Wnt Signaling Requires Retromer-Dependent Recycling of MIG-14/Wntless in Wnt-Producing Cells

Article (PDF Available)inDevelopmental Cell 14(1):140-7 · February 2008with46 Reads
DOI: 10.1016/j.devcel.2007.12.004 · Source: PubMed
Abstract
Wnt proteins are secreted signaling molecules that play a central role in development and adult tissue homeostasis. We have previously shown that Wnt signaling requires retromer function in Wnt-producing cells. The retromer is a multiprotein complex that mediates endosome-to-Golgi transport of specific sorting receptors. MIG-14/Wls is a conserved transmembrane protein that binds Wnt and is required in Wnt-producing cells for Wnt secretion. Here, we demonstrate that in the absence of retromer function, MIG-14/Wls is degraded in lysosomes and becomes limiting for Wnt signaling. We show that retromer-dependent recycling of MIG-14/Wls is part of a trafficking pathway that retrieves MIG-14/Wls from the plasma membrane. We propose that MIG-14/Wls cycles between the Golgi and the plasma membrane to mediate Wnt secretion. Regulation of this transport pathway may enable Wnt-producing cells to control the range of Wnt signaling in the tissue.
Developmental Cell
Short Article
Wnt Signaling Requires
Retromer-Dependent Recycling
of MIG-14/Wntless in Wnt-Producing Cells
Pei-Tzu Yang,
1,2
Magdalena J. Lorenowicz,
1,2
Marie Silhankova,
1
Damien Y.M. Coudreuse,
1,3
Marco C. Betist,
1
and Hendrik C. Korswagen
1,
*
1
Hubrecht Institute, Developmental Biology and Stem Cell Research, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands
2
These authors contributed equally to this work.
3
Present address: The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
*Correspondence: r.korswagen@niob.knaw.nl
DOI 10.1016/j.devcel.2007.12.004
SUMMARY
Wnt proteins are secreted signaling molecules that
play a central role in development and adult tissue
homeostasis. We have previously shown that Wnt
signaling requires retromer function in Wnt-produc-
ing cells. The retromer is a multiprotein complex
that mediates endosome-to-Golgi transport of spe-
cific sorting receptors. MIG-14/Wls is a conserved
transmembrane protein that binds Wnt and is re-
quired in Wnt-producing cells for Wnt secretion.
Here, we demonstrate that in the absence of retromer
function, MIG-14/Wls is degraded in lysosomes and
becomes limiting for Wnt signaling. We show that ret-
romer-dependent recycling of MIG-14/Wls is part of
a trafficking pathway that retrieves MIG-14/Wls from
the plasma membrane. We propose that MIG-14/
Wls cycles between the Golgi and the plasma mem-
brane to mediate Wnt secretion. Regulation of this
transport pathway may enable Wnt-producing cells
to control the range of Wnt signaling in the tissue.
INTRODUCTION
During the development of complex multicellular organisms, uni-
form fields of cells are organized to form different cell types and
anatomical structures. An important role in this process is played
by secreted morphogens such as Wnt proteins, which provide
positional information to cells in the tissue by forming a concen-
tration gradient (Cadigan, 2002). To induce a precise and repro-
ducible pattern, the shape and range of the Wnt gradient needs
to be tightly regulated. Studies on the Wnt protein Wingless in the
Drosophila wing imaginal disc have shown that the main kinetic
parameters that control the Wingless gradient are the rate of
Wingless production and the rate of Wingless diffusion and
degradation (Kicheva et al., 2007). Although much attention
has been focused on how Wnt spreading and degradation is reg-
ulated (Baeg et al., 2001; Lin, 2004; Piddini et al., 2005; Strigini
and Cohen, 2000), the mechanism of Wnt production and secre-
tion is still poorly understood (Coudreuse and Korswagen, 2007;
Hausmann et al., 2007).
Purification and biochemical characterization of secreted Wnt
has revealed that Wnt proteins are glycosylated and lipid modi-
fied (Takada et al., 2006; Willert et al., 2003). The lipid modifica-
tions are most likely attached in the endoplasmic reticulum by
the membrane-bound O-acyltransferase Porcupine and are
required for secretion and signaling activity (Kadowaki et al.,
1996; Takada et al., 2006; Zhai et al., 2004). Several lines of ev-
idence suggest that Wnt is not secreted via the default secretory
pathway. First, the lipid modification of Wnt has been reported to
function as a sorting signal that targets Wnt to specialized mem-
brane microdomains known as lipid rafts, which may partition
Wnt into a specific secretory pathway (Zhai et al., 2004). Second,
Wnt localizes to multivesicular bodies and recycling endosomes,
which may represent intermediate steps in a specialized secre-
tory route (Pfeiffer et al., 2002; van den Heuvel et al., 1989).
Finally, it has recently been shown that Wnt secretion depends
on the Wnt binding protein Wntless (Wls; also known as Even-
ness interrupted [Evi] or Sprinter) (Banziger et al., 2006; Bartsch-
erer et al., 2006; Goodman et al., 2006). Wls is a highly conserved
multipass transmembrane protein that is specifically required in
Wnt-producing cells for Wnt secretion. Based on these proper-
ties, it has been proposed that Wls functions as a chaperone
or sorting receptor for Wnt (Ching and Nusse, 2006; Coudreuse
and Korswagen, 2007; Hausmann et al., 2007).
