letters
Electrochemical cues regulate assembly of the Frizzled/
Dishevelled complex at the plasma membrane during
planar epithelial polarization
Matias Simons1, William J. Gault1, Daniel Gotthardt2, Rajeev Rohatgi3, Thomas J. Klein1, Youming Shao4,
Ho-Jin Lee4, Ai-Luen Wu5, Yimin Fang5, Lisa M. Satlin3, Julian T. Dow6, Jie Chen5, Jie Zheng4, Michael Boutros7
and Marek Mlodzik1
Dishevelled (Dsh) is a cytoplasmic multidomain protein
that is required for all known branches of the Wnt signalling
pathway1–3. The Frizzled/planar cell polarity (Fz/PCP)
signalling branch requires an asymmetric cortical localization
of Dsh, but this process remains poorly understood. Using a
genome-wide RNA interference (RNAi) screen in Drosophila
melanogaster cells, we show that Dsh membrane localization
is dependent on the Na+/H+ exchange activity of the plasma
membrane exchanger Nhe2. Manipulating Nhe2 expression
levels in the eye causes PCP defects, and Nhe2 interacts
genetically with Fz. Our data show that the binding and surface
recruitment of Dsh by Fz is pH- and charge-dependent. We
identify a polybasic stretch within the Dsh DEP domain that
binds to negatively charged phospholipids and appears to
be mechanistically important. Dsh recruitment by Fz can
be abolished by converting these basic amino-acid residues
into acidic ones, as in the mutant, DshKR/E. In vivo, the
DshKR/E(2×) mutant with two substituted residues fails to
associate with the membrane during active PCP signalling but
rescues canonical Wnt signalling defects in a dsh-background.
These results suggest that direct interaction between Fz and
Dsh is stabilized by a pH and charge-dependent interaction
of the DEP domain with phospholipids. This stabilization is
particularly important for the PCP signalling branch and, thus,
promotes specific pathway selection in Wnt signalling.
Membrane recruitment of Dsh is an important aspect of both canonical
(β-Catenin) and non-canonical (PCP) Wnt signalling1,4. In canonical signalling, Wnt ligands trigger the formation of Dsh-dependent signalosomes
at the plasma membrane5. Similarly, asymmetric membrane association of
Dsh is a key event in the Wnt-Fz/PCP signalling branch (for reviews see
refs 2 and 3). It remains unclear whether the membrane association of Dsh
in canonical and PCP signalling uses the same mechanism.
Dsh consists of three domains, DIX, PDZ and DEP, and binds weakly
to Fz via its PDZ domain through a conserved KTxxxW motif in the Fz
carboxyl terminus6. The DEP domain is also important in membrane
targeting as it is both necessary and sufficient for Fz-dependent recruitment7. However, no binding site for Fz has yet been identified in the
DEP domain. Based on the structure of the DEP domain, it has been
suggested that a polybasic amino acid stretch could interact with acidic
and negatively charged lipids in the membrane8.
To newly identify components required for the stable formation of
Fz–Dsh complexes at the plasma membrane, we developed a cell-based
assay that is applicable for high-throughput approaches (Fig. 1a–e). In
D. melanogaster S2R+ cells (as well as in other cell lines) co-expression
of Fz with Dsh–GFP led to efficient recruitment of Dsh–GFP to the
membrane9 (Fig. 1b, g). Using this assay, we performed a genome-wide
RNAi screen for factors required in Fz-mediated Dsh–GFP membrane
recruitment. Cells were plated into 57 384-well plates, with each well
containing long double-stranded RNA against approximately 90% of
all D. melanogaster genes10, and were then transfected with Dsh–GFP
and Myc-Fz plasmids (Fig. 1a, b). The plates were analysed and scored
by visual inspection using a fluorescence microscope (see Methods).
Apart from the positive controls (fz, dsh, par-111), the screen identified
several unknown genes and factors known for their role in protein
trafficking (for example SNAP). These latter factors were shown to
block Fz-transport thereby inhibiting Dsh membrane recruitment
(data not shown).
1
Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA. 2Department of
Medicine, Division of Gastroenterology, University Hospital Heidelberg, D‑69120 Heidelberg, Germany. 3Department of Pediatrics, Mount Sinai School of Medicine,
One Gustave L. Levy Place, NY, NY 10029, USA. 4Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA. 5Department
of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, 601 South Goodwin Avenue, Urbana, IL 61801, USA. 6Institute of Biomedical and Life
Sciences, Division of Molecular Genetics, University of Glasgow, Glasgow, UK. 7German Cancer Research Center, Division of Signaling and Functional Genomics, Im
Neuenheimer Feld 580, D‑69120 Heidelberg, Germany.
1,8
Correspondence should be addressed to M.M. (e-mail: [email protected]).
Received 15 October 2008; accepted 27 November 2008; published online 22 February 2009; corrected online 24 February 2009; DOI: 10.1038/ncb1836
286
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© 2009 Macmillan Publishers Limited. All rights reserved.
letters
a
RNAi library
b
c
Control dsRNA
Nhe2 dsRNA
d
e
S2R+ cells
dsRNA
bathing
Fz + Dsh–GFP
transfection
Visual scoring
*
40
20
0
j
Complete
Partial
+S3226
–NH4
g
–NH4
h
i
+NH4
0.6
0.4
0.2
– NH4 + NH4
Dsh–GFP
con siRNA
Dsh–GFP
NHE3 siRNA Dsh–GFP
k
l
Dsh–GFP
Myc–Fz
n
o
Dsh–GFP
LactC2–RFP
m
NHE3 siRNA
pH; 71
None
*
*
80
0.8
0
Con siRNA NHE3 siRNA NHE3 siRNA
+ NH4
– NH4
– NH4
f
Defective Dsh recruitment
(percentage)
+DMSO
60
∆pHi units min–1
Defective Dsh recruitment
(percentage)
f
60
40
Merge
p
20
0
Untreated
H+ load /
Na+-free
No H+ load/
Na+-free
H+ load/Na+
Merge
Figure 1 Nhe2/NHE3 activity is required for Fz-mediated Dsh recruitment.
(a) Genome-wide RNAi library screen for Fz-mediated Dsh recruitment in
D. melanogaster S2R+ cells. The cells were plated in 57 384-well plates
containing dsRNA against 90% of D. melanogaster genes, transfected
with myc–fz and dsh–GFP plasmids and analysed visually (see Methods).
(b, c) Screen hits were defined by a × 20 microscope field with >10 cells
showing defective Dsh–GFP recruitment. Control (Relish dsRNA) showed
complete recruitment (b, see enlarged cell, inset), Nhe2 dsRNA caused
defects in recruitment (c), see arrowheads and inset for defects. (d, e)
HEK293T cells expressing Myc–Fz and Dsh–GFP were treated with the
NHE3 inhibitor S3226 at 50 μM for 12 h. Treatment led to a redistribution
of Dsh–GFP (e) as opposed to the control (DMSO) (d). (f) NHE3 knockdown
caused defective Dsh recruitment (64.3% ± 2.3%, compared with
31.6% ± 4.3% in controls, this baseline defect in Dsh recruitment is due
to heterogenous Fz and Dsh expression in these cells). Treatment with
30 mM NH4Cl to increase pHi (mean ± s.e.m. from three independent
experiments, measured in f, right graph) rescued the recruitment
defect in NHE3-knockdown cells (mean ± s.d., n= three independent
experiments, *P < 0.001, measured in f left graph). (g–i) Alkalinization
was transient with gradual pH normalization within 20 min. Images
for all three conditions in f are shown. (j–p) Intracellular acidification
interferes with Dsh recruitment. S2R+ cells were prepulsed for 30 min
with 30 mM NH4Cl and then switched to a Na+-free medium for 12–16 h.
This treatment caused a stable reduction of pHi to 7.1 (measured by E2GFP
expression25 and pH calibration), resulting in defective Dsh recruitment
(k–m). Incubation in a Na+-free medium without prepulsing (pHi 7.28) or in
Na+-containing medium after prepulsing (mean ± s.d., n = 4 independent
experiments, *P < 0.001) mildly affected recruitment. For quantification
(j), only cells expressing Fz at their cell surface were counted. Note robust
recruitment in untreated cells with pHi 7.45. (n–p) In a second assay, pHi
was lowered by applying K+/nigericin for 4 h and clamping pHi according to
an extracellular buffer (pH 6.8). This did not affect membrane association
of the phosphatidylserine-binding LactC2–RFP but did affect Dsh
membrane localization. Scale bars represent 10 μm.
