LES ETAPES DU COACHING – AJC-RH – Coaching et Formation …

Loading...

Taking too long?

Reload document

| Open in new tab

Dlg3 Trafficking and Apical Tight Junction Formation Is
Regulated by Nedd4 and Nedd4-2 E3 Ubiquitin Ligases

Citation for published version:
Van Campenhout, CA, Eitelhuber, A, Gloeckner, CJ, Giallonardo, P, Gegg, M, Oller, H, Grant, SGN,
Krappmann, D, Ueffing, M & Lickert, H 2011, ‘Dlg3 Trafficking and Apical Tight Junction Formation Is
Regulated by Nedd4 and Nedd4-2 E3 Ubiquitin Ligases’, Developmental Cell, vol. 21, no. 3, pp. 479-491.
https://doi.org/10.1016/j.devcel.2011.08.003

Digital Object Identifier (DOI):
10.1016/j.devcel.2011.08.003

Link:
Link to publication record in Edinburgh Research Explorer

Document Version:
Publisher’s PDF, also known as Version of record

Published In:
Developmental Cell

General rights
Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)
and / or other copyright owners and it is a condition of accessing these publications that users recognise and
abide by the legal requirements associated with these rights.

Take down policy
The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer
content complies with UK legislation. If you believe that the public display of this file breaches copyright please
contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and
investigate your claim.

Download date: 15. Jul. 2021

Edinburgh Research Explorer Developmental Cell

Article

Dlg3 Trafficking and Apical Tight Junction
Formation Is Regulated by Nedd4
and Nedd4-2 E3 Ubiquitin Ligases

Claude A. Van Campenhout,1 Andrea Eitelhuber,2,8 Christian J. Gloeckner,4,7,8 Patrizia Giallonardo,1,8 Moritz Gegg,1
Heide Oller,1 Seth G.N. Grant,5,6 Daniel Krappmann,2 Marius Ueffing,4,7 and Heiko Lickert1,3,*
1Institute of Stem Cell Research
2Institute of Toxicology
3Institute for Diabetes and Regeneration Research
4Department of Protein Science
Helmholtz Zentrum Mu¨ nchen, 85764 Neuherberg, Germany
5Welcome Trust Sanger Institute, CB10 1SA Cambridge, UK
6Centre for Neuroregeneration Research, School of Molecular and Clinical Medicine, The University of Edinburgh, EH16 4TJ Edinburgh, UK
7Centre for Ophthalmology, Institute for Ophthalmic Research, University of Tuebingen, 72076 Tuebingen, Germany
8These authors contributed equally to this work
*Correspondence: heiko.lickert@helmholtz-muenchen.de
DOI 10.1016/j.devcel.2011.08.003

SUMMARY

The Drosophila Discs large (Dlg) scaffolding protein
acts as a tumor suppressor regulating basolateral
epithelial polarity and proliferation.
In mammals,
four Dlg homologs have been identified; however,
their functions in cell polarity remain poorly under-
the X-linked
stood. Here, we demonstrate that
mental retardation gene product Dlg3 contributes
to apical-basal polarity and epithelial junction forma-
tion in mouse organizer tissues, as well as to planar
cell polarity in the inner ear. We purified complexes
associated with Dlg3 in polarized epithelial cells,
including proteins regulating directed trafficking
and tight junction formation. Remarkably, of the
four Dlg family members, Dlg3 exerts a distinct func-
tion by recruiting the ubiquitin ligases Nedd4 and
Nedd4-2 through its PPxY motifs. We found that
these interactions are required for Dlg3 monoubiqui-
tination, apical membrane recruitment, and tight
junction consolidation. Our findings reveal an unex-
pected evolutionary diversification of the vertebrate
Dlg family in basolateral epithelium formation.