In the nematode C. elegans, the Wnt EGL-20 is produced by
a group of cells in the tail and forms a concentration gradient
along the anteroposterior body axis (Coudreuse et al., 2006;
Whangbo and Kenyon, 1999 ). Among the targets of EGL-20
are the Q neuroblasts and their descendants, which migrate in
opposite directions on the left and right side of the animal (Harris
et al., 1996). EGL-20 activates a canonical Wnt/b-catenin path-
way in the left Q neuroblast (QL) that induces the expression of
the Hox gene mab-5 and thereby directs the migration of the
QL descendants toward the posterior (Salser and Kenyon,
1992). In mutants that disrupt EGL-20 signaling, mab-5 is not
expressed and as a consequence, the QL descendants migrate
in the opposite, anterior direction. We have previously shown
that an intracellular protein sorting complex called the retromer
complex is required for the EGL-20-dependent migration of the
QL descendants (Coudreuse et al., 2006). Tissue-specific rescue
and mosaic analysis demonstrated that the retromer complex is
specifically required in EGL-20-producing cells (Coudreuse
et al., 2006; Prasad and Clark, 2006) and knockdown studies
Developmental Cell 14, 1–8, January 2008 ª2008 Elsevier Inc. 1
Please cite this article in press as: Yang et al., Wnt Signaling Requires Retromer-Dependent Recycling of MIG-14/Wntless in Wnt-Producing Cells,
Developmental Cell (2008), doi:10.1016/j.devcel.2007.12.004
in Xenopus tropicalis showed that the function of the retromer
complex in Wnt signaling is evolutionarily conserved.
The retromer consists of a core complex of Vps35, Vps26, and
Vps29 that mediates cargo recognition and the sorting nexin
accessory proteins SNX1 and SNX2, which are required for
membrane association (Seaman, 2005). Studies in yeast and
mammalian cells have shown that the retromer complex medi-
ates the retrieval of specific sorting receptors such as the car-
boxypeptidase Y receptor Vps10p and the cation-independent
mannose-6-phosphate (CI-MPR) receptor from late endosomes
to the trans-Golgi network (Arighi et al., 2004; Haft et al., 2000;
Seaman et al., 1998). In addition, the retromer complex functions
in the endocytic recycling of the Fet3p-Fet1p iron receptor in
yeast ( Strochlic et al., 2007) and in the basal-to-apical transcyto-
sis of the polymeric-immunoglobulin receptor-IgA complex in
polarized epithelial cells (Verges et al., 2004).
The function of the retromer complex in intracellular protein
trafficking suggests that it mediates a specific transport step
in Wnt-producing cells that is directly or indirectly required for
Wnt signaling. Here, we show that the Wnt binding protein
MIG-14/Wls is a target of the retromer complex. In the absence
of retromer complex function, MIG-14/Wls is degraded in lyso-
somes and becomes limiting for Wnt signaling. Our results indi-
cate that the retromer-dependent sorting step is part of a trans-
port pathway that retrieves MIG-14/Wls from the plasma
membrane. Regulation of MIG-14/Wls transport and stability
may enable Wnt-producing cells to control the rate of Wnt secre-
tion and the range of Wnt signaling in the tissue.
RESULTS
mig-14 Is Expressed in Wnt-Producing Cells
The C. elegans Wls ortholog MIG-14 has an essential function in
Wnt signaling, as is demonstrated by the wide range of Wnt phe-
notypes that can be observed in mig-14 mutants (Eisenmann
and Kim, 2000; Harris et al., 1996; Thorpe et al., 1997). Like
Wls in Drosophila and mammalian cells, MIG-14 is specifically
required in Wnt-producing cells. This has been demonstrated
by mosaic analysis in the early embryo (Thorpe et al., 1997)
and is supported by our observation that the EGL-20/Wnt-
dependent migration of the QL descendants can be restored in
mig-14 mutants by specific expression of mig-14 in EGL-20-pro-
ducing cells (Table 1). To further investigate the function of MIG-
14 in Wnt signaling, we determined the spatial and temporal
expression pattern of mig-14 by fusing the mig-14 promoter
and coding sequence to gfp. This fusion rescues the mig-14
null phenotype, demonstrating that the mig-14::gfp transgene
is expressed correctly and that the MIG-14::GFP fusion protein
remains functional (data not shown). At the comma stage, during
larval development, and in adult animals, mig-14 is mainly ex-
pressed in the posterior part of the animal (see Figures S1A
and S1B in the Supplemental Data available with this article
online). We found that the expression of mig-14 overlaps with
the known expression patterns of C. elegans Wnt genes. Thus,
mig-14 is expressed in the tail hypodermis (Figure S1C), which
expresses the Wnt gene lin-44 (Herman and Horvitz, 1994); in
cells in the anal region that express egl-20/Wnt (Whangbo and
Kenyon, 1999); and in posterior body wall muscle cells that
express cwn-1/Wnt (Gleason et al., 2006; Pan et al., 2006). In
addition, mig-14 is strongly expressed in the stomatointestinal
muscle, the mesoblast cell M and its descendants, the CAN neu-
rons, the developing vulva, the pharynx, and the pharyngeal
intestinal valve (Figures S1A, S1D, and S1E). mig-14 is also
weakly expressed in a small subset of head neurons, the ventral
nerve cord, and the seam cells, but is undetectable in the main
body hypodermis and the intestine.