One candidate with an annotated function was Nhe2 (CG9256). The
protein Nhe2 belongs to a family of sodium/proton exchangers (NHEs).
Cellular pH homeostasis is regulated by NHEs through the extrusion
of protons in exchange for sodium ions in most organellar membranes,
including the plasma membrane12,13. In D. melanogaster, there are three
NHEs, that have not yet been characterized functionally. When compared with human NHEs, D. melanogaster Nhe2 has the highest sequence
homology to human NHE3, which encodes for a plasma membrane
exchanger. These type of NHEs have been implicated in the formation of
alkaline pH and charge microenvironments at the plasma membrane14.
In polarized cell migration, localized NHEs activity leads to the activation of Rho family GTPases and actin polymerization15. As these are
important downstream events in PCP signalling, we decided to analyze
the role of Nhe2 in Dsh recruitment more closely.
nature cell biology volume 11 | number 3 | MARCH 2009
© 2009 Macmillan Publishers Limited. All rights reserved.
287
letters
288
a
c
b
sev>Nhe2
e
d
sev>Nhe2IR, Nhe21/+
f
sev>Fz at 29°C
sev>Fz
sev>Fz,>Nhe2IR, Nhe21/+
PCP defects (percentage)
First, to confirm the data from D. melanogaster S2R+ cells, we silenced
human NHE3 with short interfering RNA (siRNA) in HEK293T cells,
which also impaired Fz-mediated Dsh recruitment (Fig. 1g, h;
Supplementary Information, Fig. S1a, b). This suggests that the role of
Nhe2 in Dsh recruitment is conserved, and also rules out potential offtarget effects associated with the Nhe2 dsRNA. Consistent with other
reports16 the NHE3 knockdown did not seem to affect the global pH
within the cell (Supplementary Information, Fig. S1e). However, we
were able to partially rescue the recruitment defect seen in the NHE3
knockdown cells by transient alkalinization (Fig. 1f–i). In a similar
manner, treatment of HEK293T cells expressing Fz and Dsh with the
selective NHE3 inhibitor S3226, but not the NHE1 inhibitor cariporide,
effectively redistributed Dsh into the cytoplasm (Fig. 1d, e; data not
shown; Supplementary Information, Fig. S2c). From these results, we
can conclude that it is the proton-translocation function of NHE3 that
is required for Dsh localization. To phenocopy the absence of sodium/
proton exchange at the membrane we reduced the intracellular pH (pHi)
in S2R+ cells using two independent methods (Fig. 1j–p). Both treatments led to defects in Dsh recruitment; however they did not affect
Fz transport to the surface (Fig. 1k–m). In contrast, reduced pHi did
not inhibit the membrane recruitment of Pk, another PCP core protein,
to cell–cell contact sites (Supplementary Information, Fig. S2a, b). Nor
did reducing pHi affect the membrane association of the lactadherin
C2 domain (LactC2), which binds to phosphatidylserine in a chargeindependent manner (Fig. 1n–p). This is in accordance with the finding that the inhibitor S3226 also had no effect on LactC2 membrane
association17 (Supplementary Information, Fig. S2c).
In the D. melanogaster eye, PCP is generated in ommatidial preclusters when Fz/PCP signalling specifies the cell fates of photoreceptors
R3 and R4 (thus defining the chiral form of each ommatidium seen
in adult eyes), which is followed by a 90° rotation of each ommatidial
cluster towards the dorso-ventral midline2 (the equator). This results in
a mirror-image ommatidial arrangement across the equator (Fig. 2a).
One of the key events in Fz/PCP signalling is asymmetric Dsh membrane
association2,3. We therefore tested whether Nhe2 has a role in Fz/PCP
signalling in the D. melanogaster eye, where PCP defects manifest as
chirality inversions, symmetrical cluster formations and rotation defects
of ommatidia2. Nhe2 has two splice variants that differ in the length of
their C termini18 (Supplementary Information, Fig. S3). Overexpression
of both the short and the long form resulted in PCP defects in the eye
(Fig. 2b; data not shown). These defects were already detected during development in eye imaginal discs (Supplementary Information,
Fig. S4h–j). Consistently, overexpression of NHE3 also impaired Dsh
recruitment in HEK293T cells (Supplementary Information, Fig. S1d).
To further test for Nhe2 requirement in PCP, we generated a loss-offunction mutant allele by imprecise P-element excision. The excision
event produced a homozygous-lethal deletion of 6.1 kb (Supplementary
Information, Fig. S3). This deletion mutant, Nhe21, is a strong loss-offunction allele with no detectable Nhe2 transcript by in situ hybridization
(Supplementary Information, Fig. S3). Nhe21 is homozygous lethal and,
importantly, lethality can be rescued by uniform expression of the splice
variant UAS-Nhe2long (under control of tub>GAL4; data not shown),
confirming the specificity of the mutant allele. Nhe21 homozygous
animals die during early larval stages with defects in gut morphology
and function, and fat body development (Supplementary Information,
Fig. S3, legend). Clonal analyses showed that homozygous Nhe21 clones
60
40
*
20
0
sev>Fz
sev>Fz;
>Nhe2IR;
Nhe21/+
Figure 2 Nhe2 shows gain- and loss-of-function PCP phenotypes in
D. melanogaster eye, and interacts genetically with Fz. (a–e) show tangential
eye sections of adult eyes (upper panels) with respective schematic
representations (lower panels). Sections are around the equator (except in
b, which shows a dorsal area). Wild-type eye with ommatidia of opposing
chirality arranged around the equator and R3 positioned at the tip of the
trapezoid is shown in a. Dorsal and ventral ommatidia are depicted with
black and red arrows, respectively. Inset shows enlarged ommatidium with
numbered photoreceptor cells. Overexpression of the UAS-Nhe2short splice
variant (and also the long form, data not shown) with sev>GAL4 caused PCP
defects (b) consisting of symmetrical clusters (green arrows), rotation defects
and occasional chirality inversions. Extra photoreceptors, as well as fused
ommatidia, were also seen. Circles represent unscorable clusters. Knockdown
of Nhe2 and removal of one copy of Nhe2 (sev>Nhe2IR; Nhe21/+) led to PCP
defects (c, 7.11%±3.5%, data are mean ± s.d of three independent eyes with
> 400 ommatidia scored). In addition, general defects of eye morphology
and structure are commonly seen with this genotype. (d–f) The sev>Fz
phenotype is suppressed by co-expression of UAS-Nhe2IR and removal of
one Nhe2 copy. This experiment was performed at 29 °C to enhance Fz and
Nhe2IR expression. Total PCP defects were scored and quantified (f , data are
mean ± s.d. of six eyes with > 700 ommatidia scored, *P < 0.0001).
cannot be recovered in adult or larval tissue (data not shown), precluding PCP analysis and indicating that Nhe2 is required for cell viability.
However, expression of Nhe2 RNAi (Nhe2IR) in a heterozygous Nhe21
condition using an eye-specific driver caused PCP defects in the eye
(Fig. 2c). This genetic combination also showed phenotypes related to
general eye morphology and thus it was difficult to further enhance the
PCP defects and still maintain scorable tissue. Similarly, in other tissues,
such as the wing, Nhe2IR had pleiotropic effects and did not allow scoring
of PCP defects (data not shown).
nature cell biology volume 11 | number 3 | MARCH 2009
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letters
a
c
b
DEPwt
PA
Membrane
targeting?
PG
PC
PE
1
2
3 4
5
6 7 8 9 10 11 12 13 14 15
Protein-protein
interaction?