INTRODUCTION

During embryogenesis, acquisition of cell polarity is essential for
epithelium formation, asymmetric cell division, or directed cell
migration. Loss of cell polarity is one of the hallmarks of cancer
progression. Genetic studies conducted in Drosophila led to
the identification of
three cytoplasmic scaffolding proteins
required for both the control of cell polarity and proliferation:
Discs large (Dlg), Lethal giant larvae (Lgl), and Scribbled (Scrib).
In larvae that have a single mutation in one of these neoplastic

In vertebrate epithelial cells the apical

tumor suppressor genes, epithelial cells from the imaginal discs
and the brain lobes overgrow, whereas loss of cell polarity leads
to metastatic tumor formation (Bilder et al., 2000). Dlg, Scrib, and
Lgl are essential to establish basolateral polarity and function at
the septate junction (SJ; Woods et al., 1996). In contrast, apical
polarity is established by the Crumbs complex (Roh et al., 2003;
Tepass et al., 1990) in conjunction with the PAR-aPKC (partition-
ing defective-atypical protein kinase C) complex (Ohno, 2001),
which both counteract the activity of the Dlg-Lgl-Scrib complex.
junctional complex
(AJC) is formed by the apical tight junction (TJ) and the more
basally localized adherens junction (AJ; Shin et al., 2006; Tepass,
2003). The TJ functions as a fence separating the apical from
the basolateral membrane domain and also constitutes a barrier
against fluid diffusion. The AJ Cadherin-Catenin complex en-
sures cell-cell adhesion (Aberle et al., 1996). Both apical polarity
complexes, Crumbs and PAR-aPKC, play crucial roles in the
formation of the AJC and the maintenance of tissue architecture
(Lemmers et al., 2004; Suzuki et al., 2001). The Crumbs complex
is located apically (Tanentzapf and Tepass, 2003), whereas the
PAR-aPKC complex is localized close to the AJC (Izumi et al.,
1998). The Dlg complex is localized on the basolateral
membrane below the AJC (Naim et al., 2005). Given that the
Drosophila Dlg localizes to the SJ (Woods et al., 1996), one might
expect the vertebrate Dlgs to localize and function at the func-
tional analog TJ. This apparent discrepancy illustrates the fact
that the membrane recruitment and molecular functions of the
vertebrate Dlg complexes in apical-basal (AB) polarity and AJC
formation are far from being understood.

In mammals, four Dlgs have been identified. These belong to
the MAGUK (membrane associated guanylate kinase) family
of adaptor proteins and contain three different types of pro-
tein-protein interaction (PPI) domains: three postsynaptic den-
sity-95/DLG/zonula occludens-1 (PDZ) domain, one Src homol-
ogy domain-3 (SH3), and one guanylate kinase-like (GUK)
domain. The Dlgs act, via these PPI domains, as scaffolds to
organize membrane regions and regulate ion channels, signaling

Developmental Cell 21, 479–491, September 13, 2011 ª2011 Elsevier Inc. 479

Dlg Paralogs Functional Diversification in Mammals

Developmental Cell

molecules or other adaptor proteins (Sans et al., 2003). Several
knockout (KO) studies in mice highlighted a major role for the
Dlg homologs in central nervous system activity (Cuthbert
et al., 2007; Migaud et al., 1998), and accordingly, loss-of-func-
tion mutations in human DLG3 causes nonsyndromic X-linked
mental retardation (Tarpey et al., 2004). Several studies demon-
strated an important role for Dlg1 during embryonic and organ
development (Caruana and Bernstein, 2001; Mahoney et al.,
2006). Nevertheless, it remains unclear whether all mammalian
Dlgs function in establishing basolateral epithelial polarity or
whether they have functionally diverged during evolution.

The Nedd4 (neural precursor cell-expressed developmentally
downregulated 4) family of ubiquitin ligases (E3) consists of nine
members in mammals. The ancestral ligases Nedd4 and Nedd4-2
are most closely related to each other and consist of a N-terminal
calcium/lipid and/or protein-binding C2 domain, three to four
WW (Tryptophan Tryptophan; Bork and Sudol, 1994) PPI
domains, and a C-terminal HECT (Homologous to E6-AP C
Terminus of the human papilloma virus) ubiquitin-ligase domain
(Huibregtse et al., 1995). Both Nedd4 and Nedd4-2 have iden-
tical specificity for ubiquitin-conjugating enzymes (Fotia et al.,
2006) and bind PPxY (PY) motifs in their key substrates via their
WW domains (Kanelis et al., 2001, 2006; Staub et al., 1996). In
mammals many potential substrates and/or binding partners of
the Nedd4 family have been described, including neurotrans-
mitter channels, growth factor receptors, and signaling proteins.
Adaptor proteins contribute to the specificity, diversity, and
overall function of both Nedd4 and Nedd4-2; thus, identification
and biochemical characterization of interactors greatly facilitate
our understanding of these E3 ligases.