Mutation of the Retromer Complex Enhances
the Wnt Phenotype of mig-14
We have previously shown that EGL-20/Wnt signaling requires
retromer function in EGL-20-producing cells (Coudreuse et al.,
2006). The shared site of action and similarity in phenotype of
mig-14 and retromer mutants suggests a functional relationship
in the regulation of Wnt signaling. To investigate this possibility,
we tested whether mig-14 and retromer mutants genetically
interact. The null phenotype of mig-14 is embryonic lethal, but
the hypomorphic alleles mig-14(mu71) and mig-14(ga62) are
viable and show a range of postembryonic Wnt phenotypes (Ei-
senmann and Kim, 2000; Harris et al., 1996). We found that dou-
ble mutants of mig-14(mu71) or mig-14(ga62) with vps-35(hu68)
are mostly sterile. Double mutants with vps-29(tm1320), which
has a much weaker Wnt phenotype than vps-35(hu68) (Cou-
dreuse et al., 2006), are however viable and produce sufficient
progeny for analysis. As mig-14(mu71) and mig-14(ga62) single
mutants already show a strong defect in the EGL-20/Wnt-de-
pendent migration of the QL descendants (Table 1), we used
the polarization of the PLM mechanosensory neurons, which is
mediated by multiple, redundantly acting Wnts, as an assay for
Table 1. Rescue of the EGL-20/Wnt-Dependent Migration of the
QL Descendants
% Wild-Type QL
Descendant Migration
Wild-type 100
vps-35(hu68) 0
vps-35(hu68); Phs::mig-14 - HS 0
vps-35(hu68); Phs::mig-14 + HS 27
vps-35(hu68); mig-14::gfp 100
mig-14(mu71) 0
mig-14(ga62) 0
mig-14(mu71); Pegl-20::mig-14::gfp 35
mig-14(ga62); Pegl-20::mig-14::gfp 30
mig-14(mu71); Pric-19::mig-14::gfp 0
mig-14(ga62); Pric-19::mig-14::gfp 0
dpy-23(e840)
a
6
dpy-23(e840); Pegl-20::dpy-23 65
vps-35(hu68) mutant s carrying a Phs::mig-14 transgene were heat-
shocked for 10 min at 33
!
C at the early L1 stage (+ HS). vps-35(hu68)
was also combined with the mig-14::gfp-expressing transgene huIs71.
Tissue-specific rescue experiments using the egl-20 or ric-19 promoter
were as described (Coudreuse et al., 2006). The final positions of the
QL descendants were scored using the mec-7::gfp-expressing trans-
genes muIs32 or muIs35 or by DIC microscopy. The mean of the results
obtained in two to six independent experiments is shown (in each case,
n > 60).
a
Nontransgenic siblings from dpy-23(e840); Pegl-20::dpy-23.
Developmental Cell
Retromer-Dependent Recycling of MIG-14/Wls
2 Developmental Cell 14, 1–8, January 2008 ª2008 Elsevier Inc.
Please cite this article in press as: Yang et al., Wnt Signaling Requires Retromer-Dependent Recycling of MIG-14/Wntless in Wnt-Producing Cells,
Developmental Cell (2008), doi:10.1016/j.devcel.2007.12.004
Wnt signaling (Hilliard and Bargmann, 2006; Prasad and Clark,
2006). In wild-type and most vps-29(tm1320) or mig-14(mu71)
single mutants, the PLM neurons show a normal polarity (Table
2). In mig-14(mu71); vps-29 double mutants, however, about
a third of the animals show either reversal or loss of PLM polarity.
We observed a similar synergistic effect on PLM polarity in mig-
14(ga62); vps-29 double mutants. Taken together, these results
demonstrate that reduction of retromer function strongly en-
hances the Wnt phenotype of hypomorphic mig-14 alleles.
These experiments do not distinguish, however, between a func-
tion of mig-14 and of the retromer complex in a shared or in
parallel genetic pathways.
Human Wls Colocalizes with the Retromer Complex
at the Golgi and Endosomes
Wls localizes to the plasma membrane in Drosophila wing imag-
inal disc cells (Bartscherer et al., 2006) and to the Golgi and en-
dosomes in mammalian Vero cells (Banziger et al., 2006). We
found that in C. elegans, MIG-14 localizes to the cell periphery
as well as to distinct intracellular punctae (Figure 1A). To further
investigate the subcellular distribution of Wls and to determine
whether Wls and the retromer complex colocalize, we expressed
a human Wls-GFP fusion protein in HeLa cells. Wls-GFP local-
izes to the cell periphery (Figure 1B), indicating that Wls is pres-
ent at the plasma membran e (although localization to docked
secretory vesicles cannot be excluded). Furthermore, Wls-GFP
localizes to the endoplasmic reticulum (as indicated by the peri-
nuclear staining), to the Golgi, and to endosomal structures,
including EEA1-positive early endosomes (Figures 1B and 1C).
In agreement with other studies, we found that the retromer com-
plex localizes to endosomes (Figure 1C) (Arighi et al., 2004;
Seaman, 2004). Importantly, staining of endogenous Vps26 in
cells that express Wls-GFP showed that Wls and the retromer
complex colocalize on endosomes (Figure 1C).
MIG-14/Wls Is Targeted to Lysosomes
in Retromer Mutants
One of the principle functions of the retromer complex is the en-
dosome-to-Golgi retrieval of sorting receptors such as Vps10p in
yeast and the CI-MPR receptor in mammalian cells (Arighi et al.,
2004; Haft et al., 2000; Seaman et al., 1998). In the absence of
retromer function, Vps10p and the CI-MPR receptor are trans-
ported to lysosomes and degraded. To investigate if MIG-14 is
also a target of retromer-dependent sorting, we tested whether
the subcellular localization or stability of MIG-14 is affected in
retromer mutants. As shown in Figure 2B, there is a striking
reduction in MIG-14::GFP protein levels in vps-35(hu68) mu-
tants. Western blot analysis showed that MIG-14 protein levels
are also reduced in vps-29(tm1320) mutants (Figure 2C), but
there is a marked difference in the extent of this reduction. We
have previously shown that mutation of vps-35 produces a stron-
ger defect in EGL-20/Wnt signaling than mutation of vps-29
(Coudreuse et al., 2006). This difference is reflected in the effect
on MIG-14 protein levels, indicating that the reduction in MIG-14
correlates with the Wnt phenotype of retromer mutants. To
examine if human Wls is also a target of the retromer complex,
we knocked down Vps35 and Vps26 in human embryonic kidney
(HEK293) cells and assayed the effect on endogenous Wls
levels. Using a polyclonal antiserum that recognizes human
Wls on western blot, we found that knockdown of both Vps35
and Vps26 induces a significant reduction in endogenous Wls
protein levels (Figure 2D). These results demonstrate that the
function of the retromer complex in MIG-14/Wls recycling is
evolutionarily conserved.