Mouse Dvl1 DEP domain
d
e
30% PA
DEPwt
DEPK/E
DEPK/M
1
2
3 4
5
6 7 8
9 10 11 12 13 14 15
S1P
LPA
PI(3,4)P2
LPC
PI(3,5)P2
PI
PI(4,5)P2
PI(3)P
PI(3,4,5)P3
PI(4)P
PA
PI(5)P
PS
PE
blank
PS
His–DEPwt
∆G = –40.7 kcal mol–1
DEPK/E
His–DEPK/E
∆G = 58.5 kcal mol–1
Figure 3 The Dsh DEP domain interacts with acidic phospholipids. (a) The
solution structure of the DEP domain of mouse Dvl1 shows a polybasic
amino acid stretch positioned at a different surface location than the
electric dipole for putative protein–protein interaction (which includes
the lysine mutated in dsh1-K/M)8. (b) SUV liposomes consisting of 50%
PC and 50% of the indicated phospholipids were mixed with the Dvl1
DEP domain and separated by chromatography on a Sephacryl S‑300
column. The column fractions were analyzed by silver-staining. The lipidbound DEP domain was eluted from fractions 1 to 7, whereas free DEP
domain was eluted from fractions 10 to 15. (c) Electrostatic potentials
were calculated using the non-linear Poisson-Boltzmann equation, to
model phospholipid bilayers containing a 1:1 mixture of PC and PA. The
complexity of the structures of DEPwt, DEPK/E and the lipid membrane
model was minimized using a molecular dynamic simulation method
(see Methods). The binding energy (ΔG) between DEPwt and the acidic
lipid membrane model was predicted to be –40.7 kcal mol–1 (upper
panel). However, for the DEPK/E mutant an unfavorable binding energy
of 58.5 kcal mol–1 was calculated (lower panel). (d) SUV liposomes
containing 70% PC and 30% PA were mixed with DEPwt, DEPK/E and
DEPK/M (Lys 434, see a for location of mutations); binding to DEPK/E
was markedly decreased. The overall structure of the isolated Dvl1 DEP
domain was not affected by the K/E mutations as determined by chemical
shift patterns from NMR spectroscopy (data not shown). Full scans of b
and d are shown in Supplementary Information, Fig. S6. (e) Nitrocellulose
filters (PIP strips) spotted with several different phospholipids species were
overlayed with His-tagged DEPwt and DEPK/E (1 μg ml–1) and subjected
to western blotting with a monoclonal anti-His antibody. DEPwt bound
specifically to PA (arrow). DEPK/E did not bind to any of the spotted
phospholipids. LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine;
PI, phosphatidylinositol; PIXPn, phosphatidylinositol X phosphaten; PE,
phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine‑1phosphate; PA, phosphatidic acid; PS, phosphatidylserine.
Based on the gain- and loss-of-function eye PCP phenotypes, we
tested Nhe2 for genetic interactions with core PCP genes. Removing
one copy of Nhe2 in combination with Nhe2IR suppressed the dominant
Fz overexpression eye phenotype (sev>Fz; Fig. 2d–f)9. In addition, coexpression of Nhe2 and Fz enhanced the phenotype when compared
with Fz alone (Supplementary Information, Fig. S4a–c). No genetic
interactions of Nhe2 were detected with the PCP-phenotypes Fmi or
Stbm (Supplementary Information, Fig. S4d–f, data not shown), suggesting that Nhe2 interacts specifically with fz. Apart from the genetic
interactions, Nhe2 and Fz also interacted physically, as detected in coimmunoprecipitates from HEK293T cells (Supplementary Information,
Fig. S1c). We did not detect a genetic interaction with sev>Dsh, similar to
a lack of dominant interaction between fz and sev>Dsh. This is consistent
with Nhe2 functioning, like Fz, upstream of Dsh. These results suggest
that Nhe2 modulates Fz function in vivo and may do so by regulating a
pH-dependent Dsh recruitment.
Molecular interactions dependent on pH often involve charged
binding sites. The Dsh DEP domain contains a positively charged
amino-acid cluster that may interact with negatively charged surfaces,
such as acidic lipid headgroups, in the membrane (Fig. 3a). To test this
hypothesis, we conducted lipid-binding assays using small unilamellar
vesicles (SUVs) with various phospholipids. SUVs were made with 50%
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letters
a
b
Myc–Fz
Dshwt
d
c
e
n
Myc–Fz
Dshwt
j
k
Merge
o
i
Myc–Fz
DshKR/E
s
+ Ethanol
t
Merge
+ Sphingosine u
+ Ethanol
Dshwt
p
+ Sphingosine
Merge
l
Recruitment cells
(percentage)
m
h
Merge
f
Myc–Fz
DshKR/E
g
Myc–Fz
q
Dshwt
Merge
r
Myc–Fz
DshKR/E
v
Merge
+ Sphingosine w
DshKR/E
+ Sphingosine
+ Sphingosine
DshKR/E
Myc–Fz
60
40
20
0
x
*
80
Ethanol
Sphingosine
+ Sphingosine
Merge
Figure 4 Fz-mediated Dsh membrane recruitment is charge-dependent.
(a–f) DshKR/E(5×)–GFP, bearing five lysine or arginine substitutions for
glutamic acid, was no longer recruited by Fz in S2R+ cells (d–f). The
mutated amino acids correspond to the conserved residues mutated in
mouse Dvl1 DEP‑K/E. Fz efficiently recruited Dshwt (a–c). Fz was visualized
with a monoclonal anti-Myc antibody. Dsh-(KR/E) is expressed in higher
amounts than Dshwt, as shown by western blotting (Supplementary
Information, Fig. S5b). Merged panels (c, f) also include Hoechst 33342
(blue) as nuclear stain. (g–l) DshKR/E–GFP shows defective recruitment to
the apical junctions by Myc–Fz in third-instar wing discs in vivo. Dshwt–GFP
(g) and DshKR/E–GFP (j) were expressed ubiquitously under endogenous
dsh promoter sequences. Myc–Fz (h, k) was expressed in a stripe at the
anterior-posterior boundary using dpp>GAL4. Displayed is a x/z-stack
of wing epithelial cells with apical at the top and basal at the bottom.
Unlike Dshwt–GFP (arrows, g–i), DshKR/E–GFP is not recruited to the
apical membrane by Fz (arrowheads, j–l). Other than the dpp stripe, the
expression strength and localization patterns of Dshwt and DshKR/E(5×)
are indistinguishable (g, j). (m–r) Neutralization of negatively charged
membrane lipids (such as PA) by the membrane-permeant weak base
sphingosine (75 μM), affects Dsh–GFP localization in HEK293T cells.
The overlap of Dsh and Fz at the plasma membrane is diminished (p–r),
compared with control vehicle-treated (ethanol) cells (m–o). Partial
internalization of both proteins is also seen. The same pattern is observed
in S2R+ cells, data not shown. (s–u) Sphingosine rescues the defective
recruitment of DshKR/E–GFP by Fz. Sphingosine (75 μM) and ethanol were
applied for 3 h. Sphingosine rescue was quantified (u), data are mean ± s.d,
n = 10 different microscope fields from two experiments, *P < 0.0001.
(v–x) DshKR/E–GFP co-localizes with Fz in sphingosine-rescued cells.
phosphatidylcholine (PC) to ensure stability. The DEP domain preferentially bound to phosphatidic acid (PA) and phosphatidylglycerol
(PG), which have a negative charge at physiological pH, but not to PC
and phosphatidylethanolamine (PE) which form neutral zwitterions
(Fig. 3b). This suggests that the DEP domain interacts with lipids in an
electrostatic manner. To determine the extent to which inner surface
potential can contribute to the binding, we modelled the interaction,
using the non-linear Poisson-Boltzmann equation to calculate electrostatic potentials. Charge calculations were performed on wild-type
DEP (DEPwt) and a DEP mutant in which the polybasic amino acid
stretch was converted into a polyacidic stretch (DEPK/E). The binding
energy (ΔG) of DEPwt and the acidic lipid membrane (PC/PA at 1:1
ratio) was predicted to be favorable (ΔG = ‑ 40.7 kcal mol–1). The data
for DEPK/E, however, showed a positive binding energy (ΔG = 58.5
kcal mol–1), which does not allow interaction (Fig. 3c).
