A novel protein complex, Mesh-Ssk, is required for septate junction formation in the Drosophila midgut
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Research Article 4923
A novel protein complex, Mesh–Ssk, is required for
septate junction formation in the Drosophila midgut
Yasushi Izumi, Yuichi Yanagihashi and Mikio Furuse*
Division of Cell Biology, Department of Physiology and Cell Biology, Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki-cho, Chuo-ku,
Kobe 650-0017, Japan
*Author for correspondence (furuse@med.kobe-u.ac.jp)
Accepted 25 June 2012
Journal of Cell Science 125, 4923–4933
ß 2012. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.112243
Summary
Septate junctions (SJs) are specialized intercellular junctions that restrict the free diffusion of solutes through the paracellular route in
invertebrate epithelia. In arthropods, two morphologically different types of SJs have been reported: pleated SJs and smooth SJs (sSJs),
which are found in ectodermally and endodermally derived epithelia, respectively. However, the molecular and functional differences
between these SJ types have not been fully elucidated. Here, we report that a novel sSJ-specific component, a single-pass
transmembrane protein, which we term ‘Mesh’ (encoded by CG31004), is highly concentrated in Drosophila sSJs. Compromised mesh
expression causes defects in the organization of sSJs, in the localizations of other sSJ proteins, and in the barrier function of the midgut.
Ectopic expression of Mesh in cultured cells induces cell–cell adhesion. Mesh forms a complex with Ssk, another sSJ-specific protein,
and these proteins are mutually interdependent for their localization. Thus, a novel protein complex comprising Mesh and Ssk has an
Journal of Cell Science
important role in sSJ formation and in intestinal barrier function in Drosophila.
Key words: Drosophila, Midgut, Epithelial cell, Smooth septate junction
Introduction criteria distinguishing these two types of SJs are the arrangement of
Epithelia play important roles as barriers that separate distinct the septa visualized in negatively stained membrane preparations
compartments within the body. To accomplish these functions, and the appearance of intramembrane particles observed in freeze-
epithelial cells have specialized intercellular junctions, designated fracture images. The septa in pSJs form regular undulating rows but
as occluding junctions, that restrict the free diffusion of solutes those in sSJs are arranged in regularly spaced parallel lines. This
across the cellular sheets through the paracellular pathway. In structural difference seems to reflect differences in the molecular
vertebrates, tight junctions (TJs) act as occluding junctions in architecture of sSJs and pSJs. Among these two SJ types, the
all epithelia, including endothelial cells. These barrier/channel molecular and functional properties of pSJs have been extensively
properties are determined primarily by membrane proteins of the analyzed in Drosophila ectodermal epithelia. The molecular
claudin family (Anderson and Van Itallie, 2009; Angelow et al., components of Drosophila pSJs include: the transmembrane
2008; Furuse, 2010). proteins, Neurexin IV (Baumgartner et al., 1996), Neuroglian
In contrast to vertebrates, the epithelial cells of invertebrates (Banerjee et al., 2010), Gliotactin (Schulte et al., 2003), Contactin
generally lack TJs (although a few exceptions have been reported). (Faivre-Sarrailh et al., 2004), Fasciclin III (FasIII) (Woods et al.,
Instead, they possess different membrane specializations, called 1997), and Lachesin (Llimargas et al., 2004); an Na+/K+ ATPase
septate junctions (SJs), which perform the role of occluding (Paul et al., 2003); and the cytoplasmic proteins, Coracle (Cora)
junctions (Lane et al., 1994a; Tepass and Hartenstein, 1994). In (Lamb et al., 1998), Discs large (Dlg) (Woods et al., 1996), Lethal
ultrathin-section electron microscopy, SJs are observed as parallel (2) giant larvae (Lgl) (Bilder et al., 2000), Scribble (Scrib) (Bilder
plasma membranes between adjacent cells with ladder-like septa and Perrimon, 2000), and Varicose (Wu et al., 2007). Among them,
spanning the intermembrane space. Morphological variants of SJs it was recently reported that Dlg is unlikely to be a core pSJ
exist across the invertebrate phyla and some animals are reported component (Oshima and Fehon, 2011). The vertebrate homologs of
to possess multiple types of SJs specific to different types of Neurexin IV, Neuroglian Contactin and Cora are concentrated at the
epithelial cells (Lane et al., 1994b; Green and Bergquist, 1982). paranodal junctions (PJs) of vertebrate myelinated axons, which
However, the molecular architectures of these SJ types are largely possess ladder-like structures similar to those observed in SJs (Bhat,
unknown. In arthropods, two major classes of SJs have been 2003). Thus, the molecular organization and morphology of pSJs are
described, based on morphological appearance: pleated SJs (pSJs) similar to that of PJs. However, three claudin-like proteins,
are observed in ectodermally derived epithelia and glia, while Megatrachea (Behr et al., 2003), Sinuous (Wu et al., 2004) and
smooth SJs (sSJs) are found mainly in the endodermally derived Kune-kune (Kune) (Nelson et al., 2010), have been identified as
midgut epithelium (Lane et al., 1994a; Tepass and Hartenstein, functional pSJ components, suggesting that pSJs also have some
1994). The outer epithelial layer of the proventriculus (OELP) common features with TJs.
and the Malpighian tubules also possess sSJs, although Morphological and physiological studies have suggested that
developmentally they originate from the ectoderm. The major sSJs function to restrict or regulate the diffusion of solutes
4924 Journal of Cell Science 125 (20)
through the paracellular pathway (Skaer et al., 1987) but detailed of midgut epithelial cells, where sSJs occur (Fig. 1A).