To investigate why MIG-14/Wls protein levels are reduced, we
tested whether MIG-14 is targeted to lysosomes in the absence
of retromer function. mig-14 is expressed in posterior body wall
muscle cells, which are relatively large cells that are ideally suited
for the imaging of subcellular structures. To visualize lysosomes,
we used a fusion of the lysosomal protein LMP-1 with mCherry.
As shown in Figure 2E, there was almost no overlap between
MIG-14::GFP and LMP-1::mCherry in control animals. In vps-
35(RNAi) animals, however, most of the MIG-14::GFP protein
that remains is localized to LMP-1::mCherry-positive structures.
To investigate whether human Wls is also targeted to lysosomes,
we performed colocalization experiments with Wls-GFP and the
lysosomal marker CD63 in HeLa cells. As shown in Figure 2F,
there is a strong increase in colocalization between Wls and
CD63 when Vps35 and Vps26 are knocked down, demonstrating
that in the absence of retromer function, human Wls localizes to
lysosomes as well. To test if Wls is degraded in lysosomes, we
treated HeLa cells with the V-type ATPase inhibitor Bafilomycin
A, which prevents the endosomal acidification that is required
for the maturation of lysosomal proteases and blocks transport
from late endosomes to lysosomes (van Weert et al., 1995).
We found that treatment with Bafilomycin A results in a significant
increase in Wls protein levels (Figure 2G). These results are
consistent with the hypothesis that in the absence of retromer
function, MIG-14/Wls is degraded in lysosomes.
Reduction of MIG-14 Protein Levels Limits Wnt
Signaling in Retromer Mutants
The strong reduction in MIG-14 protein levels in retromer mutants
suggests that MIG-14 may become limiting for Wnt signaling. To
investigate this possibility, we tested whether overexpression of
MIG-14 can rescue Wnt signaling in retromer mutants. To overex-
press MIG-14, we generated a transgene that expresses mig-14
under the control of an inducible heat-shock promoter (String-
ham et al., 1992). vps-35(hu68) animals and vps-35; hs::mig-14
animals raised at the noninducing temperature show a fully pen-
etrant defect in the EGL-20/Wnt-dependent migration of the QL
descendants (Table 1). When mig-14 expression is induced by
a brief heat pulse before EGL-20 signaling commences at the
Table 2. Genetic Interaction between mig-14 and vps-29 in PLM
Polarity
% PLM Polarity Defect
Wild-type 0
vps-29(tm120) 1±2
mig-14(mu71) 0
mig-14(mu71); vps-29(tm120) 29 ± 13
mig-14(ga62) 35 ± 5
mig-14(ga62); vps-29(tm120) 67 ± 9
The polarity of the PLM neuron was scored using the mec-7::gfp-ex-
pressing transgene muIs35 as described (Hilliard and Bargmann, 2006).
Data are represented as mean ± SD (n > 100).
Developmental Cell
Retromer-Dependent Recycling of MIG-14/Wls
Developmental Cell 14, 1–8, January 2008 ª2008 Elsevier Inc. 3
Please cite this article in press as: Yang et al., Wnt Signaling Requires Retromer-Dependent Recycling of MIG-14/Wntless in Wnt-Producing Cells,
Developmental Cell (2008), doi:10.1016/j.devcel.2007.12.004
early L1 larval stage, about a third of the animals show a wild-type
pattern of QL descendant migration. In addition to heat-shock-
promoter-induced overexpression, we found that overexpres-
sion of mig-14 from its own promoter also restores EGL-20/Wnt
signaling in vps-35 mutants (Table 1). These data support the hy-
pothesis that the Wnt phenotype of retromer mutants is a result of
the reduction in MIG-14 protein levels.
Human Wls Colocalizes with the AP-2 Adaptin Complex,
but Not with Caveolin
The localization of human Wls to early endosomes (Figure 1C)
suggests that Wls may be internalized from the plasma mem-
brane. To investigate this possibility, we tested whether Wls
colocalizes with the AP-2 adaptin complex or caveolin. AP-2 is
a heterotetrameric complex that selects specific transmembrane
proteins for clathrin-mediated endocytosis (Bonifacino and
Traub, 2003), whereas caveolin is part of an alternative, clathrin-
independent endocytosis pathway (Razani and Lisanti, 2001). We
found no overlap between endogenous caveolin-1 and Wls-GFP
in HeLa cells (Figure 1C). However, staining of endogenous AP-2a
showed clear colocalization between Wls and the AP-2 complex
at distinct punctae (Figure 1C), indicating that Wls may be inter-
nalized through AP-2 and clathrin-mediated endocytosis.
EGL-20/Wnt Signaling Requires AP-2-Mediated
Internalization of MIG-14
A function of the AP-2 complex in Wnt signaling is also sug-
gested by the mutant phenotype of the AP-2 m subunit gene
Figure 1. Subcellular Localization of MIG-14 and Wls
(A) Expression of mig-14::gfp (huIs71) in the posterior half of an early L1 larva (top panel). The boxed area shows the mesoblast cell M, which is magnified in the
bottom panel. The outline of the M cell is indicated by arrowheads. Scale bar, 4 mm.
(B) HeLa cells grown on glass coverslips were transfected with Wls-GFP and fixed to determine the intracellular localization of Wl s-GFP. Bars, 20 mm. Images are
representative of at least three independent experiments. Dashed box is magnified and represented in the right panel. Arrows indicate plasma membrane local-
ization of Wls.