Here, we describe that Dlg3 contributes to the establishment
of AB and planar cell polarity (PCP) in the mouse embryo. We
further identify Nedd4 and Nedd4-2 as Dlg3 interactors and
establish that this interaction results in Dlg3 monoubiquitination
and apical membrane recruitment. Importantly, we demonstrate
that the Dlg3-Nedd4(-2) PPI promotes TJ formation and has
contributed to paralog diversification among Dlgs.

RESULTS

Homozygous Dlg3 Mutations Cause Midgestational
Embryonic Lethality
We recently identified Dlg3 in a screen for X-linked genes
required for mouse embryonic development (Cox et al., 2010).
A hemizygous male (XY) mouse embryonic stem cell (mESC)
line with a gene-trap (GT)
insertion in intron 10 of Dlg3
(GtP038A02) was used to generate completely mESC-derived
embryos via the tetraploid complementation method (Nagy
et al., 1993). At embryonic day (E) 9.0, Dlg3GtP038A02/Y embryos
displayed an array of phenotypic severity that ranged from
morphologically normal (data not shown) to a failure of embry-
onic turning (n = 5 out of 18, Figure 1C), which was associated
in rare cases with lack of anterior neural induction (n = 1 out of
18; Figure 1D).

To confirm our findings, we intercrossed hemizygous male
and heterozygous female mice carrying a null Dlg3tm1Grnt allele
on the inbred C57BL/6 background (Cuthbert et al., 2007). The
examination of Dlg3 mutant embryos at various stages of
development revealed no discernible defects prior to E8.0 (see

Figure S1A available online). From E8.5 onward, Dlg3 mutants
are statistically underrepresented, and recovered mutants dis-
played incompletely penetrant defects in embryonic turning,
failure of chorioallantoic fusion, posterior truncations (n = 16
out of 38), and lack of anterior neural induction (n = 6 out of 38;
Figures 1A and 1B). Dlg3tm1Grnt null embryos also show occa-
sionally an open brain phenotype (n = 8 out of 38; Figures S1B
and S1C). Taken together, both the Dlg3GtP038A02 and Dlg3tm1Grnt
mutant alleles cause embryonic lethality with low penetrance.

Dlg3 Contributes to AB Polarity in the Mesendodermal
Lineage and PCP in the Inner Ear
In a mESC <-> tetraploid embryo chimera wild-type (WT) tetra-
ploid cells contribute only to extraembryonic tissues, such as
yolk sac and placenta, and to the gut tube of the early embryo
(Figure 1E; Kwon et al., 2008; Tam and Rossant, 2003). Surpris-
ingly, when tetraploid complementation experiments were per-
formed using the Dlg3GtP038A02/Y mutant mESC line (n = 29
chimeras generated in three independent experiments), WT
tetraploid cells contributed more extensively to the epithelial
lineages of the endoderm, including the fore- (n = 17), mid-
(n = 22), and hindgut (n = 28; Figures 1F and 1H–1K), when
compared to control experiments (Figures 1E and S1F). In a
subset of chimeras, WT tetraploid cells were also found in axial
mesendoderm tissues, namely the prechordal plate, notochord,
and ventral node (n = 11; Figures 1F, 1G, 1K, and S1F). This
contribution is specific because it was never observed in
chimeras produced with 68 other X-linked GT mutant mESC
lines (Cox et al., 2010) and rescues the Dlg3 mutant phenotype.
Our chimera analysis suggests that Dlg3 acts cell autonomously
in the axial mesendoderm and definitive endoderm lineages:
organizer tissues required for neural
induction, neural tube
patterning and closure, as well as embryonic turning.

Lack of Dlg3 may result in defects in definitive endoderm and
axial mesendoderm specification and/or morphogenesis. To
discriminate between these possibilities, we examined differen-
tiation of these tissues using the Forkhead transcription factor
Foxa2 (Ang and Rossant, 1994) as a marker. Embryos were
also stained with an antibody directed against the apical cilia
marker, Arl13b (Caspary et al., 2007), to distinguish the axial
mesendoderm (notochord) from the definitive endoderm. Before
E8.0, the axial mesendoderm and definitive endoderm lineages
were established at comparable ratios, and no obvious morpho-
logical defects were discernible between WT and Dlg3 mutant
embryos (data not shown). At E8.5, we frequently observed an
enlarged ventral node region and kinked axial midline in mutant
embryos (8 out of 14; Figures 1L–1O), suggesting that the axial
mesendodermal elongation movement had failed.