To confirm these models experimentally, we tested the ability of DEPwt
and DEPK/E to bind phospholipid SUVs. The overall structure of DEPK/E
was unchanged relative to DEPwt as determined by chemical shift patterns
in NMR spectroscopy (data not shown). As predicted, binding of DEPK/E
to PA/PC SUVs was markedly impaired (Fig. 3d). In an independent assay,
we confirmed that DEPwt specifically interacted with PA, which was spotted among an array of phospholipids, whereas DEPK/E failed to interact
with any lipids (Fig. 3e).
Next, we asked whether the same charge-dependent mechanism
also has a role in the recruitment of Dsh by Fz in live cells. We introduced the same lysine- or arginine-to-glutamic acid changes in the
DEP domain of full-length D. melanogaster Dsh–GFP, which did not
compromise protein stability (Supplementary Information, Fig. S5b).
These mutations in the resulting mutant, DshKR/E, dramatically
interfered with Fz-mediated membrane recruitment in S2R+ cells
(Fig. 4a–f), HEK293T cells (data not shown), and importantly, also
in vivo in native wing-disc epithelia (Fig. 4k–l). When up to five basic
amino acids were mutated, DshKR/E(5×), Dsh recruitment by Fz was
abolished, suggesting that negative charge on the DEP domain surface
counteracts membrane recruitment (Fig. 4a–l). To further confirm this
hypothesis we tested the effect of charges on the membrane. Strikingly,
290
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letters
Dsh–GFP
a
Merge
h
i
30 hrs APF
dshV26; dsh>dshwt–GFP
Phalloidin
g
b
dshV26; dsh>dshwt–GFP
dshV26; dsh>dshKR/E–GFP
j
k
l
dshV26; dsh>dshKR/E[2x]–GFP
c
m
n
o
34 hrs APF
dshV26; dsh>dshwt–GFP
f– dshV26; dsh>dshwt–GFP
p
d
q
r
dshV26; dsh>dshKR/E[2x]–GFP
s
f– dshV26; dsh>dshKR/E[2x]–GFP
e
f
H+
Nhe2
Fz
–––
+++
f– dshV26; dsh>dshwt–GFP
DEP
PDZ
DIX
Dsh
Fz
Fz
f– dshV26; dsh>dshKR/E[2x]–GFP
–––
H+
H+
+++
DEP
Figure 5 The polybasic stretch in the Dsh DEP domain is essential for PCP
signalling in vivo. Dshwt–GFP and DshKR/E–GFP were expressed under a
dsh promoter in a dshV26 null background (dshV26; dsh>dsh–GFP). (a–r) Flies
that rescued the dshV26 lethality were scored for phenotypes in adult eyes
(a, b, schematic representations lower panels, dorsal and ventral ommatidia
are depicted with black and red arrows respectively, the green arrow depicts
a symmetrical cluster), adult wings (c–f) and pupal wings (g–r). In the eye,
DshKR/E(2x) caused mild phenotypes, including symmetrical clusters,
chirality inversions and rotation defects (b). In adult wings, DshKR/E(2x)
caused severe defects in wing-hair orientation, whereas the complete wing
margin was intact (d). The anterior wing margin bristles are shown for
DshKR/E(2×) (f), and Dshwt (e). Rescue with Dshwt was complete with fully
wild-type appearance (a, c, e). (g–r) Pupal wings were examined at a stage
before wing-hair formation (~30 h APF, g–l) and during wing hair formation
(~34 hours APF, m–r). Dshwt–GFP co-localized with phalloidin-stained actin
at the cell cortex at the early stage (g–i). In contrast, DshKR/E(2x)–GFP
showed diffuse cytoplasmic localization (j–l). At the later stage (~34 h APF),
H+
PDZ
Low pH
DIX
Dsh
–––
DEP
PDZ
DIX
Dsh-K/E
Dshwt–GFP showed a typical cortical distribution with enrichment in the
proximo-distal axis, and wing hairs showed a normal proximo-distal orientation
(m–o, as reflected by Phalloidin, red). DshKR/E(2x)–GFP also failed at this
stage to associate with the membrane (p–r). The resulting phenotypes are
misoriented wing hairs and a multiple wing hair phenotype (q–r). Wing hairs
appear shorter than shown in n, as the misoriented hairs point up and out
of the confocal plane. (s) Suggested model of Fz-mediated Dsh membrane
recruitment. Dsh binds weakly via its PDZ domain to the Fz C terminus.
In addition, the DEP domain binds directly to acidic phospholipids. The
interaction is dependent on local pH and charge. With Dshwt , proximity of
the Na+/H+ exchanger Nhe2 to Fz maintains a slightly basic local pH (upper
panel). When local pH is lower (Nhe2 mutant), the lipid headgroups are
protonated, losing their negative charge (green) and leading to repulsion of
Dsh (lower left panel). Similarly, mutations in the polybasic stretch of the
DEP domain cause cytoplasmic Dsh localization (lower right panel), which
can be rescued by lowering surface negativity (Fig. 4s–u). It is possible that
another surface of the DEP domain also interacts with Fz directly.
nature cell biology volume 11 | number 3 | MARCH 2009
© 2009 Macmillan Publishers Limited. All rights reserved.
291
letters
the application of sphingosine, a cationic lipid that reduces the negative charges of the inner surface, impaired Dsh co-localization with
Fz at the membrane (Fig. 4m–r; it did not affect LactC2 membrane
localization, Supplementary Information, Fig. S2c)19,17. In contrast,
sphingosine treatment allowed effective association of the DshKR/E
mutant with Fz at the membrane, suggesting that the neutralization
of surface charge rescues repulsion of the DshKR/E mutant from the
membrane (Fig. 4s–y).
For the most stringent test, we investigated whether the DshKR/E
mutations affect Dsh function in vivo by using rescue assays of the dsh–
genotype. We expressed various Dsh mutants, mutated at the polybasic
stretch, under the endogenous dsh promoter in a dsh null background
(dshV26; dsh>dsh–GFP). Dsh is required for both the canonical and the
non-canonical Wnt pathway, which govern distinct developmental processes. Therefore, the expression of Dsh constructs in this background
allowed us to assess the importance of polybasic stretch-mediated
membrane recruitment for either Wnt/β-cat signalling or the Fz/PCP
pathway. Using this assay, we found that complete conversion of the polybasic stretch, DshKR/E(5x), failed to rescue dshV26 lethality and therefore
canonical Wnt/β-cat signalling. However, the replacement of only two
basic residues with acidic ones (see Methods), generating DshKR/E(2×),
produced viable flies with no apparent defects in the canonical Wnt/
β-cat pathway. In contrast, these flies showed mild PCP defects in the
eye (Fig. 5a–b) and severe PCP defects in the wing (Fig. 5c–d, q–r). As
acute overexpression of Fz can still recruit DshKR/E(2×) to the membrane (Supplementary Information, Fig. S5a), membrane localization of
this mutant, unlike DshKR/E(5×), is not fully defective, which probably
explains the absence of canonical Wnt phenotypes (Fig. 5e–f; data not
shown). However, immunostaining of the pupal wing epithelium showed
that during critical phases of active PCP signalling, dsh>dshKR/E(2×)
fails to associate with the cell cortex (Fig. 5g–r). Therefore, we conclude
that membrane association of Dsh mediated by the polybasic stretch in
the DEP domain is necessary for proper PCP signalling in vivo, but seems
to be dispensable for canonical Wnt signalling.
Taken together, our data demonstrate that Dsh membrane recruitment during PCP signalling is dependent on electrostatic interactions
of the DEP domain with negatively charged phospholipids (such as PA).
According to the biophysical properties of these lipids, their negative
charge should be maintained by a more alkaline pH20. Therefore we
speculate that proximity to a Nhe should facilitate the interaction by
maintaining a low proton concentration below the membrane. In our
model, Dsh binds to the KTxxxW motif of Fz via its PDZ domain, and
directly to the membrane via its DEP domain (Fig. 5s). Under lower pH
conditions or after neutralization of the membrane surface charge, the
positively charged Dsh DEP domain is repelled from the membrane.
Similarly, when the DEP domain bears a polyacidic amino acid cluster,
Dsh is also repelled from the membrane (Fig. 5s).