(C) HeLa cells grown on glass coverslips were transfected with Wls-GFP, fixed, and stained for endogenous Golgin-97, EEA1, Vps26, caveolin-1, or AP-2a. Scale
bars, 10 mm. Images are representative of at least three independent experime nts. Dashed boxes are magni fied and represented in the lower panels. White
arrows indicate colocalization of Wls with EEA1, Vps26, or AP-2a.
Developmental Cell
Retromer-Dependent Recycling of MIG-14/Wls
4 Developmental Cell 14, 1–8, January 2008 ª2008 Elsevier Inc.
Please cite this article in press as: Yang et al., Wnt Signaling Requires Retromer-Dependent Recycling of MIG-14/Wntless in Wnt-Producing Cells,
Developmental Cell (2008), doi:10.1016/j.devcel.2007.12.004
dpy-23 (G. Garriga, personal communication; see also Pan et al.,
2008). dpy-23(e840) mutants are viable and show a highly pene-
trant defect in the EGL-20/Wnt-dependent migration of the QL
descendants (Table 1). To investigate if dpy-23 function is re-
quired in EGL-20-producing cells, we tested whether specific
expression of wild-type dpy-23 from the egl-20 promoter can
restore EGL-20 signaling in dpy-23(e840) mutants. As shown in
Table 1, expression of dpy-23 in EGL-20-producing cells is suf-
ficient to rescue the migration of the QL descendants in dpy-
23(e840) mutants. This demonstrates that AP-2 function is
specifically required in EGL-20-producing cells.
To examine whether MIG-14 is a target of AP-2-mediated endo-
cytosis, we testedthe effectof RNAi-mediated knockdown of differ-
ent AP-2 subunits on the subcellular localization of MIG-14::GFP. In
control animals, MIG-14 was visible throughout the cell and at the
plasma membrane (Figures 3A and 3B). However, in animals that
were treated with RNAi against the AP-2 subunits dpy-23, apa-2,
or aps-2 or the shared AP-2 and AP-1 subunit apb-1 (Boehm and
Bonifacino, 2001), there was a striking accumulation of MIG-14
on the cell membrane, indicating that internalization of MIG-14 is
impaired in the absence of a functional AP-2 complex.
Inhibition of AP-2-Mediated Endocytosis Increases
MIG-14/Wls Protein Levels
We found that inhibition of AP-2 function results in an increase in
MIG-14/Wls protein levels. Thus, MIG-14 protein levels were
increased when AP-2 function was disrupted by apa-2 or apb-
1 RNAi (Figure 3C). Furthermore, human Wls protein levels
were increased when AP-2 function was inhibited by siRNA-me-
diated knockdown of AP-2a in HeLa cells (Figure 3D). Inhibition
of AP-2 function may increase MIG-14/Wls protein levels by pre-
venting degradation of MIG-14/Wls. Even in a wild-type back-
ground, incomplete recycling of MIG-14/Wls by the retromer
complex leads to MIG-14/Wls degradation, as shown by the
increase in Wls protein levels when lysosomal degradation is
inhibited (Figure 2G). We propose that inhibition of AP-2 function
prevents MIG-14/Wls degradation because AP-2-mediated
internalization occurs before the retromer-dependent recycling
of MIG-14/Wls. When internalization is blocked, the balance
between de novo synthesis and degradation of MIG-14/Wls is
shifted, leading to the observed increase in MIG-14/Wls protein
levels. An important prediction of this model is that inhibition of
MIG-14 internalization should also increase MIG-14 protein
levels in a retromer mutant background. We found that this is
indeed the case (Figure 3E). The increase in MIG-14 protein
levels is however smaller than in wild-type animals, which is likely
the result of incomplete inhibition of apa-2 and apb-1 function by
RNAi. Taken together, these results suggest that MIG-14/Wls is
first internalized through AP-2-mediated endocytosis and is then
recycled by the retromer complex.
DISCUSSION
Wnt proteins are lipid-modified signaling molecules that can
form long-range concentration gradients to pattern developing
tissues. A fundamental question is how the hydrophobic Wnt
Figure 2. Reduction of MIG-14 and Wls Protein Levels
and Lysosoma l Targeting in the Absence of Retromer
Function
(A and B) MIG-14::GFP (huIs71) in wild-type and vps-35(hu68).
Images were taken using identical camera settings.
(C) Western blot analysis of MIG-14:: GFP (huIs71) protein
levels in wild-type, vps-35(hu68), and vps-29(tm1320) mu-
tants. a-tubulin is used as a loading control. The ratio between
MIG-14::GFP and tubulin levels is shown.
(D) Western blot detection of endogenous human Wls in
HEK293 cells stably expressing a doxycycline (DOX)-inducible
Vps35 siRNA construct (Coudreuse et al., 2006). HEK293 cells
were nonstimulated or stimulated with DOX and additionally
transfected with a control siRNA or a combination of Vps26
siRNA and Vps35 siRNA for more efficient knockdown of ret-
romer expression. a-tubulin was used as a control for equal
loading. The ratio between Wls or Vps35 and tubulin is shown.
Representative results of at least three independent experi-
ments are shown.
(E) Subcellular localization of MIG-14::GFP (huIs72, green) and
the lysosomal marker LMP-1::mCherry (red) in posterior body
wall muscle cells of adult animals. Images show a single mus-
cle cell. Scale bar, 10 mm. Arrow heads show colocalization of
MIG-14::GFP and LMP-1::mCherry.
(F) HeLa cells grown on glass coverslips were transfected with
Wls-GFP and control siRNA or siRNAs directed against Vps35
and Vps26, fixed, and stained for endogenous CD63. Scale
bar, 10 mm.
(G) HeLa cells were treated with 100 nM Bafilomycin A for
30 min before cells were lysed to detect endogenous Wls.
