
Lebovitz, R. M., Takeyasu, K. and Fambrough, D. M. (1989). Molecular characterization and expression of the (Na+ K+)-ATPase alpha-subunit in Drosophila melanogaster. EMBO J. 8,193 -202.[Medline]
Llimargas, M., Strigini, M., Katidou, M., Karagogeos, D. and
Casanova, J. (2004). Lachesin is a component of a septate
junction-based mechanism that controls tube size and epithelial integrity in
the Drosophila tracheal system. Development
131,181
-190.
Lowery, L. A. and Sive, H. (2005). Initial
formation of zebrafish brain ventricles occurs independently of circulation
and requires the nagie oko and snakehead/atp1a1a.1 gene products.
Development 132,2057
-2067.
Luschnig, S., Batz, T., Armbruster, K. and Krasnow, M. A. (2006). serpentine and vermiform encode matrix proteins with chitin binding and deacetylation domains that limit tracheal tube length in Drosophila. Curr. Biol. 16,186 -194.[CrossRef][Medline]
Manning, G. and Krasnow, M. A. (1993). Development of the Drosophila tracheal system. In The Development of Drosophila Melanogaster (ed. M. Bate and A. Martinez-Arias), pp. 609-685. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Muller-Husmann, G., Gloor, S. and Schachner, M.
(1993). Functional characterization of beta isoforms of murine
Na,K-ATPase. The adhesion molecule on glia (AMOG/beta 2), but not beta 1,
promotes neurite outgrowth. J. Biol. Chem.
268,26260
-26267.
Noguchi, S., Mutoh, Y. and Kawamura, M. (1994). The functional roles of disulfide bonds in the beta-subunit of (Na,K)ATPase as studied by site-directed mutagenesis. FEBS Lett. 341,233 -238.[CrossRef][Medline]
Ohtsubo, M., Noguchi, S., Takeda, K., Morohashi, M. and Kawamura, M. (1990). Site-directed mutagenesis of Asp-376, the catalytic phosphorylation site, and Lys-507, the putative ATP-binding site, of the alpha-subunit of Torpedo californica Na+/K(+)-ATPase. Biochim. Biophys. Acta 1021,157 -160.[Medline]
Palladino, M. J., Bower, J. E., Kreber, R. and Ganetzky, B.
(2003). Neural dysfunction and neurodegeneration in Drosophila
Na+/K+ ATPase alpha subunit mutants. J. Neurosci.
23,1276
-1286.
Paul, S. M., Ternet, M., Salvaterra, P. M. and Beitel, G. J.
(2003). The Na+/K+ ATPase is required for septate junction
function and epithelial tube-size control in the Drosophila tracheal system.
Development 130,4963
-4974.
Peifer, M., Sweeton, D., Casey, M. and Wieschaus, E. (1994). wingless signal and Zeste-white 3 kinase trigger opposing changes in the intracellular distribution of Armadillo. Development 120,369 -380.[Abstract]
Rajasekaran, S. A., Palmer, L. G., Moon, S. Y., Peralta Soler,
A., Apodaca, G. L., Harper, J. F., Zheng, Y. and Rajasekaran, A. K.
(2001a). Na,K-ATPase activity is required for formation of tight
junctions, desmosomes, and induction of polarity in epithelial cells.
Mol. Biol. Cell 12,3717
-3732.
Rajasekaran, S. A., Palmer, L. G., Quan, K., Harper, J. F.,
Ball, W. J., Jr, Bander, N. H., Peralta Soler, A. and Rajasekaran, A. K.
(2001b). Na,K-ATPase betasubunit is required for epithelial
polarization, suppression of invasion, and cell motility. Mol.
Biol. Cell 12,279
-295.
Samakovlis, C., Hacohen, N., Manning, G., Sutherland, D. C., Guillemin, K. and Krasnow, M. A. (1996). Development of the Drosophila tracheal system occurs by a series of morphologically distinct but genetically coupled branching events. Development 122,1395 -1407.[Abstract]
Schmalzing, G., Gloor, S., Omay, H., Kroner, S., Appelhans, H. and Schwarz, W. (1991). Up-regulation of sodium pump activity in Xenopus laevis oocytes by expression of heterologous beta 1 subunits of the sodium pump. Biochem. J. 279,329 -336.
Shoshani, L., Contreras, R. G., Roldan, M. L., Moreno, J.,
Lazaro, A., Balda, M. S., Matter, K. and Cereijido, M.
(2005). The polarized expression of Na+,K+-ATPase in epithelia
depends on the association between beta-subunits located in neighboring cells.
Mol. Biol. Cell 16,1071
-1081.
Shu, X., Cheng, K., Patel, N., Chen, F., Joseph, E., Tsai, H. J.
and Chen, J. N. (2003). Na,K-ATPase is essential for
embryonic heart development in the zebrafish.
Development 130,6165
-6173.
Tepass, U. and Hartenstein, V. (1994). The development of cellular junctions in the Drosophila embryo. Dev. Biol. 161,563 -596.[CrossRef][Medline]
Tepass, U., Tanentzapf, G., Ward, R. and Fehon, R. (2001). Epithelial cell polarity and cell junctions in Drosophila. Annu. Rev. Genet. 35,747 -784.[CrossRef][Medline]
Van Itallie, C. M. and Anderson, J. M. (2006). Claudins and epithelial paracellular transport. Annu. Rev. Physiol. 68,403 -429.[CrossRef][Medline]
Wang, S., Jayaram, S. A., Hemphala, J., Senti, K. A., Tsarouhas, V., Jin, H. and Samakovlis, C. (2006). Septate-junction-dependent luminal deposition of chitin deacetylases restricts tube elongation in the Drosophila trachea. Curr. Biol. 16,180 -185.[CrossRef][Medline]
Wodarz, A., Hinz, U., Engelbert, M. and Knust, E. (1995). Expression of crumbs confers apical character on plasma membrane domains of ectodermal epithelia of Drosophila. Cell 82,67 -76.[CrossRef][Medline]
Wu, V. M. and Beitel, G. J. (2004). A junctional problem of apical proportions: epithelial tube-size control by septate junctions in the Drosophila tracheal system. Curr. Opin. Cell Biol. 16,493 -499.[CrossRef][Medline]
Wu, V. M., Schulte, J., Hirschi, A., Tepass, U. and Beitel, G.
J. (2004). Sinuous is a Drosophila claudin required for
septate junction organization and epithelial tube size control. J.
Cell Biol. 164,313
-323.
Xie, Z. and Askari, A. (2002). Na(+)/K(+)-ATPase as a signal transducer. Eur. J. Biochem. 269,2434 -2439.[Medline]
Zhang, Z., Devarajan, P., Dorfman, A. L. and Morrow, J. S.
(1998). Structure of the ankyrin-binding domain of
alpha-Na,K-ATPase. J. Biol. Chem.
273,18681
-18684.
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