molecular and genetic analyses of sSJs are lacking. In Drosophila, Immunoprecipitation of the midgut membrane fraction with these
Ankyrin, a/b-spectrin, FasIII (Baumann, 2001) and Dlg (Maynard mAbs identified a ,80 kDa protein (Fig. 1B). Mass spectrometry
et al., 2010) are localized at the apicolateral region of midgut revealed this protein to be silkworm BGIBMGA009402-PA
epithelial cells, and Lgl is localized at the sSJs of the (supplementary material Fig. S1). The primary structure of this
proventriculus (Strand et al., 1994). protein contains a domain characteristic of a transmembrane-
Recently, we identified a novel protein with four membrane- spanning segment close to the C-terminus, a signal peptide, a NIDO
spanning domains, Snakeskin (Ssk), which specifically localizes at domain, an Ig-like E set domain, an AMOP domain, a vWD domain,
sSJs and is required for the organization and function of sSJs and a sushi domain (supplementary material Fig. S1). These
(Yanagihashi et al., 2012). Here, we identify a previously extracellular domains are found in cell adhesion proteins playing
uncharacterized putative membrane protein, which we have important roles in cell–cell and/or cell–matrix adhesion (Bork et al.,
named ‘Mesh’. Mesh specifically localizes at sSJs, induces cell– 1994; Ciccarelli et al., 2002; Colombatti et al., 1993; Ichinose et al.,
cell adhesion in cultured cells, and is required for the formation 1990; Mayer et al., 1998). To further investigate the function of this
and function of sSJs in Drosophila. We also found that Mesh and protein in Drosophila, we looked for its Drosophila ortholog by
Ssk display mutually dependent localizations at sSJs and form a database searching and found the CG31004 gene (Fig. 1D;
complex with each other. Therefore, we conclude that Mesh acts supplementary material Fig. S1), which is located on the right arm
together with Ssk to organize sSJs. of the third chromosome. We named CG31004 protein ‘Mesh’ for
its immunofluorescence staining images in Drosophila midgut (see
Results below). Proteins characterized by similar domain compositions exist
Mesh is a candidate for a novel sSJ-associated membrane in other invertebrates, including Caenorhabditis elegans (K03H1.5)
protein and sea urchins (LOC580458). In vertebrates, the mouse Susd2/Svs-
To further identify sSJ-specific molecules, we generated 1 ortholog is the sole protein containing the AMOP, vWD, and sushi
monoclonal antibodies (mAbs) in rats against an sSJ-containing domains (supplementary material Fig. S1), suggesting that Susd2/
Journal of Cell Science
membrane fraction obtained from the midgut of silkworm (Bombyx Svs-1 is a vertebrate ortholog of Mesh (Sugahara et al., 2007).
mori) fifth-instar larvae, and finally isolated two mAb clones that The Flybase predicts that Mesh transcripts are translated into
specifically recognized the apical region of the lateral membrane three isoforms with different C-terminal cytoplasmic regions
Fig. 1. Mesh is a candidate for a novel sSJ-localizing protein. (A) Immunofluorescence staining of a frozen section of silkworm larval midgut using a mAb of
hybridoma clone 75. The signals were observed in the lateral regions of the epithelial cells. Arrows indicate the apex of the lateral plasma membrane. Basal
membranes are delineated by dots. An asterisk indicates the lumen of the midgut. Scale bar: 50 mm. (B) The membrane fractions of silkworm larval midguts (+) or
control buffer (2) were subjected to immunoprecipitation with mAb of clone 75. The immunoprecipitate was separated on a 12% SDS-polyacrylamide gel, and
the gel was stained with Coomassie Brilliant Blue. Mass spectrometry revealed a protein of relative molecular mass of 80,000 Da (asterisk) to be silkworm
BGIBMGA009402-PA. (C) Physical map of genomic region containing the mesh gene in Drosophila. Three kinds of the splicing variants are predicted in Flybase.
The piggyBac (pBac{WH}meshf04955) was inserted into the coding sequence of mesh transcripts as shown in the figure. Gray bar: untranslated regions of the mesh
transcript. Black boxes: coding sequences of the mesh transcripts. (D) Schematic representation of Mesh structure. The three Mesh isoforms share a large
extracellular region and differ in the cytoplasmic region. The domains in the extracellular region and the piggyBac insertion in the protein are shown. The Mesh
protein is hypothesized to be cleaved at the GDPH proteolytic site in the vWD domain. TM, putative transmembrane domain.
Septate junction formation in Drosophila gut 4925
(Fig. 1C,D). A piggyBac insertion, pBac{WH}CG31004f04955 is Mesh localizes at sSJs in Drosophila
located in the region shown in the schematic drawing of the mesh To determine the expression pattern and subcellular localization
gene and the protein (Fig. 1C,D). Embryos homozygous for the of Mesh in Drosophila, anti-Mesh antibodies were generated
meshf04955 chromosome hatched into first-instar larvae but died at against the C-terminal cytoplasmic region. Western blot analysis
this stage. Df(3R)Excel6218 or Df(3R)tll-e, both of which lack the revealed that Mesh was mainly detected as a protein of relative
mesh locus, failed to complement the lethality of meshf04955. The molecular mass 90,000 in embryos, in third-instar larvae, and in
lethality of meshf04955 homozygotes was rescued by precise extracts of S2 cells expressing Mesh (supplementary material
excision of pBac{WH}CG31004f04955 and the expression of a Fig. S2A). Mesh-PA/PB consists of 1431 amino acids with a
mesh-RNAi using the 48Y-GAL4 driver at 25 ˚C caused lethality at calculated molecular mass of 162,400, suggesting that the protein
the first-instar larval stage (data not shown), demonstrating that the is processed at a specific region. Indeed, higher-molecular-mass
lethality is attributable to the piggyBac insertion in the mesh bands (,200,000) were detected (supplementary material Fig.
gene. In addition, transheterozygotes for meshf04955 and S2A), and a putative GDPH cleavage site, an autocatalytic
Df(3R)Excel6218 showed identical phenotypes to the phenotype proteolysis site in some mucins that cleaves between GD and PH
of meshf04955 homozygotes (see below; also supplementary residues (Hollingsworth and Swanson, 2004), is located in the
material Fig. S4A,B), and expression of Mesh with a 48Y-GAL4 vWD domain (a.a. 827–830 of Mesh-PA/PB) (Fig. 1D).
driver in meshf04955 homozygotes rescued their phenotype Immunofluorescence microscopic analyses revealed that the
regarding sSJ organization (Fig. 3C9,F9). We confirmed that expression of Mesh protein was first observed in the
meshf04955 eliminated the immunostaining of Mesh and that the endodermally derived tissues at embryonic stage 12 (Fig. 2A).
expression of a UAS-mesh in meshf04955 rescued the Mesh staining In late-stage embryos and third-instar larvae, Mesh was
(Fig. 3B,C,E,F). Taken together, these observations indicate that expressed in the midgut, OELP and Malpighian tubules
mesh is an essential gene and that meshf04955 is a null or strong (Fig. 2A–E), but was not expressed in the foregut and hindgut
loss-of-function allele of mesh. (Fig. 2B,D,E; supplementary material Fig. S2C), demonstrating
Journal of Cell Science
Fig. 2. Mesh localizes to sSJs. (A) Double immunofluorescence staining of wild-type embryos using anti-Mesh (green) and anti-Dlg (red) antibodies. The
expression of Mesh protein was first observed in the endodermally derived epithelial cells at embryonic stage 12 (arrow). At stage 16, Mesh was exclusively
expressed in the midgut, the OELPs and the Malpighian tubules. Dlg was expressed in both endodermally and ectodermally derived epithelial cells.