The ratio between Wls and tubulin is shown. Representative
results of two independent experiments are shown.
Developmental Cell
Retromer-Dependent Recycling of MIG-14/Wls
Developmental Cell 14, 1–8, January 2008 ª2008 Elsevier Inc. 5
Please cite this article in press as: Yang et al., Wnt Signaling Requires Retromer-Dependent Recycling of MIG-14/Wntless in Wnt-Producing Cells,
Developmental Cell (2008), doi:10.1016/j.devcel.2007.12.004
protein is secreted and released at the cell membrane. We have
previously shown that Wnt signaling requires retromer function in
Wnt-producing cells (Coudreuse et al., 2006). In this study, we
show that in the absence of retromer function, the Wnt binding
protein MIG-14/Wls is degraded in lysosomes and becomes
limiting for Wnt signaling.
The retromer complex mediates the endosome-to-Golgi
retrieval of specific cargo proteins such as Vps10p and the CI-
MPR receptor (Arighi et al., 2004; Haft et al., 2000; Seaman
et al., 1998). In the absence of retromer-dependent sorting,
these proteins are degraded in lysosomes. Since MIG-14 is
also targeted to lysosomes in retromer mutants, it is likely that
the retromer complex functions in a similar endosome-to-Golgi
retrieval step for MIG-14/Wls. Cargo recognition by the retromer
complex is mediated by the Vps35 subunit, which directly binds
to the cargo protein (Arighi et al., 2004; Nothwehr et al., 2000).
Studies on the retromer-dependent transport of the CI-MPR
receptor have shown that the interaction with cargo depends
on a highly conserved W/F-L-M/V tripeptide motif in the target
protein (Seaman, 2007). Interestingly, such a motif (F-L-M) is
also present at the end of the third intracellular domain of MIG-
14/Wls. Although we have not been able to demonstrate an inter-
action between MIG-14/Wls and the retromer in coimmunopre-
cipitation experiments, the presence of a conserved retromer
sorting motif and the colocalization between human Wls and
the retromer on endosomes suggests that MIG-14/Wls may be
a direct target of the retromer complex.
Several models have been proposed for the function of MIG-
14/Wls in Wnt secretion, including a role of MIG-14/Wls in Wnt
folding and maturation and a function of MIG-14/Wls as a specific
sorting receptor for Wnt ( Ching and Nusse, 2006; Coudreuse and
Korswagen, 2007; Hausmann et al., 2007). The subcellular local-
ization of Wls at the Golgi, endosomes, and the plasma mem-
brane and the requirement of MIG-14/Wls transport for Wnt
signaling favor the latter possibility. We therefore propose
a model in which MIG-14/Wls functions as a sorting receptor
that transports Wnt from the Golgi to the plasma membrane for
release. To maintain sufficient levels of MIG-14/Wls in the Golgi,
MIG-14/Wls is recycled from the plasma membrane through
an AP-2-dependent and retromer complex-dependent pathway.
Figure 3. MIG-14 Internalization Requires AP-2-Mediated Endocytosis
(A) MIG-14::GFP (huIs71) expression in animals treated wit h control, dpy-23, or apa-2 RNAi. Arrowheads indicate plasma membrane accumulation of
MIG-14::GFP in body wall muscle cells. The dotted line indicates the EGL-20/Wnt-producing cells, which express a higher level of MIG-14::GFP. Scale bar,
20 mm. (B) Quantification of the effect of AP-2 RNAi on the subcellular distribution of MIG-14::GFP. In all cases n > 100, except apa-2 (n = 58). (C and E) Western
blot detection of MIG-14::GFP in wild-type or vps-29(tm1320) animals treated with control, apa-2, or apb-1 RNAi. The ratio between MIG-14::GFP and tubulin
levels is shown. (D) Western blot detection of endogenous human Wls in HeLa cells transfected with control siRNA or siRNA directed against AP-2a. a-tubulin was
used as a control for equal loading. The ratio between Wls or AP-2a and tubulin is shown. Representative results of at least three independent experiments are
shown.
Developmental Cell
Retromer-Dependent Recycling of MIG-14/Wls
6 Developmental Cell 14, 1–8, January 2008 ª2008 Elsevier Inc.
Please cite this article in press as: Yang et al., Wnt Signaling Requires Retromer-Dependent Recycling of MIG-14/Wntless in Wnt-Producing Cells,
Developmental Cell (2008), doi:10.1016/j.devcel.2007.12.004
In the absence of the first, AP-2-dependent step, MIG-14/Wls is
trapped on the plasma membrane. When the second, retromer-
dependent recycling step is disrupted, MIG-14/Wls is targeted to
lysosomes and degraded. In both cases, only a limited pool of
MIG-14/Wls is available in the Golgi to mediate Wnt transport
and secretion. As predicted by this model, overexpression of
mig-14 rescues the Wnt phenotype of retromer mutants. Al-
though this model explains the function of AP-2-mediated endo-
cytosis and retromer-dependent recycling in Wnt signaling, the
function of MIG-14/Wls in Wnt transport and release remains
to be established. It is not known at what stage in the trafficking
cycle Wnt binds to MIG-14/Wls, and it is also not clear how Wnt
is released.
An important aspect of the retromer mutant phenotype is that
the effect on Wnt signaling increases with distance from the
source of Wnt (Coudreuse et al., 2006). We initially hypothesized
that the retromer complex is required for the formation of a spe-
cific long-range acting pool of Wnt; for example, by enabling the
interaction of Wnt with lipoprotein particles (Coudreuse and
Korswagen, 2007; Coudreuse et al., 2006). Our results suggest
that the effect on the range of Wnt signaling may also be a con-
sequence of the decrease in MIG-14 protein levels. According to
our model, the decrease in MIG-14 protein levels will lead to a re-
duction in the rate of Wnt secretion. Such a reduction in secretion
rate will result in a shallower concentration gradient (Kicheva
et al., 2007), explaining the observed defect in long-range Wnt
signaling. As MIG-14 is decreased, but not eliminated, in the
absence of retromer function, Wnt secretion will only be partially
affected, indicating why short-range signaling by EGL-20 and
other Wnt proteins is not, or only weakly, affected in retromer
mutants. In conclusion, our results show that transport and recy-
cling of MIG-14/Wls in Wnt-producing cells is essential for Wnt
signaling. Future studies will examine whether regulation of this
process provides Wnt-producing cells with a mechanism to
control the range of Wnt signaling in the tissue.
EXPERIMENTAL PROCEDURES
Cell Culture and Transfection
HeLa and HEK293 cells were maintained in RPMI 1640 medium (GIBCO) con-
taining 10% heat-inactivated FCS (GIBCO), 2 mM L-glutamine, 100 U/ml pen-
icillin, and 100 mg/ml streptomycin. HeLa cells were transfected with control
siRNA (Dharmacon) or siRNA against AP-2a (Motley et al., 2003) using Oligo-
fectamine (Invitrogen). Cells were transfected three times with 24 hr intervals
between each transfection. HEK293 cells stably transfected with different
pools of Vps35 siRNA constructs cloned into the pTER vector were stimulated
with doxycycline to induce Vps35 siRNA expression (Coudreuse et al., 2006).
Forty-eight hours after stimulation, cells were additionally transfected with
a combination of siRNA against Vps26 (He et al., 2005 ) and siRNA against
Vps35 (CUGUAGGGAUGCUUUGGCU) to induce more efficient knockdown
of retromer expression. The samples were analyzed by western blot 48 hr after
the last transfection.
Western Blot Analysis
Cells were lysed in Laemmli sample buffer and cell lysates were separated on
10% SDS-P AGE gels, transferred onto PVDF membranes (Bio-Rad), and
stained with antibodies against the indicated proteins. To quantify MIG-
14::GFP protein levels, synchronized L1 larvae were lysed in 2 volumes of
10 mM Tris (pH 8), 150 mM NaCl, and 0.1% NP-40 containing protease inhib-
itors (Roche). For AP-2 RNAi experiments, synchronized egg populations were
hatched on dsRNA expressing bacteria and grown until the L4 stage before the
animals were collected for western blot analysis. Densitome tric analysis was
performed on scanned images using GeneTools (Syngene) analysis software.
Immunofluorescence
HeLa cells were plated on glass coverslips and transfected with plasmid
containing Wls-GFP (Wls-pEGFP, 1 mg) using FuGENE Transfection Rea gent
(Roche). Twenty four hours after transfection, cells were fixed in 0.1 M Phos-
phate Buffer containing 4% paraformaldehyde for ten minutes on ice and
permeabilized with 0.1% Triton X-100 for five minutes. Thereafter, cells were
incubated with 0.5% BSA for 30 min followed by incubation with the indicated
primary antibodies and subsequent incubation with a chicken-anti-mouse-Ig
or chicken-anti-rabbit-Ig antibody labeled with Alexa 594 (Molecular Probes).
Images were recorded with a Bio-Rad Radiance 2100MP confoc al and multi-
photon system (Zeiss/Bio-Rad).
Supplemental Data
The Supplemental Data include Supplemental Experimental Procedures, Sup-
plemental References, and one figure and can be found with this article online
at http://www.developmentalcell.com/cgi/content/full/14/1/---/DC1/.
ACKNOWLEDGMENTS
We thank Hans Clevers for critically reading the manuscript, Raul Rojas and
Juan Bonifacino for retromer antibodies, Shohei Mitani (National Bioresource
Project for the Nematode, Tokyo, Japan) for deletion mutants, Richard Wub-
bolts and Willem Stoorvogel for help with confocal analysis, Andrew Fire for
different vectors, and the Caenorhabditis Genetics Center (University of Min-
nesota, Minneapolis) for strains. This work was supported by the Dutch Cancer
Foundation, the EU FP6 program Cells into Organs, and an NWO VIDI grant
(H.C.K.).