Despite the strong Fz/Dsh association at the membrane, the interaction of the Dsh-PDZ domain with Fz has been reported to be weak
(Kd~10 μM)6. It is therefore likely that other interacting surfaces and
modes exist that stabilize the interaction. Although it cannot be excluded
that the DEP domain can also bind directly to Fz in a pH- and chargedependent manner, our studies show that reducing the amount of negatively charged lipids in the plasma membrane (using neutralizing agents
such as sphingosine) has strong effects on the localization of wild-type
and mutant Dsh (Fig. 4m–y). Dsh may first bind to Fz.This interaction
292
could then be stabilized by the attraction of the Dsh DEP domain to the
negatively charged lipids of the plasma membrane, although the opposite
is equally possible. In either case, the phospholipid-binding DEP domain
would cooperate with the specific (but low-affinity) Fz-binding PDZ
domain to recruit Dsh specifically to membranes that are enriched in
acidic phospholipids and Fz. Such cooperative binding is used by many
proteins to increase the specificity of their membrane association, and
allows for integration of complex signalling input21.
A characteristic feature of Dhs function is that it is able to signal in
different Wnt pathways. What determines the signalling specificity
remains so far unknown. Most canonical Wnt-signalling mutations in
D. melanogaster Dsh are not found in the DEP domain22. Unlike the DIX
domain, the DEP domain has also been shown to be dispensable for LRP6
phosphorylation occurring in signalosomes at the membrane23. In contrast, all PCP-specific mutations (including dsh1) are located in the DEP
domain and impair stable Dsh membrane recruitment. It is therefore
possible that specific Wnt pathway activation requires conformational
changes in the Dsh protein that lead to differential domain exposure
with different effects on membrane recruitment. Here, we describe a
mechanism for a DEP-mediated membrane recruitment that is particularly important for PCP signalling. We propose that distinct membrane
lipids can promote a stable Fz/Dsh interaction at the surface, which is
pH- and charge-dependent, and regulated by local Na+/H+-exchange
activity. Future studies will be directed at determining whether local
electrochemical cues could serve as general determinants in the spatial
control of signalling events that lead to cell and tissue polarization.
Methods
Cell Culture. S2R+ cells were maintained at 25 °C in Schneider’s medium
(Invitrogen) and HEK293T cells were maintained at 37 °C in Dulbecco’s MEM
(GIBCO) in a humidified atmosphere with 5% CO2. both media were supplemented
with 10% fetal calf serum (PAA) and 50 μg ml–1 penicillin/streptomycin.
Genetics and phenotypic analysis. Overexpression and transgenic RNAi studies were performed using the GAL4/UAS system (RNAi crosses grown at 29 °C,
other crosses at 25 °C; w1118 was control). Nhe21 was generated by P‑element excision of EY11323 and mapped by PCR. UAS-fz, UAS-dsh, UAS-stbm and UAS-fmi
strains were as described previously1,24. sev-GAL4 was an eye-specific driver and
dpp>GAL4 was for expressing Myc–Fz at the A/P boundary in the wing. Flip-out
clones were generated with hsflp; act5C>CD8>GAL4; mδlacZ. For dshV26 rescue:
male flies transformed with pCaSpeR dsh>dshwt4 or the respective dshKR/E isoforms were crossed with y,w,dshV26 f36A/FM6 females. For assessment of rescue,
male y,w larvae or male f36A non-FM6 flies were selected and processed for pupal
wing immunohistochemistry, and adult eye and wing analysis. For each genotype, 3–6 independent eyes were scored. Third- instar eye and wing discs were
processed for immunohistochemistry as described previously24. Antibodies: rat
anti-Elav (DSHB) and rabbit anti-βgal (Cappel) for eye discs and mouse anti-Myc
9E10 (Santa Cruz) and rabbit anti-GFP (Molecular Probes) for wing discs . Pupal
wings were also stained with TRITC-labelled phalloidin.
Molecular cloning. D. melanogaster Nhe2 has two transcripts with differing
lengths of C termini. Nhe2short was amplified from EST AT11019 by PCR.
For Nhe2long, the C‑terminal coding region was amplified from cDNAs and
fused to the partial EST clone RE21674. Complete Nhe2long and short coding
sequences were cloned into the UAS-based Gateway vector pTWV (DGRC). For
in-vivo RNAi hairpin expression, two identical 455 bp Nhe2 sequences (Probe
ID: HFA03172 at http://rnai.dkfz.de/) were cloned in opposite orientations into
UAS-based pWIZ vector.
For cell culture experiments, Dsh–GFP and Myc–Fz were subcloned into
pAc5.1/V5-His, pMT/V5-His (Invitrogen) or pCS2+ vectors. E2–GFP (used for
pH calibration) was amplified from pcDNA3.1 E2–GFP (a gift from R. Bizzarri,
Scuola Normale Superiore, Pisa)25 and cloned into pAc5.1 with Dsh–GFP replacing
nature cell biology volume 11 | number 3 | MARCH 2009
© 2009 Macmillan Publishers Limited. All rights reserved.
letters
GFP. DshKR/E mutations in pAc5.1 Dsh–GFP were made by Quickchange-PCR
(Stratagene). They included R422E, R425E, R426E, R433E, K443E for DshKR/
E(5x) and R426E and R433E for DshKR/E(2×). K/E mutations in mouse Dvl1
DEP were made in the pQE vector (Qiagen), including K408E, K458E, K465E,
K472E and K482E. Primer sequences available on request.
RNAi experiments. For RNAi experiments in D. melanogaster S2R+ cells, dsRNAs
were generated from DNA templates by in vitro transcription as described previously10 (sequence information available at http://rnai.dkfz.de/). RNAi screening was
performed as reported10. Each screen plate included one negative control (Relish
dsRNA) and three positive controls (fz, par‑1 and dsh). Relish knockdown did not
affect Dsh recruitment, fz knockdown resulted in no Dsh recruitment (Dsh–GFP
being localized to cytoplasmic aggregates26), par‑1 knockdown in severely impaired
recruitment, and dsh knockdown in no detectable Dsh–GFP. Control genes were
also identified with their respective phenotypes among the RNAi library. Primary
screen hits were re-screened three times in a blinded fashion.
For RNAi in HEK293T cells, the cells were transfected with a total of 100 nM
siRNA (in the case of NHE3 4 × 25 nM SMARTPool siRNA) using Oligofectamine
(Invitrogen). After 24 h, cells were transfected with plasmids encoding Xdsh–GFP
and HA–Fz4 (a gift from T. Kirchhausen, Harvard Medical School, Boston) or
pCS2+ DmDsh–GFP and pCS2+ Myc–DmFz respectively (Dm represents D.
melanogaster). NHE3 knockdown was confirmed by western blotting after mannitol lysis using a rabbit anti-NHE3 (Chemicon)27. The protein band pattern
was compared with the pattern of transfected rabbit NHE3–HA (provided by J.
Orlowski, McGill University, Montreal).
Intracellular acidification/alkanilization and pH measurements. For intracellular acidification, cells were transfected with pMT Dsh–GFP and pAc5.1
Myc–Fz. Six hours after induction of Dsh–GFP expression with CuSO4 (500 mM),
cells were prepulsed with NH4Cl (30 mM) for 30 min and kept for 12–16 h in
KCl or NaCl (0.14 M), Hepes (20 mM), CaCl2 (2 mM) and MgCl2 (1 mM) at
pH 7.0 (ref. 28). For analysis, cells were stained with anti–Myc 9E10 antibody
(Santa Cruz) after fixing with 4% paraformaldehyde and permeabilizing with
0.1% Triton-X100. Complete, partial or no recruitment was scored in cells by
measuring Myc–Fz expression at the cell surface. To determine the effects of
these treatments on pHi, S2R+ cells were transfected with pAc5.1 E2–GFP and
imaged with Leica SP3 confocal microscope. Emission ratios were obtained using
an excitation wavelength of 476 nm and two emission wavelength intervals of
480–515 nm and 515–600 nm as described25. The pHi was clamped for calibration with high K+/nigericin (Biomol) at pH 6.8, 7.6 and 8.2 after determining
emission ratios at normal conditions. Ratio images were made with ImageJ
software. Longer treatments with the same calibration solutions were used as
a second method to lower pHi. For this, pH 6.8 calibration solutions, with or
without nigericin, were applied to S2R+ cells (transfected with pAc5.1 Dsh–GFP
and pAc5.1 Myc–Fz) for 4 h and processed for immunocytochemistry.