To understand the origin of the node cell accumulation, we
examined the localization of AB polarity and AJC markers in
the mutant embryos. In WT posterior notochord cells, the AJ
protein E-cadherin is localized along the basolateral regions of
the PM (Figures 2A–2C) but is mislocalized at the apical PM in
mutant cells (Figures 2D–2F and S2G–S2I). Similar results were
observed with the TJ marker ZO-1 (Figures 2G–2L and S2I)
and apical polarity marker aPKC (Figures 2M–2R and S2I).
Loss of Dlg3 slightly, but significantly, increases the overall cell
proliferation, whereas the apoptosis rate is unchanged (Figures
S2A–S2F). Because no changes in proliferation rate were

480 Developmental Cell 21, 479–491, September 13, 2011 ª2011 Elsevier Inc.

Developmental Cell

Dlg Paralogs Functional Diversification in Mammals

Figure 1. Homozygous Dlg3 Mutations Cause Midgestational Embryonic Lethality
(A–D) Lateral views of (A) WT and (B–D) Dlg3 homozygous mutant embryos at E9.0–E9.5. Note the similarity of the phenotype observed with the two different Dlg3
mutant alleles: (B) Dlg3tm1Grnt and (C and D) Dlg3GtP038A02. White arrowheads indicate the tail bud (tb) truncation, absence of embryo turning, unfused allantois
(all), and in some embryos forebrain (fb) and midbrain (mb) deletions, whereas the hindbrain (hb) remains intact. At E9.0, the GT b-galactosidase (lacZ) reporter
gene is expressed ubiquitously.
(E–K) WT tetraploid embryos expressing a DsRed transgene were aggregated with (E) WT or (F–K) Dlg3GtP038A02/Y mESCs and isolated at E8.75–E9.0. (E–G) Red
arrowheads indicate WT tetraploid cells (red) contributing to (E) the yolk sac (ys) or to (F and G) the node (n), midgut (mg), foregut (fg), notochord (nc), and
prechordal plate (pcp). (H–K) Chimeras were immunostained with anti-RFP (red) in combination with anti-Foxa2 or anti-T (green) antibodies. DAPI (blue) has been
used to mark all nuclei. Optical sections were taken at the level (H) of the foregut, (I) midgut, (J) hindgut (node outlined by broken oval), and (K) notochord. White
arrows indicate tetraploid WT cells, expressing (H–J) the endoderm marker Foxa2 or (K) the notochord marker T.
(L–O) Whole-mount immunofluorescence combined with confocal imaging. Ventral views of the hindgut and node region in (L and M) WT and (N and O) Dlg3 homozygous
mutant embryos at E8.5. White dashed lines and ovals mark the midline and the node, respectively. Embryos were stained with DAPI (blue) and with Foxa2 (green) and
Arl13b (red) antibodies. At the level of the node, some Dlg3 mutants present a lateral expansion of Foxa2 and Arl13b-positive cells compared to the WT (compare i and ii).
Scale bars represent 300 mm in (A)–(G), 50 mm in (H)–(K), and 75 mm in (L)–(O).
See also Figures S1 and S2.

Developmental Cell 21, 479–491, September 13, 2011 ª2011 Elsevier Inc. 481

Dlg Paralogs Functional Diversification in Mammals

Developmental Cell

observed in the axial mesendoderm (data not shown), enlarge-
ment of the node region in Dlg3 mutants could result from
abnormal anterior-posterior (AP) elongation of the notochord,
which depends on PCP-mediated convergence and extension
movements. To seek evidence for a possible function of Dlg3
in PCP, we investigated tissue polarity in the inner ear, which
revealed clear defects in Dlg3 mutant embryos at E18.5 (Figures
S1D and S1E). Thus, analogous to Drosophila Dlg, mammalian
Dlg3 influences both AB polarity (Woods et al., 1996) and PCP
(Bellaı¨che et al., 2001).

The Mammalian Dlg1–Dlg4 Show Different
Tissue-Specific and Subcellular Localization
Within the Dlg family, Dlg3 shows the earliest reported polarity
phenotype. To dissect possible functional differences and
redundancies between individual Dlgs, we investigated the
mRNA expression of Dlg1–Dlg4 during early mouse develop-
ment. Reverse transcriptase-polymerase chain reaction (RT-
PCR) analysis revealed that all four Dlgs are expressed from
gastrulation to the onset of organogenesis (Figure 3A). Whole-
mount in situ hybridizations showed that at E8.5, Dlg1, Dlg3,
and Dlg4 are expressed in the tail bud region at the junction
where presomitic mesodermal cells undergo mesenchymal-
epithelial transition to condense and form segmented somites
(Figure 3B). All Dlgs are expressed in epithelial neuroectodermal
cells of the head region, whereas Dlg2, Dlg3, and Dlg4 are
also expressed in the epithelial lining of the gut along the AP
axis (Figure 3B). Thus, the tissue-specific expression of the
different Dlg family members suggests a partially redundant
function in epithelial polarization in all three germ layers.

Next, we characterized the tissue distribution and the subcel-
lular localization of the Dlg proteins during gastrulation at E7.5.
Dlg1 is localized to all three germ layers, whereas Dlg2, Dlg3,
and Dlg4 show higher abundance in the mesoderm and endo-
derm compartment (Figure S3). The four Dlgs are found at cell
junctions in epithelial cell types, but interestingly, these proteins
are also present at clearly distinct intracellular localizations
(Figures 3C–3F). In ventral node cells only Dlg1 expression is
restricted to the basolateral membrane of epithelial cells, analo-
gous to Drosophila Dlg (Figure 3C). In contrast, Dlg2 and Dlg3 are
located along the cell membrane with a peak in distribution at the
level of the apical membrane (Figures 3D and 3E). Dlg4 is found
along the PM and is not restricted to a particular compartment
(Figure 3F).

In stable transfected and polarized Madine Darby canine
kidney (MDCK) cells, a N-terminal
tagged Strep-Flag-Dlg3
(SF-Dlg3) localizes to both cytoplasm and membrane, compa-
rable to endogenous Dlg3 in mouse embryos (compare Figures
3E and 3G). Whereas Dlg1 is strictly localized to the basolateral
membrane (Figure 3H), Dlg3 membrane localization extends to
the apical apex and AJC, where it colocalizes with TJ markers
(Figures 3I ad 3J). These unexpected findings suggest that
during evolution the different Dlgs have acquired paralog-
specific functions in the consolidation of polarized membrane
domains.

Scale bars represent 25mm.
See also Figures S1 and S2.

Figure 2. Loss of AB Cell Polarity in Dlg3 Mutant
WT and Dlg3 mutant embryos were whole-mount immunostained with the
indicated antibodies, and nuclei were marked with DAPI (blue) at E8.5. White
dashed ovals indicate the node. The levels of transverse optical sections (i and
ii) are marked by white dashed lines. Pictures show ventral views on the mouse
node with the anterior facing up.
(A–F) Expression of the cilia marker Arl13b (red) and the AJ marker E-cadherin
(green) in WT and Dlg3 mutant embryos. Note the loss of basolateral localization
of the AJ marker E-cadherin in Dlg3 mutant cells indicated by arrowheads (i and ii).
(G–L) Overlapping expression of cilia (Arl13b; red) and TJ (ZO-1; green)
markers in Dlg3 mutant embryos in comparison with WT. Loss of cell polarity is
visible in higher magnification images (compare i and ii).
(M–R) Expression of apical epithelium (aPKC; red) and AJ (E-cadherin; green)
in WT and Dlg3 mutant embryos. Note the ectopic localization of E-cadherin in
the apical PM (compare i and ii). Cells losing AB polarity are positive for the
axial mesendoderm and definitive endoderm marker Foxa2 (data not shown;
Figures S2G and S2H).

482 Developmental Cell 21, 479–491, September 13, 2011 ª2011 Elsevier Inc.

Developmental Cell

Dlg Paralogs Functional Diversification in Mammals

Identification of the Dlg3 Interactome in Polarized
Epithelial Cells
With the goal of understanding Dlg paralog diversification, we
screened for Dlg3 interaction partners in polarized MDCK cells
that establish fully functional
junctions when grown at high
density. We performed tandem affinity purification (TAP) using
SF-Dlg3 in conditions optimized for the native purification of
protein complexes (Gloeckner et al., 2007). As a control, TAPs
were performed in parallel with untransfected MDCK cells. The
purified proteins were identified by liquid chromatography
coupled with tandem mass spectrometry (LC-MS/MS).