Received: October 5, 2007
Revised: November 15, 2007
Accepted: December 7, 2007
Published online: December 20, 2007
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Developmental Cell
Retromer-Dependent Recycling of MIG-14/Wls
8 Developmental Cell 14, 1–8, January 2008 ª2008 Elsevier Inc.
Please cite this article in press as: Yang et al., Wnt Signaling Requires Retromer-Dependent Recycling of MIG-14/Wntless in Wnt-Producing Cells,
Developmental Cell (2008), doi:10.1016/j.devcel.2007.12.004
    • "Cell biologically, the retromer complex is essential for retrieval of transmembrane proteins or receptors from endosome-to-Golgi apparatus (Bonifacino and Hurley, 2008; McGough and Cullen, 2011; Seaman et al., 2013; Seaman et al., 1997). A growing list of retromer cargos has been identified, such as VPS10/sortilin/SorLA family proteins(Seaman, 2005), cation-independent M6P receptor (CI-M6PR) (Seaman, 2004), mammalian iron transporter DMT1(Tabuchi et al., 2010 ), amyloid precursor protein (APP) (Vieira et al., 2010), APP processing β1 secretase (BACE1) (Wen et al., 2011 ), Caenorhabditis elegans phagocytosis receptor Ced1(Chen et al., 2010), receptor activator of nuclear factor kappa-B (RANK) (Xia et al., 2013b), and seven transmembrane receptors such as Wntless (Belenkaya et al., 2008; Pan et al., 2008; Yang et al., 2008 ), β2- adrenergic receptor (Choy et al., 2014; Temkin et al., 2011), and PTH1R (type 1 receptor for parathyroid hormone) (Feinstein et al., 2011). Thus, VPS35/retromer is likely to be involved in various cellular functions or processes via its regulation of different cargos. "
    [Show abstract] [Hide abstract] ABSTRACT: Parathyroid hormone (PTH) plays critical, but distinct, roles in bone remodeling, including bone formation (anabolic response) and resorption (catabolic response). Although its signaling and function have been extensively investigated, it just began to be understood how distinct functions are induced by PTH activating a common receptor, the PTH type 1 receptor (PTH1R), and how PTH1R signaling is terminated. Here, we provide evidence for vacuolar protein sorting 35 (VPS35), a major component of retromer, in regulating PTH1R trafficking, turning off PTH signaling, and promoting its catabolic function. VPS35 is expressed in osteoblast (OB)-lineage cells. VPS35-deficiency in OBs impaired PTH(1-34)-promoted PTH1R translocation to the trans-Golgi network, enhanced PTH(1-34)-driven signaling, and reduced PTH(1-34)’s catabolic response in culture and in mice. Further mechanical studies revealed that VPS35 interacts with not only PTH1R, but also protein phosphatase 1 regulatory subunit 14C (PPP1R14C), an inhibitory subunit of PP1 phosphatase. PPP1R14C also interacts with PTH1R, which is necessary for the increased endosomal PTH1R signaling and decreased PTH(1-34)’s catabolic response in VPS35-deficient OB-lineage cells. Taken together, these results suggest that VPS35 deregulates PTH1R-signaling likely by its interaction with PTH1R and PPP1R14C. This event is critical for the control of PTH(1-34)-signaling dynamics, which may underlie PTH-induced catabolic response and adequate bone remodeling.
    Full-text · Article · May 2016
    • "doi:10.1371/journal.pone.0149314.g006 Retrograde Transport in C. elegans secretion into the extracellular space [36,383940414243. Retrograde transport of the Wntless/MIG-14 sorting receptor allows it to return to the Golgi to associate with a nascent Wnt cargo molecule for another round of secretion. "
    [Show abstract] [Hide abstract] ABSTRACT: Retrograde transport is a critical mechanism for recycling certain membrane cargo. Following endocytosis from the plasma membrane, retrograde cargo is moved from early endosomes to Golgi followed by transport (recycling) back to the plasma membrane. The complete molecular and cellular mechanisms of retrograde transport remain unclear. The small GTPase RAB-6.2 mediates the retrograde recycling of the AMPA-type glutamate receptor (AMPAR) subunit GLR-1 in C. elegans neurons. Here we show that RAB-6.2 and a close paralog, RAB-6.1, together regulate retrograde transport in both neurons and non-neuronal tissue. Mutants for rab-6.1 or rab-6.2 fail to recycle GLR-1 receptors, resulting in GLR-1 turnover and behavioral defects indicative of diminished GLR-1 function. Loss of both rab-6.1 and rab-6.2 results in an additive effect on GLR-1 retrograde recycling, indicating that these two C. elegans Rab6 isoforms have overlapping functions. MIG-14 (Wntless) protein, which undergoes retrograde recycling, undergoes a similar degradation in intestinal epithelia in both rab-6.1 and rab-6.2 mutants, suggesting a broader role for these proteins in retrograde transport. Surprisingly, MIG-14 is localized to separate, spatially segregated endosomal compartments in rab-6.1 mutants compared to rab-6.2 mutants. Our results indicate that RAB-6.1 and RAB-6.2 have partially redundant functions in overall retrograde transport, but also have their own unique cellular- and subcellular functions.
    Full-text · Article · Feb 2016
    • "The triple and quadruple Wnt mutants were scored with maternal contribution of both egl-20 and cwn-2, so we cannot be certain that maternal Wnt function remains in these mutants. MIG-14/ Wntless is required for Wnt processing and function and affects QL descendant migrations [25]. Two mig-14 mutants had less severe defects than the Wnt triple and quadruple mutants (Fig 1) . "
    [Show abstract] [Hide abstract] ABSTRACT: Directed neuroblast and neuronal migration is important in the proper development of nervous systems. In C. elegans the bilateral Q neuroblasts QR (on the right) and QL (on the left) undergo an identical pattern of cell division and differentiation but migrate in opposite directions (QR and descendants anteriorly and QL and descendants posteriorly). EGL-20/Wnt, via canonical Wnt signaling, drives the expression of MAB-5/Hox in QL but not QR. MAB-5 acts as a determinant of posterior migration, and mab-5 and egl-20 mutants display anterior QL descendant migrations. Here we analyze the behaviors of QR and QL descendants as they begin their anterior and posterior migrations, and the effects of EGL-20 and MAB-5 on these behaviors. The anterior and posterior daughters of QR (QR.a/p) after the first division immediately polarize and begin anterior migration, whereas QL.a/p remain rounded and non-migratory. After ~1 hour, QL.a migrates posteriorly over QL.p. We find that in egl-20/Wnt, bar-1/β-catenin, and mab-5/Hox mutants, QL.a/p polarize and migrate anteriorly, indicating that these molecules normally inhibit anterior migration of QL.a/p. In egl-20/Wnt mutants, QL.a/p immediately polarize and begin migration, whereas in bar-1/β-catenin and mab-5/Hox, the cells transiently retain a rounded, non-migratory morphology before anterior migration. Thus, EGL-20/Wnt mediates an acute inhibition of anterior migration independently of BAR-1/β-catenin and MAB-5/Hox, and a later, possible transcriptional response mediated by BAR-1/β-catenin and MAB-5/Hox. In addition to inhibiting anterior migration, MAB-5/Hox also cell-autonomously promotes posterior migration of QL.a (and QR.a in a mab-5 gain-of-function).
    Full-text · Article · Feb 2016
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