For intracellular alkanilization, HEK293T cells treated with control and Nhe3
siRNA and transfected with Dsh–GFP and Myc–Fz were kept for 90 min in isotonic
solution (140mEq NaCl and 5mEq KCl) with or without 30 mM NH4Cl. Ratiometric
dye BCPCF (Molecular Probes) was used to monitor pHi, which was subsequently
clamped for calibration using the high K+/nigericin method at pH 6.8 and 7.8. A
pHi rise to more than pH 8.0 was shown, before gradual normalization.
Sphingosine and Nhe inhibitor treatments. Synthetic Sphingosine (AvantiPolar-Lipids) was applied for 6–12 h (75 μM) to Dsh/Fz-transfected HEK293T or
S2R+ cells before fixing and immunostaining with anti-Myc antibody. For rescue
of DshKR/E membrane recruitment sphingosine was applied for 3 h.
The Nhe inhibitors S3226 (more specific for NHE3) and cariporide (more
specific for Nhe1) were provided by J. Puenter (Sanofi-Aventis). S3226 affected
Dsh recruitment in HEK293T cells at > 30 μM. In depicted experiments, S3226
and cariporide were applied for 12 h at 50–100 μM.
All statistical analyses were performed using two-tailed Student’s t‑tests to
calculate P values (Microsoft Excel).
Protein expression, small unilamellar vesicles (SUV) lipid-binding assays
and computational methodology for electrostatic potential calculations.
Purification of the mouse Dvl1(Dsh homologue) DEP domain was performed as
described previously8. The HSQC spectra of mutant DEP domains were obtained
to determine whether site-directed mutagenesis affected the structure. The NMR
samples consisted of 0.3 mM of the DEP domain (0.3 mM) in phosphate buffer
(0.1 M, pH 6.8), EDTA (0.5 mM) and 1,4-dithiothreitol (DTT, 3 mM). We observed
that both mutant DEP domains showed a similar chemical shift pattern to wildtype DEP, indicating that the structure of mutant DEP domains was not affected.
Generation of SUV liposomes was as described previously29. We generated
SUVs with various phospholipids. Vesicles containing PA, PG, PC, and PE were
made with 50% PC to ensure stability. For lipid binding assays, vesicles (~5 mM
phospholipids) were mixed with purified wild-type or mutant DEP domains in
column buffer (40 mM Tris-Cl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA
and 1 mM DTT), incubated at room temperature for 30 min, and separated by
chromatography on a Sephacryl S‑300 column. Fractions were analyzed in 13%
SDS gels by silver-staining.
To determine whether PA‑binding to DEP was specific, PIP (phospho-inosidephosphate) strips (Echelon) were overlayed with purified His-tagged DEPwt and
K/E domains in 3%BSA/PBS‑0.1% Triton-X100 for 3 h at room temperature.
After washing steps with 3%BSA/PBS‑0.1% Triton-X100, strips were subjected
to standard western blotting with an anti-His antibody (Qiagen).
For computational methodology for electrostatic potential calculations see
Supplementary Information.
Note: Supplementary Information is available on the Nature Cell Biology website.
Acknowledgements
We thank Bloomington Stock Center for fly strains, S. Grinstein, J. Orlowski, T.
Kirchhausen and R. Bizzarri for cDNA constructs. We are grateful to A. Jenny
and Y. Wang for the cloning of cDNA constructs, and U. Weber for analysis of the
embryonic/early larval lethal phenotype. We thank C. Iomini, R. Krauss, S. Sokol
and D. del Alamo for reading the manuscript, members of the Mlodzik laboratory
for discussions and S. Okello, G. Garcia and M. Stricker for technical support.
The work has been supported by NIH grant to M.M. RO1 GM62917. M.S. was a
recipient of EMBO and DFG long-term fellowships.
Author contributions
M.S. coordinated the project, designed and conducted the RNAi screen, performed
the experimental work and data analysis and wrote the manuscript. W.J.G.
performed the pupal dissections. W.J.G. and D.G. assisted with experiments. T.J.K.
helped with the RNAi screen assay. R.R. and L.S.M. performed pH measurements.
Y.S., H.-J.L. and J.Z. purified the DEP domain and performed electrostatic
potential calculations. A.-L.W., Y.F. and J.C. performed SUV lipid binding assays.
J.T.D. provided Nhe2 tools. M.B. designed and conducted the RNAi screen. M.M.
coordinated the project, assisted with planning the experiments and data analysis
and wrote the manuscript.
Competing financial interests
The authors declare no competing financial interests.
Published online at http://www.nature.com/naturecellbiology/
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/
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s u p p l e m e n ta r y i n f o r m at i o n
DOI: 10.1038/ncb1836
Supplementary information:
Figure S1: Nhe3 knockdown and overexpression affects Dsh surface expression but not global intracellular pH.
(a) Examples are shown for HEK293T cells, which were transfected with XDsh-GFP/HA-Fz4 and silenced with control
co-immunoprecipitated by Fz (right panel). Expression of Fz-FLAG and
Figure S1 Nhe3 knockdown and overexpression affects Dsh surface expression
(top
row) or
siRNA
row). Fz4
remainsDHAL-FLAG
at the surface
after
Nhe3
knockdown.
The
in the lysates
is shown
in both
lower panels.(b)
Cells
werepartial
lysed in
butsiRNA
not global
intracellular
pH.Nhe3
(a) Examples
are (bottom
shown for HEK293T
cells,
M2 Buffer (50mM Tris-HCl, pH 8.0, 150mM NaCl, 1mM EDTA, 1% Triton
which were transfected with XDsh-GFP/HA-Fz4 and silenced with control
knockdown of Nhe3 was confirmed by Western Blotting with an anti-Nhe3 antibody. Nhe3 runs in two bands in SDS-PAGE
X-100 + Phospatase and Protease Inhibitor Cocktails). (d) HEK293T cells
siRNA (top row) or Nhe3 siRNA (bottom row). Fz4 remains at the surface
Nhe3 show
impaired aDsh
recruitment.
after
Nhe3
(b) The
partial at
knockdown
Nhe3 waskDa
confirmed
gels
at knockdown.
80 kDa and
a larger
aroundof160-200
(asterisks).overexpressing
The upper band
is possibly
dimer
and isHigh
alsomagnification
seen when
in the upper panel shows a single cell expressing Dsh-GFP, myc-Fz and
by Western Blotting with an anti-Nhe3 antibody. Nhe3 runs in two bands in
exogenous
expressed
(third
lanekDa
of (asterisks).
left panel).
bands are
by in
Nhe3
siRNA
comparedcell
to
Low reduced
magnification
the lower
paneltreatment
shows a representative
SDS-PAGE
gels atNhe3-HA
80 kDa andisa larger
at around
160-200
TheBothNhe3-HA.
population.
The
cell
population
with
Nhe3-HA
expression
contains
more
cells
upper
band
is
possibly
a
dimer
and
is
also
seen
when
exogenous
Nhe3-HA
is
GFP siRNA treatment (asterisks). Equal loading was confirmed by aPKC Western blotting. The same human Nhe3 siRNAs
with accumulation of intracellular Dsh-GFP (arrowheads) compared to the cell
expressed (third lane of left panel). Both bands are reduced by Nhe3 siRNA
population
serum response factor (SRF)-HA
as a in
negative
control.
can also
reduce
thesiRNA
expression
exogenous
panel).expressing
(c) Fz co-immunoprecipitates
Nhe3
HEK293T
treatment
compared
to GFP
treatmentof(asterisks).
Equalrabbit
loadingNhe3
was (right
(e) Nhe3 knockdown and overexpression does not alter global intracellular pH
confirmed by aPKC Western blotting. The same human Nhe3 siRNAs can
lysates.
FLAG-tagged
Fz andrabbit
HA-tagged
Nhe3
were
in HEK293T
cells
and co-immunoprecipitated with antiof HEK293T
cells. pH
also
reduce the
expression of exogenous
Nhe3 (right
panel).