The identified Dlg3 interactome consists of polarity-associ-
ated proteins such as the TJ-associated protein 1 (TJAP1/Pilt),
a known interaction partner of Dlg1 (Kawabe et al., 2001) and
Dlg2, which shows similar apical localization as Dlg3 (Figure 4A).
Furthermore, we identified the protein phosphatase 1 that has
been recently described as being part of an AJC regulating
Par-3/aPKC activity (Hendrickx et al., 2009; Traweger et al.,
2008) and the motor protein Dynein required for vectorial trans-
port of vesicles to the apical surface (Lafont et al., 1994). We also
interactions of Dlg3 with Nedd4-2, an E3-
identified potential
ubiquitin ligase involved in protein trafficking, and the Nedd4-
binding protein 3 (N4BP3; Murillas et al., 2002), a scaffolding
protein that recruits Nedd4. These observations, in combination
with our in vivo findings, strongly suggest a function of Dlg3 and
possibly also Nedd4(-2) in cell polarity and AJC formation.

Dlg3 Interaction with the E3-Ubiquitin Ligases Nedd4
and Nedd4-2 Increases during Cell Polarization
To characterize the Dlg3-Nedd4(-2) interaction, we immunopre-
cipitated recombinant SF-Dlg3 from polarized MDCK cells.
Using antibodies specific to the Nedd4 ubiquitin ligases
(Nedd4 and Nedd4-2) on immunoblots of the Strep eluate, we
detected endogenous Nedd4 and Nedd4-2, confirming our
TAP findings (Figure 4B). The interaction of Dlg3 with the
Nedd4 protein was confirmed in vivo by coimmunoprecipitation
(coIP) of the endogenous Nedd4 ligases from brain tissues
together with Dlg3 (Figure 4C). During epithelial polarization sub-
confluent cells first establish E-cadherin mediated contacts that
correspond to the ensuing AJs. This is followed by recruitment of
TJ-associated proteins. Evidence from our interactome studies
suggests that Dlg3 and Nedd4(-2) E3 ligases may establish
polarization-dependent protein associations (Figures 4A and
4B). To test this hypothesis, we immunoprecipitated SF-Dlg3
from stable MDCK transfectants grown under unpolarized or
polarized conditions. We observed that higher levels of endoge-
nous Nedd4(-2) ligases were pulled down from polarized cells
compared to unpolarized cells (Figures 4D and 4E).

Immunolocalization studies in MDCK transfectants revealed
that Nedd4(-2) and Dlg3 colocalize in the cytoplasm (Figure 4F

(G–J) Immunolocalization studies of stably expressed SF-Dlg3 in polarized
MDCK cells. Cells were stained against Flag-epitope (red) in combination
with antibodies specific for polarity markers (green). Note that, contrary to
Dlg1, SF-Dlg3 is not restricted to the basolateral membrane and is also found
at the TJ marked by ZO-1 and Sec8 (yellow arrowheads) and at the apical
membrane (white arrowheads).
Scale bars represent 500 mm in (B), 25 mm in (C)–(F), and 10 mm in (G–J).
See also Figure S3.

Developmental Cell 21, 479–491, September 13, 2011 ª2011 Elsevier Inc. 483

Figure 3. The Mammalian Dlg1–Dlg4 Show Distinct mRNA Expres-
sion and Protein Localization
(A) RT-PCR analysis of the Dlg mRNA expression at E7.5–E9.5. Expression of
Actin mRNA has been used for normalization.
(B) Whole-mount in situ hybridization analysis of the four Dlgs’ mRNA at E8.5.
Note that the Dlgs are expressed in regions of epithelial-mesenchymal and
mesenchymal-epithelial transition. fb, forebrain; mb, midbrain; hb, hindbrain;
ba, branchial arches; st, septum transversum; mg, midgut; s, somites; pm,
presomitic mesoderm; hg, hindgut.
(C–F) Whole-mount localization studies of the Dlg proteins and the AJ marker
b-catenin in ventral (vn) and dorsal (dn) node cells. (C) Dlg1 colocalizes with
b-catenin at the basolateral PM and cell junctions. Note the absence of Dlg1 at
the apical PM (green arrowheads). (D and E) Dlg2 and Dlg3 are expressed in
the cytoplasm and along the PM with a higher expression at the apical region
(red arrowheads). (F) Dlg4 is present all around the PM, including the apical
domain (red arrowheads).

Dlg Paralogs Functional Diversification in Mammals

Developmental Cell

Figure 4. Dlg3 Interacts with Cell Polarity and TJ-Associated Proteins
(A) Interactome of Dlg3-binding proteins in polarized MDCK cells.
(B) Confirmation of the identified Dlg3 interactome. The SF-tagged Dlg3 was immunoprecipitated from stably transfected MDCK cells using Streptavidin affinity
beads (IP Strep). The total lysate (right panel) and the IP (left panel) were split and subjected to anti-Nedd4, anti-Nedd4-2, anti-PP1, anti-TJAP1, anti-Dynein
intermediate chain (IC), and anti-Flag via western blotting. Note the specific detection of endogenous Nedd4 (two isoforms), Nedd4-2 (two isoforms), PP1, TJAP1,
and Dynein IC in the IP. MDCK cells expressing the SF tag alone were used as negative control.
(C) The Nedd4 ubiquitin ligases interact with Dlg3 in vivo. Brain tissues were used to specifically immunoprecipitate (IP) endogenous Nedd4. The lysate and IP
were split and subjected to anti-Dlg3 (top panel) and anti-Nedd4 (bottom panel). An anti-GFP antibody was used as a specificity control.
(D and E) Dlg3 and Nedd4 interaction increases with polarization. SF-Dlg3 was immunoprecipitated (IP Strep) from unpolarized (Unpol.) or polarized (Pol.) MDCK
cells. Equivalent amounts of SF-Dlg3 bait were pulled down from the different samples. Note that the amount of Nedd4 ligases immunoprecipitated is higher in
polarized cells in comparison with unpolarized cells. Five percent input of the total lysate is shown as a loading and specificity control. (E) Data from three
independent experiments were compiled and quantified using Photoshop. Interaction strength in the unpolarized conditions is arbitrarily set to 1. Errors bars
indicate standard deviation in the graphs (mean ± SD; p < 0.05). (F) Colocalization studies of Dlg3 and the Nedd4 ligases during epithelial polarization. In unpolarized MDCK cells, SF-Dlg3 and Nedd4/Nedd4-2 localize mainly in the cytoplasm (top panels; scale bar, 10 mm). In polarized MDCK cells, Dlg3 and Nedd4/Nedd4-2 colocalize at the PM and in the cytoplasm (middle panel; scale bar, 10 mm). Transverse cryosection stained for Dlg3 and Nedd4(-2) at the level of the node at E8.5 (bottom panel; scale bar, 25 mm). Note the colocalization of Dlg3 and Nedd4 at the apical PM (white arrowheads). ne, neurectoderm; vn, ventral node. See also Figure S4. and S4A–S4F), whereas polarization led to an increased colocal- ization at the apical membrane and AJC (Figure 4F). This is consistent with the colocalization of Dlg3 and Nedd4(-2) at the apical PM of neural ectoderm and ventral node cells at E8.5 (Figure 4F). Taken together with the fast recruitment of Dlg3 to the apical PM and TJ in a repolarization assay (Figures S4G–S4L), these data suggest that the Dlg3-Nedd4(-2) in- teractions might be important for membrane recruitment during cell polarization. Importantly, both the PPI with the apical trans- port motor protein Dynein IC (Figure 4B) and the Sec8 com- ponent of the exocyst complex (Figure 6B) depend on functional Dlg3-Nedd4(-2) interactions (see below). 484 Developmental Cell 21, 479–491, September 13, 2011 ª2011 Elsevier Inc. Developmental Cell Dlg Paralogs Functional Diversification in Mammals Dlg3 Is the Only Dlg Family Member Containing PY Motifs for Nedd4(-2) Ligase Binding To map the Dlg3 interaction domain with the Nedd4(-2) ligases, we used a series of different Dlg3 deletion mutants in coIP exper- iments (Figure 5A). We observed that the PDZ domain of Dlg3 weakly binds to the endogenous Nedd4(-2) ligases, whereas Dlg3DPDZ does not. In contrast the deletion mutants of Dlg3 that were missing either the SH3 or GUK domain remained capable of binding the endogenous Nedd4 and Nedd4-2 (Figure 5A). A large number of Nedd4 interactors contain PY motifs that mediate direct interaction with the WW domains of Nedd4 proteins (Kanelis et al., 2001, 2006). Sequence analysis of Dlg proteins revealed that Dlg3 contains two PY motifs, which are located before and in between the Nedd4(-2) interacting PDZ domains (Figure 5C). Point mutations that resulted in an amino acid substitution of Tyrosine for Alanine in both PY motifs com- pletely abolished the interaction of Dlg3 with endogenous Nedd4(-2) proteins (Figure 5B). Another remarkable feature was the conservation of the Dlg3 PY motifs in vertebrates (Fig- ure 5C), even though these motifs are absent in other mammalian Dlgs and in Drosophila Dlg (Figure S5). We observed that only Dlg3 is capable of binding to the Nedd4 and Nedd4-2 proteins (Figure 5D). Nedd4 Directly Catalyzes Dlg3 Monoubiquitination Given the known function of the E3-ubiquitin ligase Nedd4 in protein trafficking by monoubiquitination and in targeted protein degradation by polyubiquitination, we tested the capacity of In the presence of NEDD4 to mediate Dlg3 ubiquitination. NEDD4 and after SF-Dlg3 IP after very stringent SDS boiling conditions, we observed a slightly retarded band that is de- tected with an anti-Flag as well as an anti-ubiquitin antibody, providing evidence that NEDD4 can catalyze Dlg3 monoubiqui- tination (Figure 6A). A catalytically inactive form of NEDD4, NEDD4(C-A)-Myc, which still associates with Dlg3, cannot promote SF-Dlg3 monoubiquitination (Figures 6A and S6). We used a series of Dlg3 mutants to map the region targeted for monoubiquitination by NEDD4 (Figure S6A). The Dlg3 mutant for the NEDD4-binding motif (SF-Dlg3YA1+2) cannot be monoubi- quitinated, providing further evidence that Nedd4 directly targets Dlg3. The SF-PDZ alone is not modified by ubiquitin, which could either be due to an inefficient association with Nedd4 (Figure 5A), or it may indicate that ubiquitination takes place outside of this region. Thus, we determined ubiquitination in C-terminal Dlg3 deletion mutants that are efficiently interacting with Nedd4 (Fig- ure 5A). Whereas deletion of the GUK did not affect the extent of monoubiquitination, removal of the SH3 domain caused a com- plete loss of NEDD4-mediated ubiquitin attachment (Figure S6A). These results suggest that lysines within the SH3 domain are either directly targeted for ubiquitin modification by NEDD4, or the removal of the SH3 domain exerts a conformational change that prevents ubiquitin attachment to Dlg3. Dlg3-Nedd4(-2) Association Regulates Intracellular Trafficking via the Exocyst Complex to Contribute to Polarity and TJ Consolidation During epithelialization the targeting of membrane-associated proteins to cell junctions is controlled by the exocyst pathway (Hsu et al., 1999). Dlg3 interacts with the exocyst component Sec8 to regulate microtubule (MT)-dependent neurotransmitter intracellular trafficking from the endoplasmic reticulum to the synaptic membrane (Sans et al., 2003). We found that the inter- action of Dlg3 with the exocyst and Dynein apical transport motor protein complexes also occurs in polarized MDCK cells and specifically depends on the capacity of SF-Dlg3 to bind the Nedd4(-2) ligases, whereas interaction with PP1 was not affected by the PY mutations (Figure 6B). To test whether Dlg3-Nedd4(-2) binding is involved in targeted delivery of proteins to the apical membrane and TJ, we derived cysts from untransfected MDCK cells and from MDCK cells that stably expressed SF-Dlg3 or SF-Dlg3YA1+2. We observed that SF-Dlg3YA1+2 overexpression results in abnormal polariza- tion of the MDCK cysts, whereas SF-Dlg3 overexpression does not (Figure S6C). In contrast to SF-Dlg3, SF-Dlg3YA1+2 mislocal- izes to the basolateral PM of MDCK cells and is absent from both the apical membrane and TJ (Figure S6C). Using a combination of different siRNAs designed to knock down Nedd4 and/or Nedd4-2 (Figures 6C and 6E–6G), we could confirm that Nedd4 and Nedd4-2 are required for TJ formation in polarized MDCK cells (Figures 6D, 6H, and 6I). Finally, we tested the functional impact of the Dlg3-Nedd4(-2) interaction in vivo. We stably expressed equal levels of SF-Dlg3 and SF-Dlg3YA1+2 in embryonic stem cells (ESCs) (Figure 7A) and performed tetraploid complementation to generate completely ESC-derived embryos.