(B–E) Antibody-stained wild-type third-instar larvae analyzed in the anterior midgut (B), the middle midgut (C), the posterior midgut (D) and the Malpighian
tubules (E) using anti-Mesh antibody. Mesh was expressed in the midgut, the OELPs and the Malpighian tubules and was localized at cell–cell contact regions in
their epithelial cells. Mesh signals were not detected in the foregut (B) and hindgut (E). (F) Immunoelectron microscopy of wild-type first-instar larval midguts
using anti-Mesh antibody. Immunolabels were detected at the bicellular contacts where the septa were observed. F9 is an enlarged view of F. (G) Antibody-stained
stage-16 embryos showing the proventriculus, which includes the boundary between the ectodermally derived foregut and endodermally derived midgut. Embryos
were double-stained for Mesh (G) and Kune (G9) as markers for sSJs and pSJs, respectively. The weak Kune expressions in the OELP are indicated by open
arrows in G9. G0 shows the merged image, in which dots delineate basal membranes of epithelial cells. The boundary cells (asterisk) expressing both Mesh
(arrows) and Kune (arrowheads) are identified. Scale bars: 100 mm (A, B–E); 500 nm (F); 5 mm (G–G0).
4926 Journal of Cell Science 125 (20)
Journal of Cell Science
Fig. 3. Mesh is required for the localization of sSJ
components. (A–R) Immunofluorescence microscopic
analysis was performed in the OELPs and the anterior
midguts of first-instar larvae. In wild-type OELPs (A,G) and
midguts (D,I), Mesh was concentrated in the apicolateral
region of bicellular contacts and colocalized with Ssk
(A9, A90, D9 and D90). Dlg (A0, D0, G0 and I0), Lgl
(G9,I9), Cora (K,L) and FasIII (O,P) localized at the
apicolateral region of bicellular contacts where Mesh
colocalized with Dlg and Lgl (A90, D90, G90, I90). In
meshf04955, Ssk was mislocalized to apical and basolateral
membrane in the OELP (B9) and the midgut (E9). Dlg was
localized at the apicolateral region (B0, E0 H0 and J0). Lgl
was distributed along the lateral membrane in the meshf04955
OELP (H9) and midgut (J9) with partial concentration in the
apicolateral region (J9, arrowheads). Cora was observed at
the apicolateral region, but it spread into more basolateral
membrane regions in the meshf04955 OELP (M) and was
distributed to the cytoplasm in midgut epithelial cells (N). In
the meshf04955 OELP, FasIII was localized at the apicolateral
region (Q) and to the apical membrane (Q, arrowheads), and
it was observed as large aggregates in the apicolateral region
of the midgut (R). Expression of the UAS-mesh construct
with 48Y-GAL4 rescued Mesh (C,F) and Ssk
(C9,F9) localization in meshf04955 larvae. Scale bars: 5 mm.
that the expression of Mesh is specific for tissues bearing sSJs. localization of Mesh correspond with those of Ssk, a previously
The immunoreactivities of these antibodies were diminished in identified sSJ-specific protein (supplementary material Fig. S3A;
mesh mutant embryos and first-instar larvae, indicating the Fig. 3A–A90,D–D90). To confirm Mesh localization at sSJs, we
specificity of our anti-Mesh antibodies (supplementary material carried out immunoelectron microscopy using anti-Mesh
Fig. S2B,D). The expression pattern, timing, and subcellular antibody. As shown in Fig. 2F, immunolabels were detected at
Septate junction formation in Drosophila gut 4927
bicellular contacts where septa were observed, indicating that observed in the apicolateral region in the wild-type OELP and
Mesh specifically localizes at sSJs in larval midgut epithelial midgut epithelial cells but it was spread more in the basal
cells. Expression of Mesh was also observed in the apicolateral direction in the mesh mutant OELP and was distributed
region of epithelial cells in the adult midgut, OELP, and throughout the cytoplasm of the midgut epithelial cells
Malpighian tubules (supplementary material Fig. S3B), (Fig. 3M,N). In the mesh mutant OELP, FasIII was localized in
indicating that Mesh is a component of sSJs in Drosophila the apicolateral region but also mislocalized to the apical
from the embryo through to adulthood. membrane (Fig. 3Q). In contrast, it was observed as large
aggregates in the apicolateral region of the midgut epithelial cells
Cells at the foregut–midgut boundary possess both pSJs (Fig. 3R). Expression of the UAS-mesh construct with 48Y-
and sSJs GAL4 rescued Ssk localization (Fig. 3C9,F9). In addition, mesh-
Epithelia derived from ectoderm and endoderm possess pSJs and RNAi (12074-R1 generated by NIG-FLY) induced by 48Y-GAL4
sSJs, respectively, raising an intriguing question of how the SJs at on meshf04955/+ backgrounds decreased the level of Mesh at sSJs
their boundary are organized. The specific localization of Mesh and caused mislocalization of Ssk in the midgut epithelial cells
at sSJs enabled us to investigate this issue. Stage-16 embryos (supplementary material Fig. S5). Taken together, these results
were double-stained with antibodies to Mesh and Kune as indicate that Mesh determines the proper localization of several
markers for sSJs and pSJs, respectively. Their localizations sSJ proteins.
were closely examined in the proventriculus, which includes
the boundary between the ectodermally derived foregut and Mesh is required for proper sSJ organization
endodermally derived midgut. As shown in Fig. 2G, we To further characterize the nature of the sSJ defect in mesh
identified boundary cells expressing both Mesh and Kune mutants, ultrastructural analysis of the first-instar larvae was
(Fig. 2G–G0, asterisk). In these cells, Kune localized at the performed. In wild-type midgut epithelial cells, typical sSJs were
apicolateral membrane on the foregut side and Mesh localized on observed at cell–cell contacts (Fig. 4A, brackets). In mesh
the midgut side (Fig. 2G0), suggesting that individual boundary mutants, which were transheterozygotes for meshf04955 and
cells possess both pSJs and sSJs depending on which cells they
Journal of Cell Science
Df(3R)Excel6218, large gaps between the lateral membranes of
are adjacent to. adjacent epithelial cells were frequently observed compared with
the wild-type (Fig. 4B,C, asterisks). However, a few septa were
Mesh is required for proper localization of sSJ components
still observed at the cell–cell contacts of the mesh mutant midgut
As described above, the mesh mutant animals hatched into the
epithelial cells (Fig. 4B,C, brackets). pSJs in the mesh mutant
first-instar larvae, but died within 1 day. However, sSJs are not
completed until late stage 17 (Tepass and Hartenstein, 1994).
Therefore, in the present study, we analyzed sSJ formation in the
first-instar larvae. We focused on the OELP and the anterior
midgut epithelial cells because sSJ organization is clearest in
these columnar cells. Several pSJ proteins including Dlg, Lgl,
and FasIII have been reported to localize at the apicolateral
region of bicellular contacts in Drosophila midgut (Baumann,
2001; Maynard et al., 2010; Strand et al., 1994). We confirmed
that these proteins all colocalized with Mesh in the apicolateral
region of wild-type OELP and midgut epithelial cells
(Fig. 3A0,D0,G9,G0,I9,I0,O,P; and data not shown). We also
checked whether other pSJ proteins localize at sSJs and
observed that Cora colocalized with Mesh at the apicolateral
region (Fig. 3K,L; and data not shown). These results indicate
that Dlg, FasIII, Lgl, and Cora, at least, are both sSJ components
and pSJ components.