(c) Fzexpressed
i was measured in different cells within a cell population
(cells
with
no
visible
Dsh-GFP,
cells with Dsh-GFP atprotein
the membrane
cells
co-immunoprecipitates
Nhe3
in
HEK293T
lysates.
FLAG-tagged
Fz
and
HAFLAG beads. Nhe3 was efficiently co-immunoprecipitated by Fz, whereas the control transmembrane
DHALand
was
with most of the Dsh-GFP in the cytoplasm) using BCPCF and subsequent K+/
tagged Nhe3 were expressed in HEK293T cells and co-immunoprecipitated
not.
Lysates
indicate
theefficiently
input ofco-immunoprecipitated
Nhe3. The C-terminus
of Drosophila
Nhe2 was
also
co-immunoprecipitated
byanalysis
Fz (right
nigericin calibration.
Only
the whole
cell area was used for the
in order
with
anti-FLAG
beads.
Nhe3 was
by
assess global pHi changes. There appeared to be no significant differences in
Fz, whereas the control transmembrane protein DHAL was not. Lysates
panel). Expression of Fz-FLAG and DHAL-FLAG in the lysates
is
shown
in
both
lower
panels.
Cells
were
lysed
in
M2
pHi in the different cell types and cell populations (data are mean ± s.e).
indicate the input of Nhe3. The C-terminus of Drosophila Nhe2 was also
Buffer (50mM Tris-HCl, pH 8.0, 150mM NaCl, 1mM EDTA, 1% Triton X-100 + Phospatase and Protease Inhibitor
Cocktails). (d) HEK293T cells overexpressing Nhe3 show impaired Dsh recruitment. High magnification in the upper panel
shows a single cell expressing Dsh-GFP, myc-Fz and Nhe3-HA. Low magnification in the lower panel shows a
representative cell population. The cell
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population with Nhe3-HA expression contains more cells with accumulation of
© 2009 Macmillan Publishers Limited. All rights reserved.
intracellular Dsh-GFP (arrowheads) compared to the cell population expressing serum response factor (SRF)-HA as a
1
s u p p l e m e n ta r y i n f o r m at i o n
Figure S2: Negative controls for membrane localization.
Myc-tagged Pk (green) co-localizes with endogenous Fmi (red) at cell-cell contact sites (yellow color indicates coFigure S2
Negative controls
for membrane
Myc-tagged
Pk
experiments
(b; data
mean ± s.d., n=3ofindependent
localization,
arrow)
in S2R+ localization.
cells (a, left).
This localization
is enhanced
by are
overexpression
Fmi (red),experiments).
which also
(green) co-localizes with endogenous Fmi (red) at cell-cell contact sites
In vivo both Fmi and Stbm are required for Pk membrane recruitment (Tree
broadens
the
cell-cell contact
zone cells
(arrowheads).
Overall
plasma
membrane
recruitment
of 2004,
Pk isEMBO
alsoJ. slightly
(yellow color
indicates
colocalization,
arrow) in S2R+
(a, left).
et al.,
2002, Cell
109, 371-381;
Jenny et al.,
22, 4400This localization is enhanced by overexpression of Fmi (red), which also
4420). In S2R+ cells, Stbm is not transported
+ efficiently to the plasma
enhanced by Fmi (red; a, middle). Prepulsing with NH Cl and subsequent incubation in Na -free medium, which
broadens the cell-cell contact zone (arrowheads). Overall plasma membrane 4 membrane. Therefore, we used Fmi for the Pk recruitment assay (see
recruitment
of Pkinis intracellular
also slightly enhanced
by Fmi (red;
middle).
enlarged
contact
zone in “a” (middle
panel).
(c) Synthetic
Sphingosine
results
acidification,
doesa, not
affect the cell-cell
contact
localization
of Pk (a,
right),
as compared
with was
incubation in Na+-free medium,
Prepulsing with NH4Cl and subsequent
applied for 12 hours in a concentration of 75μM to Dsh-GFP/Fztransfected
+
incubation
in Na acidification,
-free medium
without
prepulsing (a, HEK293T
middle).orPk
localization
cell-cell
contact sites
which results
in intracellular
does but
not affect
the cell-cell
S2R+
cells before at
fixing
and immunostaining
withinanFmianti-myc
contact localization of Pk (a, right), as compared with incubation in Na+antibody. S3226 was applied for 12 hours in a concentration of 100μM.
expressing cells was scored in three independent experiments (b; data are mean ± s.d., n=3 independent
free medium but without prepulsing (a, middle). Pk localization at cell-cell
Neither treatment affected the membrane association of LactC2, which
contact experiments).
sites in Fmi expressing
cells
was Fmi
scored
in three
binds
to phosphatidylserine
in a(Tree
non-electrostatic
manner.
In vivo
both
and
Stbmindependent
are required for Pk
membrane
recruitment
et al., 2002,
Cell 109, 371-
381; Jenny et al., 2004, EMBO J. 22, 4400-4420). Although in cell culture neither Fmi nor Stbm localize efficiently to
the plasma membrane, Fmi works better (see enlarged contact zone in “a” (middle panel) and so we used Fmi for the
Pk recruitment assay. (c) Synthetic Sphingosine was applied for 12 hours in a concentration of 75µM to Dsh-GFP/Fztransfected HEK293T or S2R+ cells before fixing and immunostaining with an anti-myc antibody. S3226 was applied
for 12 hours in a concentration of 100µM. Neither treatment affected the membrane association of LactC2, which
binds to phosphatidylserine in a non-electrostatic manner.
2
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s u p p l e m e n ta r y i n f o r m at i o n
chrom. 2L (39B)
1
2
3
4
5
6
7
8
9
GTA
CG12050
CG9257
ATG
5’UT
R
5’UT
RR
ATG
Nhe2-RA
ATG
Nhe2-RB
EY11323
Nhe21
6.1 kb
1
Figure S3: Imprecise P-element excision results in a putative Nhe2 null allele, Nhe2 .
Genomic organization of Nhe2 locus. Nhe2 possesses two 5’UTRs that drive the transcription of Nhe2-RA and Nhe2-RB. In
is also
spliced
differentially
more
downstream
giving
rise also
to aremoved
long anda very
a short
transcript
thatgene
differ
in the length
deletion
small
neighboring
(CG9257).
However,
Figure S3addition,
ImpreciseNhe2
P-element
excision
results
in a putative
Nhe2
null
1 lethality was rescued by uniform expression of UAS-Nhe2 long
1. Genomic organization of Nhe2 locus. Nhe2 possesses two
Nhe2
allele, Nhe2
of their C-termini (not shown). The P-element EY11323 was excised with the aid of the !2-3 transposase, which resulted in a
(under tub>GAL4) indicating that lethality is solely caused by Nhe2 and
5íUTRs that drive the transcription of Nhe2-RA and Nhe2-RB. In addition,
homozygous
lethal excision
line. The molecular
mapping
that
the excision
eventofproduced
a 6.1kbtodeletion
notrevealed
by removal
of CG9257.
The cause
lethality appears
be a defect in
Nhe2 is also
spliced differentially
more downstream
giving rise to
a long analysis
stof their C-termini (not shown).
1
gut
morphology
and
function,
and
also
the
absence
of a fatby
body.
Cuticle
and a short
transcript
that
differ
in
the
length
removing both promoters and 1 exons/5’UTRs, resulting in undetectable transcript in homozygous Nhe2
embryos
in situ
patterning is normal suggesting that Nhe21 does not affect1canonical Wg
The P-element EY11323 was excised with the aid of the δ2-3 transposase,
hybridization
(lower left
panel).
The
deletion
also removed
small neighboring
gene
(CG9257).
However,
Nhe2
Due to the gut
defects
it was not
possible to
assesslethality
whether leftwhich resulted
in a homozygous
lethal
excision
line.
The molecular
mappinga verysignaling.
right
asymmetry
was
normal
in
these
animals.
This
is
of
interest
as pH has
analysis revealed
that
the
excision
event
produced
a
6.1kb
deletion
removing
was rescued by uniform expression of UAS-Nhe2 long (under tub>GAL4) indicating that lethality is solely caused
by Nhe2
been suggested to control left-right differentiation in vertebrates (e.g. Levin
both promoters and 1st exons/5’UTRs, resulting in undetectable transcript in
andNhe2
not 1by
removal
ofsitu
CG9257.