To examine the role of Mesh in the molecular organization of
sSJs, we analyzed the subcellular localization of the sSJ proteins
in mesh mutants. Ssk was mislocalized to the apical and
basolateral membranes of the OELP and midgut epithelial cells
in mesh mutant larvae (Fig. 3B9,E9), and was often observed as
aggregates in the cytoplasm (Fig. 3E9; supplementary material
Fig. S4B). The intensity of Ssk signals in the apical membrane
was much higher than that of the basolateral membrane. In Fig. 4. Mesh is required for sSJ organization. Transmission electron
contrast, Dlg was still localized at the apicolateral region microscopy of wild-type (A) and meshf04955/Df(3R)Exel6218 (B,C) first-
instar larval midguts. In wild-type midgut, the typical sSJs were observed at
(Fig. 3B0,E0,H0,J0), indicating that Mesh is not required for the
the bicellular contacts (A, brackets). In meshf04955/Df(3R)Exel6218 midguts,
localization of Dlg. Moreover, the polarized distribution of Dlg in
large gaps between the lateral membranes of adjacent epithelial cells were
mesh mutants suggests that Mesh does not have a significant role frequently observed (B,C, asterisks). A few septa were still observed at the
in specifying the apical-basal polarity of either OELP or midgut bicellular contacts of the meshf04955/Df(3R)Exel6218 midgut (B,C, brackets).
epithelial cells. Lgl was distributed along the lateral membrane (D,E) In contrast to the sSJs, pSJs in the epidermis were intact in meshf04955/
with partial concentration in the apicolateral region in the mesh Df(3R)Exel6218 midguts (E, bracket), as seen in the wild-type (D, bracket).
mutant OELP and midgut epithelial cells (Fig. 3H9,J9). Cora was Scale bars: 500 nm.
4928 Journal of Cell Science 125 (20)
epidermis (Fig. 4E, bracket) were indistinguishable from those in Localization of Mesh to sSJs depends on Ssk but not on
the wild-type (Fig. 4D, bracket). These results indicate that Mesh Dlg, Lgl, Cora and FasIII
is specifically required for proper sSJ organization. Since Ssk was mislocalized in mesh mutant sSJs, we next
investigated whether the localization of Mesh would be affected
Mesh is involved in the barrier function of the midgut by suppression of Ssk. As described in our previous report
epithelium (Yanagihashi et al., 2012), animals expressing ssk-RNAi with the
We speculated that Mesh is involved in the barrier function of the 48Y-GAL4 driver exhibited a reduction in Ssk expression, while
midgut epithelium. However, mesh mutant larvae fail to form the those homozygous for Df(3L)ssk showed no expression of Ssk in
three-layered structure of the proventriculus (supplementary the midgut epithelial cells (Fig. 6B9,C9). In these cells, Mesh no
material Fig. S4D), although the structure is formed correctly longer localized to the apicolateral region but was distributed
in stage-16 embryos, suggesting that mesh mutant animals cannot diffusely and formed some aggregates in the cytoplasm
maintain the proper structure of the proventriculus. Consistent (Fig. 6B,C). Thus, Mesh and Ssk are mutually dependent on each
with this observation, colored yeast fed to mesh mutant larvae did other for their proper localization; Mesh is required for the
not accumulate in the gut, whereas it was observed throughout accumulation of Ssk at sSJs, and Ssk is required for the
their gut in wild-type larvae (data not shown). This phenotype translocation of Mesh from cytoplasm to sSJs. As observed in
hampered the dye permeability assay used to examine the mesh mutants, Lgl (Fig. 6E), Cora (Fig. 6G) and FasIII (Fig. 6I)
integrity of the paracellular barrier, by feeding with a fluorescent were mislocalized in Ssk-deficient cells. However, Dlg was still
dye tracer. To overcome this problem, we generated mesh weak localized at apicolateral region (Fig. 6B0,C0). Taken together, these
loss-of-function conditions using mesh-RNAi (12074-R1) induced results suggest that Mesh acts together with Ssk to organize sSJs.
by 48Y-GAL4 on meshf04955 heterozygous backgrounds. Uninduced Next, we investigated the localization of Mesh in dlg, lgl, cora
control first-instar larvae (UAS-mesh-RNAi/meshf04955) and and fasIII null mutants. In dlgm52 and lgl4 zygotic mutants, Mesh
mesh-RNAi-induced first-instar larvae on wild-type (48Y-GAL4 and Ssk accumulated at the apicolateral region in the OELP and
.UAS-mesh-RNAi/+) or meshf04955 heterozygous (48Y-GAL4 midgut epithelial cells (data not shown), suggesting that Dlg and
.UAS-mesh-RNAi/meshf04955) backgrounds were fed fluorescent-
Journal of Cell Science
Lgl are not required for the maintenance of sSJs. Since the
labeled dextran of 10 kDa and observed by confocal microscopy. In maternally supplied Dlg and Lgl are thought to be adequate for the
control larvae, the midgut was well contrasted, with the fluorescent establishment of cell polarity and sSJs organization, we examined
tracer confined within the midgut (Fig. 5, upper panel). In contrast, the phenotype of dlgm52 and lgl4 maternal/zygotic mutant sSJs.
the tracer was detected in various parts of the body cavity in mesh- When eggs from wild-type animals were allowed to develop for
RNAi-expressing meshf04955 heterozygous larvae (Fig. 5, middle 24 h at 25 ˚C, they hatched into first-instar larvae and their midguts
panel), indicating leakage of the tracer from the lumen of the developed a tube-like structure. In contrast, dlgm52 and lgl4
midgut. These observations indicate that Mesh is required for the maternal/zygotic mutants exhibited a hypertrophied midgut
barrier function of the midgut epithelium in Drosophila. However, phenotype (Manfruelli et al., 1996). In the midgut epithelial cells
we did not observe significant leakage of the tracer on the mesh- of these mutants, Mesh and Ssk accumulated in the apicolateral
RNAi-induced wild-type background (Fig. 5, lower panel), region with faint leakage to the lateral membrane (Fig. 6J,K). In
suggesting insufficient RNAi-mediated suppression of Mesh on fasIIIE25 mutants, Mesh was localized to the apicolateral region in
the wild-type background. the midgut epithelial cells (Fig. 6L). Since cora5 mutant animals
Fig. 5. Mesh is required for barrier functions in midgut. Dye permeability assays of larvae with a weak mesh loss-of-function achieved by using mesh-RNAi
(12074-R1) induced by 48Y-GAL4. Uninduced control first-instar larvae (UAS-mesh-RNAi/meshf04955, upper panel) and mesh RNAi-induced first-instar larvae on
wild-type (48Y-GAL4 .UAS-mesh-RNAi/TM6B, lower panel) or meshf04955 heterozygous (48Y-GAL4 .UAS-mesh-RNAi/meshf04955, middle panel)
backgrounds were fed Alexa-Fluor-555-labeled 10 kDa dextran. In the control and mesh-RNAi-induced wild-type larvae, the midgut was defined clearly, with the
fluorescent tracer confined within the midgut (upper and lower panels). In contrast, the tracer was detected in various parts of the body cavity in mesh-RNAi-
expressing meshf04955 heterozygous larvae (middle panel). In the right panels, the background signals of green fluorescence excited by 488-nm laser irradiation
were used to trace the larval shape. GFP signals (arrow) are derived from the TM6B Ubi-GFP balancer in the mesh-RNAi-induced wild-type background larvae
(48Y-GAL4 .UAS-mesh-RNAi/TM6B). The images were taken in the same visual field. Scale bar: 100 mm.Septate junction formation in Drosophila gut 4929
Fig. 6. Ssk is required for sSJ localization of Mesh.
Immunofluorescence microscopic analyses were performed
for the anterior midguts of the first-instar larvae or the
embryos. (A,D,F,H) In control (UAS-ssk-RNAi/TM6B, ssk-
RNAi-uninduced larvae) midguts, Mesh was concentrated in
the apicolateral region of bicellular contacts (A,A90). Ssk
(A9,A90), Dlg (A0,A90), Lgl (D), Cora (F) and FasIII (H) were
localized at the apicolateral region of bicellular contacts in
control midguts. (B,C,E,G,I) The first-instar larvae
expressing ssk-RNAi with the 48Y-GAL4 driver (48Y-
GAL4.UAS-ssk-RNAi/TM6B) exhibited a reduction in Ssk
expression in the midgut (B9). In these cells, Mesh was
distributed diffusely and formed aggregates in the cytoplasm
(B). In Df(3L)ssk midguts, in which Ssk signals were not
observed (C9), Mesh was distributed diffusely and formed
aggregates in the cytoplasm (C). Dlg was localized at the
apicolateral region in the ssk-RNAi (B0) and Df(3L)ssk
(C0) midguts. Lgl (E), Cora (G) and FasIII (I) were
mislocalized in the ssk-RNAi midguts. (J,K) In dlgm52 (J) and
lgl4 (K) maternal/zygotic mutants, Mesh was accumulated in
Journal of Cell Science
the apicolateral region of the midgut. (L) In fasIIIE25 mutants,
Mesh was localized to the apicolateral region of the midgut.
(M,N) In cora5 mutants, Mesh was localized to the
apicolateral region of the stage-16 OELP (N), as seen in the
wild-type (M). Scale bar: 5 mm.
fail to hatch into larvae, we observed Mesh localization in the apicolateral region of the stage-16 OELP in cora5 mutants. Taken
stage-16 OELP, by which time Mesh as well as Cora had together, these results indicate that the accumulation of Mesh
accumulated in the apicolateral region of the wild-type (Fig. 6M, within the apicolateral region of the plasma membrane depends on
data not shown). As shown in Fig. 6N, Mesh was localized to the Ssk, but not on Dlg, Lgl, Cora or FasIII.
Mesh forms a complex with Ssk
Mesh and Ssk were mutually dependent for their localization at sSJs
(Figs 3,6), raising the possibility that Mesh is physically associated
with Ssk. When the embryonic and larval extracts of Drosophila
were subjected to immunoprecipitation with anti-Mesh antibodies,
Ssk coprecipitated with Mesh (Fig. 7A,B). Consistently, Mesh
coprecipitated with Ssk during immunoprecipitation from
embryonic extracts with anti-Ssk antibodies (Fig. 7C). Neither
Mesh nor Ssk was precipitated by the pre-immune sera (Fig. 7A–C).
These results indicate that Mesh forms a complex with Ssk in vivo.
Mesh mediates the cell–cell adhesion
To investigate the possible role of Mesh as a cell–cell adhesion
molecule, we transfected Drosophila S2 cells with a Mesh–EGFP
expression vector and carried out an aggregation assay to
examine their adhesive properties. When S2 cells expressing
Mesh–EGFP were co-cultured with those expressing mCherry,
only Mesh–EGFP-expressing cells formed the cell aggregation
(Fig. 8A–C). Since S2 cells appear to lack endogenous Mesh
Fig. 7. Mesh forms a complex with Ssk. Mesh co-immunoprecipitated with
(supplementary material Fig. S2A), this experiment shows that
Ssk. The embryonic (A) and larval (B) extracts were subjected to
immunoprecipitation (IP) with anti-Mesh antibodies. Mesh was Mesh expression leads to cell aggregation in a homophilic
immunoprecipitated with anti-Mesh antibodies, but not with pre-immune manner. Furthermore, Mesh accumulated at cell–cell contact
serum (A,B, upper panel). The immunoprecipitates of Mesh contained Ssk regions between two cells expressing Mesh–EGFP (Fig. 8E,F,
(A,B, lower panel). (C) Ssk immunoprecipitates from embryonic extracts also arrows). These results suggest that Mesh organizes sSJs by
contained Mesh (upper panel). mediating cell adhesion via its homophilic interaction.4930 Journal of Cell Science 125 (20)
Fig. 8. Ectopic expression of Mesh induces cell-cell adhesion in
S2 cells. S2 cells transfected with the expression vectors for Mesh–
EGFP (A,C) and mCherry (B,C) were co-cultured. The S2 cell
aggregations were formed in Mesh–GFP-expressing cells
(A,C, arrow) but not in mCherry-expressing cells (B,C). (C) The
brightfield image was merged with the images of A and B.
(D–F) Mesh–EGFP (E,F, arrow) but not EGFP (D) accumulated at
the cell–cell contact region. Scale bars: 50 mm (A–C); 5 mm (D–F).
Discussion expression in the OELP but not in the midgut (Fig. 2G0).
We have identified a novel membrane-spanning protein, Mesh, Therefore, the boundary cell may have the ability to form either
which is specifically localized at sSJs and has cell adhesion sSJs or pSJs according to the SJ type of adjacent cells. The
activity. Mesh is required for the formation of sSJs and occurrence of such ‘SJ-boundary cells’ seems to be crucial because
paracellular diffusion barriers in the Drosophila midgut. This they connect the ectodermally and endodermally derived epithelia
study, together with the recent identification of Ssk (Yanagihashi into a tandem tube while maintaining the continuity of the
et al., 2012), whose interaction with Mesh was shown in the paracellular barrier. However, we cannot completely exclude the
present study, provides a key starting point for understanding possibility that small amounts of pSJs and sSJs are also contained
sSJs, which must play crucial roles in the gut and renal functions in the sSJs on the midgut side and pSJs on the foregut side of the
SJ-boundary cells, respectively, to form hybrid junctions.
Journal of Cell Science
of arthropods, at the molecular level.
Implication of Mesh in the ultrastructure of sSJs Interdependency between Mesh and Ssk for their sSJ
Electron microscopic observations have shown that sSJs and pSJs localization
can be distinguished morphologically. Obliquely sectioned pSJs Our analyses of Mesh and Ssk have clarified their interaction,
and sSJs are visualized as regular undulating rows and regularly interdependency in their localizations, and requirements for the
spaced parallel lines, respectively (Lane et al., 1994b), while both organization and barrier function of sSJs, suggesting that Mesh–
types of SJs have ladder-like structures in the intermembrane Ssk is a key system for sSJ formation. In mesh mutants, Ssk failed
space. Of the two sSJ-specific integral membrane proteins, Ssk is to localize at sSJs, but mislocalized to the apical and basolateral
unlikely to be the structural element of the septa in sSJs, because plasma membrane domains. In ssk-RNAi and Df(3L)ssk fly, Mesh
its extracellular loops are both too short (25 and 22 a.a., no longer localized at the sSJs, but was distributed in the
respectively) to bridge the intercellular space. In contrast, Mesh cytoplasm. Ssk may translocate Mesh from the cytoplasm to sSJs
induces cell–cell adhesion, implying that it may be one of the or to the plasma membrane. However, how the Mesh–Ssk complex
components of the septa observed in ultrathin section electron recognizes and localizes to sSJ regions remain elusive. Mesh
microscopy. Faint ladder-like structures were still observed in the expression in S2 cells leads to cell aggregation without Ssk
mesh mutants, suggesting that other membrane proteins also expression, suggesting that there is a mechanism by which Mesh
contribute to the septal structures. FasIII is such a candidate translocates to the cell membrane and induces cell–cell adhesion
because it shows cell–cell adhesion activity (Snow et al., 1989) and independently of Ssk in S2 cells. Detailed analysis of the dynamics
was still distributed to the apicolateral region, as well as the apical of Mesh–Ssk distribution will shed light on the mechanisms of sSJ
region, in the mesh mutants (Fig. 3Q,R). However, fasIII null formation and the sorting systems for sSJ proteins.
mutant flies are viable (Whitlock, 1993) and both Mesh and Ssk are
normally localized at their sSJs, indicating that FasIII is dispensable A complicated hierarchy among sSJ components
for sSJ formation. FasIII may provide robustness to the Mesh–Ssk- By using Mesh and Ssk as specific markers for sSJs, we
mediated sSJ organization via its cell–cell adhesion activity. confirmed that Dlg, Lgl and FasIII localize at sSJs in the larval
OELP and midgut epithelial cells. In addition, we found that Cora
SJ-boundary cells at foregut–midgut boundary is also concentrated into sSJs. Among these proteins that are
The issue of how SJs are organized in cells at the boundary generally known as pSJ components, Lgl, Cora and FasIII were
between pSJ- and sSJ-bearing epithelia is intriguing. Interestingly, mislocalized in mesh mutants and ssk-RNAi lines. On the other
we observed boundary cells in which the pSJ marker Kune and sSJ hand, Lgl, Cora and FasIII were not required for the localization
marker Mesh were concentrated in the anterior and posterior of Mesh and Ssk at the apicolateral membrane. These
regions, respectively, of the apicolateral membranes. This result observations imply a possible hierarchy in the molecular
suggests that individual cells possess both pSJs and sSJs depending constituents of sSJs; Mesh-Ssk might act as a platform for the
on the orientation of their plasma membranes. The proventriculus assembly of Lgl, Cora and FasIII in endodermal epithelia. Such a
is originally derived from ectoderm (Tepass and Hartenstein, feature in sSJs is in sharp contrast to that in pSJs where each
1994). However, the OELP bears sSJs and expresses Mesh and molecular component is interdependent. Mutations in most of the
Ssk, suggesting that the OELP has both ectodermal and genes encoding pSJ-associated proteins result in disruption of the
endodermal characters. In fact, we observed weak Kune barrier function and mislocalization of other pSJ proteins (FehonSeptate junction formation in Drosophila gut 4931
et al., 1994; Baumgartner et al., 1996; Behr et al., 2003; Genova Bryant), cora5 (a gift from R. G. Fehon), UAS-ssk-RNAi and Df(3L)ssk
(Yanagihashi et al., 2012). Germline clones of lgl4 and dlgm52 were made by the
and Fehon, 2003; Paul et al., 2003; Schulte et al., 2003; Faivre- FLP-DFS technique (Chou and Perrimon, 1992). For the phenotype rescue
Sarrailh et al., 2004; Llimargas et al., 2004; Wu et al., 2004; Wu experiment, pUAST vectors (Brand and Perrimon, 1993) containing mesh were
et al., 2007; Nelson et al., 2010). constructed and a fly strain carrying this construct was established. 48Y-GAL4,
which drives GAL4 expression in the anterior and posterior midgut primordium
Interestingly, in mesh mutants and ssk-RNAi lines, Dlg still from embryonic stage 10 (Martin-Bermudo et al., 1997), was used to express UAS-
localized at the apicolateral region of the OELP and midgut mesh in meshf04955 for the rescue experiment.
epithelial cells, although sSJs were disrupted at the ultrastructural
level. Furthermore, Mesh and Ssk were distributed to the Membrane fraction from silkworm midgut
apicolateral region in dlg mutants, suggesting that Mesh-Ssk The membrane fraction was prepared from midguts of silkworm 5th-instar larvae
according to the method described previously (Yanagihashi et al., 2012).
and Dlg are independent in their localizations. This is consistent
with a recent report that Dlg is probably not a core pSJ Production of monoclonal antibodies and identification of the antigens
component (Oshima and Fehon, 2011). Nevertheless, a functional Rat mAbs against membrane fractions of silkworm fifth-instar larval midguts were
relationship exists between Dlg and Lgl in determining cell generated as described previously (Yanagihashi et al., 2012). For identification of
the antigens, the membrane fractions (,500 mg) were centrifuged at the maximum
polarity in ectodermally derived epithelia. Therefore, in the speed in a microcentrifuge for 20 min and the pellet was resuspended in 500 ml of
absence of Mesh and Ssk, Dlg may be unable to function properly lysis buffer [25 mM Tris-HCl, pH 8, 27.5 mM NaCl, 20 mM KCl, 25 mM
because of an inadequate level of Lgl in the apicolateral regions. sucrose, 10 mM EDTA, 10 mM EGTA, 1 mM DTT, 10% (v/v) glycerol, 1% NP40
In fact, dlgm52 and lgl4 maternal/zygotic mutants exhibited a and protease inhibitor cocktail (Nakarai, Kyoto, Japan)] for 30 min at 4 ˚C. The
lysates were centrifuged at the maximum speed for 20 min, and the supernatants
similar hypertrophied midgut phenotype (data not shown), were used for immunoprecipitation with protein G sepharose (GE Healthcare)
suggesting that these proteins may function together in conjugated with the mAbs. The sepharose preparations were incubated with the
endodermal epithelia, as well as in ectodermal epithelia (Bilder supernatants for 4 h at 4 ˚C and were washed five times in lysis buffer. Bound
proteins were separated by SDS-PAGE and analyzed by Coomassie Brilliant Blue
et al., 2003; Tanentzapf and Tepass, 2003). G-250 (Wako) staining. Mass spectrometry analyses of the tryptic peptide mass
The functions of Dlg, Lgl, Cora and FasIII at sSJs remain data were carried out by the Integrated Center for Mass Spectrometry (Kobe
unknown. Dlg may act together with Lgl to regulate the apical- University Graduate School of Medicine). The resulting tryptic peptide mass data
basal polarity in the early stage of epithelial development. In the were matched against the NCBInr database using the Mascot program.
Journal of Cell Science
late developmental stage, compensation mechanisms for the Dlg
Production of polyclonal Abs
function may rescue the apicolateral localization of Mesh, as noted A region of the Mesh PA/PB protein (amino acids 1211–1431) was cloned into
in ectodermally derived epithelial cells of dlgm/z and lglm/z mutants pGEX-6P (GE Healthcare) to produce a GST-fusion protein. The proteins were
(Bilder et al., 2003; Tanentzapf and Tepass, 2003). As larval expressed in Escherichia coli. Polyclonal antibodies were generated in rabbits
(995-1 and -2) and rats (8002) by MBL (Nagoya, Japan).
midgut sSJs are completed at the end of embryogenesis (stage 17)
in Drosophila (Tepass and Hartenstein, 1994), the organization of Immunohistochemistry
sSJ may not be influenced by early polarity defects of dlgm/z and Embryos were fixed with 3.7% formaldehyde in PBS for 20 min. Larvae were
lglm/z mutants. Alternatively, Dlg and Lgl may be important for the dissected in Hanks’ Balanced Salt Solution and fixed with 3.7% formaldehyde in
regulation of the epithelial cell shape change that induces the PBS with 0.4% Triton X-100. The following antibodies were used: rabbit and rat
anti-Mesh, rabbit anti-Ssk (6981-1; 1:1000) (Yanagihashi et al., 2012), rabbit anti-
midgut tube-like structure (Manfruelli et al., 1996). In ectodermally Kune (1:1000) (Nelson et al., 2010), mouse anti-Dlg 1:50 [Developmental Studies
derived epithelia, Cora acts together with Yurt to regulate the Hybridoma Bank (DSHB)], mouse anti-coracle C615.16 1:50 (DSHB), mouse anti-
apicobasal polarity (Laprise et al., 2006; Laprise et al., 2009). Thus, FasIII 1:20 (DSHB), rabbit anti-Lgl 1:1000 (provided by F. Matsuzaki, RIKEN
CDB). Alexa Fluor 488-conjugated (Invitrogen), and Cy3- and Cy5-conjugated
Cora and a Yurt-like molecule may function together to organize (Jackson ImmunoResearch Laboratories) secondary antibodies were used at 1:400.
sSJs and/or to regulate the endodermal epithelial polarity. Samples were mounted in Vectashield (Vector Laboratories). Images were acquired
with a confocal microscope (model TCS-SPE; Leica) with its accompanying
software using HC PLAN Apochromat 206NA 0.7 and HCX PL Apochromat
Homologous proteins of Mesh in vertebrates
636NA 1.4 objective lens (Leica). Images were processed with Adobe PhotoshopH.
Homologous proteins, characterized by similar extracellular domains
to Mesh, are present in vertebrates (e.g. mouse Susd2/SVS-1), Electron microscopy
implying that this family of proteins shares functions conserved First-instar larvae of wild-type or mesh mutants were dissected and fixed overnight at
across species. Mouse Susd2/SVS-1 has been suggested as a tumor- 4 ˚C with a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M
cacodylate buffer (pH 7.4). The specimens including the midguts were prepared as
reversing gene product, because it inhibited the growth of cancer cell described previously (Yanagihashi et al., 2012). For immunoelectron microscopy,
lines (Sugahara et al., 2007). Susd2/SVS-1 was distributed in the first instar larvae were dissected and fixed for 2 h at room temperature with 4%
apical membrane of the epithelial cells in renal tubules and bronchial paraformaldehyde in 0.1 M sodium phosphate buffer (PB) (pH 7.4). The specimens
were washed three times with 50 mM glycine in PB and incubated with 0.1% saponin
tubes, suggesting that it does not contribute to the cell–cell adhesion in PB. After blocking with 10% normal goat serum in PB for 1 h, they were incubated
and/or paracellular barrier function in vertebrate epithelial cells. for 2 days at 4 ˚C with anti-Mesh antibody (995-2; 1:1000) diluted in the blocking
However, expressing Susd2/SVS-1 in HeLa cells induces the cell solution. After six washes with PB, the specimens were incubated for 2 h with a
aggregation (Sugahara et al., 2007), implying that this protein family secondary antibody that had been conjugated with both the 1.4 nm NANOGOLD
particles (1:100; Nanoprobes, Inc.), followed by six washes. The specimens were
conserves the cell–cell adhesion activity. Further studies of the fixed for 15 min with 2.5% glutaraldehyde in PB, washed with 50 mM glycine in PB,
functions of Mesh–Susd2/SVS-1 family proteins in vertebrates and and again four times with 50 mM HEPES, pH 5.8 for 15 min. Signals were silver-
in invertebrates will lead to a better understanding of the conserved enhanced by use of an HQ-silver kit (Nanoprobes, Inc.) for 14 min in the dark. After
thorough washing with distilled water, they were fixed with 0.5% osmium oxide in
physiological functions in these proteins and of the evolution of PB for 1.5 h on ice and washed again with distilled water. Subsequently the
intercellular junctions across species. specimens were embedded with Epon 812. The ultrathin sections (50–100 nm) were
stained doubly with 4% hafnium (IV) chloride and lead citrate, and observed with a
Materials and Methods JEM-1011 electron microscope (JEOL) at an accelerating voltage of 80 kV.
Fly stocks and genetics
The fly strains meshf04955, Df(3R)Exel6218, fasIIIE25, lgl4 and 48Y-GAL4, were Co-immunoprecipitation and western blotting
obtained from the Bloomington Stock Center, and the mesh-RNAi strains, 12074- Wild-type fly embryos and third-instar larvae were mixed with a 5-fold volume of
R1 was obtained from NIG-FLY. We also used the strains dlgm52 (a gift from P. J. lysis buffer [25 mM Tris-HCl pH 8, 27.5 mM NaCl, 20 mM KCl, 25 mM4932 Journal of Cell Science 125 (20)
Sucrose, 10 mM EDTA, 10 mM EGTA, 1 mM DTT, 10% (v/v) glycerol, 0.5% neurexin is required for septate junction and blood-nerve barrier formation and
NP40 and protease inhibitor cocktail from Sigma] and homogenized using a pestle function. Cell 87, 1059-1068.
for 1.5 ml microfuge tubes. The method for immunoprecipitation was essentially Behr, M., Riedel, D. and Schuh, R. (2003). The claudin-like megatrachea is essential in
the same as described above. Anti-Mesh (995-1 and -2) and anti-Ssk (6981-1 septate junctions for the epithelial barrier function in Drosophila. Dev. Cell 5, 611-
and -2) antibodies were used for the immunoprecipitation and the 620.
immunocomplexes were separated by SDS-PAGE, transferred to polyvinylidene Bhat, M. A. (2003). Molecular organization of axo-glial junctions. Curr. Opin.
difluoride membranes and probed with an anti-Ssk (6981-1; 1:1000) and an anti- Neurobiol. 13, 552-559.
Mesh (995-1; 1:1000) antibodies. Bilder, D. and Perrimon, N. (2000). Localization of apical epithelial determinants by
the basolateral PDZ protein Scribble. Nature 403, 676-680.
Bilder, D., Li, M. and Perrimon, N. (2000). Cooperative regulation of cell polarity and
Cell culture and aggregation assay growth by Drosophila tumor suppressors. Science 289, 113-116.
For the ectopic expression of Mesh in S2 cells, Mesh–EGFP and mCherry Bilder, D., Schober, M. and Perrimon, N. (2003). Integrated activity of PDZ protein
(Clontech) cDNA were subcloned into pMT-V5His (Invitrogen) and EGFP cDNA complexes regulates epithelial polarity. Nat. Cell Biol. 5, 53-58.
was subcloned into pAC-V5His (Invitrogen). S2 cells were cultured at 25 ˚C in Bork, P., Holm, L. and Sander, C. (1994). The immunoglobulin fold. Structural
Schneider medium containing 10% fetal bovine serum and antibiotics. DNAs classification, sequence patterns and common core. J. Mol. Biol. 242, 309-320.
(pAC-EGFP, pMT-mCherry, and pMT-Mesh-EGFP) were transfected into cells Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering
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immunostaining or aggregation assay. To induce the expression from pMT vectors, Chou, T. B. and Perrimon, N. (1992). Use of a yeast site-specific recombinase to
copper sulfate (final concentration: 500 mM) was added to the culture medium at produce female germline chimeras in Drosophila. Genetics 131, 643-653.
24 h after transfection. For immunostaining, the cells were transferred onto Ciccarelli, F. D., Doerks, T. and Bork, P. (2002). AMOP, a protein module
concanavalin A-coated coverslips and incubated for 2 h before fixation with alternatively spliced in cancer cells. Trends Biochem. Sci. 27, 113-115.
methanol-acetone (1:1) for 10 min at 220 ˚C. After washing with PBS with 0.05% Colombatti, A., Bonaldo, P. and Doliana, R. (1993). Type A modules: interacting
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temperature, followed by incubation with Alexa Fluor 488-conjugated secondary Faivre-Sarrailh, C., Banerjee, S., Li, J., Hortsch, M., Laval, M. and Bhat, M. A.
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dissociated by repeated pipetting and the cell concentrations were readjusted with Fehon, R. G., Dawson, I. A. and Artavanis-Tsakonas, S. (1994). A Drosophila
cell culture medium to 16106 cells/ml. The cells were shaken at 100 rpm on a homologue of membrane-skeleton protein 4.1 is associated with septate junctions and
rotation platform at room temperature. Aggregation of the cells was analyzed after is encoded by the coracle gene. Development 120, 545-557.
Furuse, M. (2010). Molecular basis of the core structure of tight junctions. Cold Spring
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Acknowledgements Lane, N. J., Campiglia, S. S. and Lee, W. M. (1994a). Junctional types in the tissues of
We are grateful to S. Yonemura, A. Nagafuchi, and all the members an onychophoran: the apparent lack of gap and tight junctions in Peripatus. Tissue
of Furuse laboratories for helpful discussions. We also thank F. Cell 26, 143-154.
Lane, N. J., Dallai, R., Martinucci, G. and Burighel, P. (1994b). Electron microscopic
Matsuzaki for the antibody and the fly stocks, and R. G. Fehon, P. J. structure and evolution of epithelial junctions. In Molecular Mechanisms of Epithelial Cell
Bryant, the Bloomington Stock Center, the Drosophila Genetic Junctions: From Development to Disease (ed. S. Citi), pp 23-43. R. G. Landes Co.: Austin,
Resource Center at Kyoto Institute of Technology and the fly stocks TX.
of National Institute of Genetics (NIG-Fly) for fly stock. Laprise, P., Beronja, S., Silva-Gagliardi, N. F., Pellikka, M., Jensen, A. M.,
McGlade, C. J. and Tepass, U. (2006). The FERM protein Yurt is a negative
regulatory component of the Crumbs complex that controls epithelial polarity and
Funding apical membrane size. Dev. Cell 11, 363-374.
This work was supported in part by grants from the Japan Society for Laprise, P., Lau, K. M., Harris, K. P., Silva-Gagliardi, N. F., Paul, S. M., Beronja,
the Promotion of Science (JSPS) [grant number 09009170 to Y. I.]; S., Beitel, G. J., McGlade, C. J. and Tepass, U. (2009). Yurt, Coracle, Neurexin IV
and the Na(+),K(+)-ATPase form a novel group of epithelial polarity proteins. Nature
Takeda Science Foundation (to M. F. and Y. I.); Hyogo Science and 459, 1141-1145.
Technology Association (to Y. I.); and by the ‘‘Funding Program for Llimargas, M., Strigini, M., Katidou, M., Karagogeos, D. and Casanova, J. (2004).
Next Generation World Leading Researchers (NEXT Program)’’ of Lachesin is a component of a septate junction-based mechanism that controls tube
JSPS, initiated by the Council for Science and Technology Policy size and epithelial integrity in the Drosophila tracheal system. Development 131, 181-
190.
[grant number LS084 to M. F.].
Manfruelli, P., Arquier, N., Hanratty, W. P. and Sémériva, M. (1996). The tumor
suppressor gene, lethal(2)giant larvae (1(2)g1), is required for cell shape change of
Supplementary material available online at epithelial cells during Drosophila development. Development 122, 2283-2294.
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.112243/-/DC1 Martin-Bermudo, M. D., Dunin-Borkowski, O. M. and Brown, N. H. (1997).
Specificity of PS integrin function during embryogenesis resides in the alpha subunit
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