The cause
of lethality
appearsMtoand
be Palmer
a defect
gut morphology
and function, and also the
AR in
, Bioessays.
2007 Mar;29(3):271-87).
embryos
by in
hybridization
(lower left
panel). The
homozygous
1
absence of a fat body. Cuticle patterning is normal suggesting that Nhe2 does not affect canonical Wg signaling. Due to the
gut defects it was not possible to assess whether left-right asymmetry was normal in these animals. This is of interest as pH
has been suggested to control left-right differentiation in vertebrates (e.g. Levin M and Palmer AR , Bioessays. 2007
Mar;29(3):271-87).
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3
© 2009 Macmillan Publishers Limited. All rights reserved.
s u p p l e m e n ta r y i n f o r m at i o n
Figure S4 Genetic interactions between Nhe2 and PCP core components
and clonal analysis of Nhe2 overexpression in the eye imaginal disc. (a-e) All
panels show tangential section of adult eyes with the indicated genotypes.
Arrows are as in Figure 2. sev>Fz and sev>Fmi are heterozygous in all
genotypes. (a-c) sev>Fz at 25°C (c) shows typical PCP phenotypes with
symmetrical clusters (green), chirality inversions and rotation defects. Coexpression of UAS-Nhe2short enhances the frequency of these phenotypes,
in particular the amount of symmetrical clusters (b). Total PCP defects were
scored and quantified in (c; data are mean ± s.d., n=3 eyes). (d-f) Nhe2
does not modify the sev>Fmi phenotype. Like in a-c sevGal4 was used to
drive Fmi expression (sev>Fmi; d), but unlike above the phenotype was not
affected by the co-expression of UAS-Nhe2IR and simultaneous removal of
4
one Nhe2 copy (e). Total PCP defects were scored and quantified in f (data
are mean ± s.d., n=4 eyes). (g) Schematic diagram of eye imaginal disc with
the dorsal-ventral midline or equator in yellow. Anterior is left, dorsal is up.
PCP signaling determines the R3 and R4 fates and regulates a 90-degree
rotation towards the equator. (h-j) PCP defects can also be seen at early
stages of eye development in eye imaginal discs. Nhe2 was overexpressed in
flip-out clones under act>GAL4. Clones are marked with GFP (green). mδlacZ (stained with anti-βGal, red) is normally expressed high in R4 and very
low in R3 precursors. This asymmetry is a result of correct Fz/PCP signaling.
The asterisk highlights a cluster with aberrant Fz/PCP establishment,
showing equal low expression of mδ-lacZ in both R3/R4 precursors. Elav
(blue) marks all photoreceptor precursors.
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s u p p l e m e n ta r y i n f o r m at i o n
a
b
Figure S5: DshKR/E [2x] can be recruited to membrane by overexpression of Fz.
(a) Acute Fz overexpression recruits DshKR/E [2x]-GFP to the membrane in 293T cells and wing discs. In wing discs
DshKR/E[2x] was expressed uniformly at low levels under dsh promoter sequences. Myc-Fz was overexpressed in a
DshKR/E-GFP (2x, 4x, 5x) were expressed in S2R+ cells and expression levels
Figure S5 DshKR/E [2x] can be recruited to membrane by overexpression of
stripe at the anterior-posterior boundary using
(b) Dsh-wt-GFP
DshKR/E-GFP
(2x, 4x, 5x) were
weredpp>GAL4.
confirmed by Western
blotting using aand
monoclonal
anti-GFP antibody
Fz. (a) Acute Fz overexpression recruits DshKR/E [2x]-GFP to the membrane in
(Roche).
isoforms,
are notblotting
(4x or 5x) using
or poorlya(2x;
only when Fz anti-GFP
293T cells and wing discs.
In wing discsin
DshKR/E[2x]
expressed
uniformly
expressed
S2 cellswas
and
expression
levels
wereDshKR/E
confirmed
by which
Western
monoclonal
is overexpressed) recruited to the membrane by Fz are more stable than Dsh-wt.
at low levels under dsh promoter sequences. Myc-Fz was overexpressed in a
antibody
(Roche).
DshKR/E
are
notloading
(4x or
5x)
or poorly
(2x; only
when Fz is overexpressed) recruited
Equal
was
confirmed
by probing
for α−tubulin.
stripe at the anterior-posterior
boundary
using dpp>GAL4.
(b) isoforms,
Dsh-wt-GFP which
and
to the membrane by Fz are more stable than Dsh-wt. Equal loading was confirmed by probing for #tubulin.
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5
© 2009 Macmillan Publishers Limited. All rights reserved.
s u p p l e m e n ta r y i n f o r m at i o n
Figure S6: Unedited blots from Figure 3.
Figure S6 (a)
Unedited
blots from
3. (a) Unedited
for Figure
3b.for
(b) Figure
Unedited3d.
blots for Figure 3d.
Unedited
blotsFigure
for Figure
3b. (b) blots
Unedited
blots
6
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© 2009 Macmillan Publishers Limited. All rights reserved.
Supplemental Methods
Computational methodology for electrostatic interactions
We built a simple all-atom molecular model of the bilayer membrane using Membrane Package
implemented in the VMD software. First, we constructed a bilayer with a mixture of PC/PA in a
1:1 ratio. The bilayer model consisted of a total of 131 lipids, 63 in upper membrane leaflet and
2
68 in bottom leaflet with a square dimension of 70x70 . The DEP domain of mouse Dvl1 was
placed on top of the upper membrane leaflet. Based on this construction, 1000 steep
minimizations were run followed by 5000 gradient minimizations to relax the DEP-membrane
model system by using the SANDER module in AMBER8 program. A modified Generalized Born
(GB) implicit water model was used to mimic the solvable environment. Charges and other
parameters used in the minimization for PA and PC lipid molecules were obtained from the
ANTECHAMBER module in AMBER8. For PA, a charge of -2 was estimated. The Amber99 force
field was used for the DEP domain during minimization. The same procedures were used to
prepare the K/E mutant DEP domain/membrane modeling.
To facilitate the calculations, the relaxed DEP-membrane bilayer structure was changed into a
single layer model by removing the bottom membrane leaflet. The binding energy of DEP to
single layer membrane was evaluated by using the MM/PBSA module in AMBER8. The free
energy change in solution was taken as the sum of
!Gel (polar part) and !Gnp (non polar part) in
Equation (1):
(1)
!G sol = !Gel + !Gnp
!Gel was calculated by solving the nonlinear Poisson-Boltzmann (PB) equation using DelPhi, a
three-dimensional finite difference program, and
!Gnp was calculated by using an empirical
expression (Equation (2)), which mainly involves an accurate determination of molecular surface
!A of the membrane, protein and resulting complex.
(2)
!Gnp = "!A
The contribution of entropy to the binding free energies was not considered, given the difficulty to
estimate entropy for large protein/membrane systems and given the fact that the confirmation
difference between wild-type DEP/membrane and K/E DEP/membrane systems was negligible.
All calculations were carried out in a 420-cpu P4 cluster system located at the Hartwell Center, St
Jude Children’s Research Hospital.
siRNA sequences
against GFP
(5’- P GGCUACGUCCAGGAGCGCACCUUCCGAUGCAGGUCCUCGCGU P-5’).
© 2009 Macmillan Publishers Limited. All rights reserved.
against human Nhe3
duplex 1: sense: GGACAGAUCGGCAUAAUUUU, antisense: AAUUGUGCCCGAUCUGUCCUU;
duplex
2:
sense:
UGUGAAGGACGCGAUGUGGUU;
antisense:
CCACAUCGCGUCCUUCACAUU,
duplex
3:
sense:
UGUUUCUCGUCCUCCGUGGUU;
antisense:
CCACGGAGGACGAGAAACAUU,
duplex
4:
GCACCACCAUCAUCGUAGUUU, antisense: ACUACGAUGAUGGUGGUGCUU)
were obtained from Dharmacon.
© 2009 Macmillan Publishers Limited. All rights reserved.
sense: