1 Division of Immunology, Children's Hospital, Harvard Medical School, Boston,
MA 02115, USA
2 Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
3 Dipartimento di Scienze Cliniche e Biologiche, Università degli Studi
di Torino, Ospedale San Luigi Gonzaga, Orbassano 10043 TO, Italy
4 Division of Experimental Medicine, Brigham and Women's Hospital, Harvard
Medical School, Boston, MA 02115, USA
5 Department of Medicine, Harvard Medical School, Boston, MA 02115, USA
* Author for correspondence (e-mail: ines.anton{at}unito.it)
Accepted 20 February 2003
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Summary |
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Key words: Actin, PDGF, WIP, Circular ruffle
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Introduction |
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Actin reorganization mediates changes in cell shape associated with cell
motility, migration and chemotaxis in response to platelet-derived growth
factor (PDGF) in a range of cell types including 3T3 fibroblasts
(Hooshmand-Rad et al., 1997;
Wennstrom et al., 1994b
;
Westermark et al., 1990
). PDGF
binds to the PDGF receptor (PDGFR) and induces receptor dimerization,
stimulation of its receptor tyrosine kinase activity and rapid
autophosphorylation. The phosphorylated residues recruit phosphatidylinositol
3-kinase (PI3K), Nck-1, and Nck-2 through Src-homology domain 2 (SH2).
Activation of PI3K leads to Rac1 activation and actin polymerization at the
plasma membrane to produce edge ruffles and lamellipodia
(Chen et al., 2000
;
Kazlauskas, 1994
;
Ridley et al., 1992
;
Wennstrom et al., 1994a
).
The WASP-interacting protein, WIP, is broadly expressed
(Ramesh et al., 1997;
Vetterkind et al., 2002
) and
regulates actin polymerization and spatial organization in different cell
types. WIP interacts in vitro with globular and filamentous actin (G- and
F-actin, respectively) and stabilizes actin filaments
(Martinez-Quiles et al.,
2001
). WIP also interacts with profilin, a regulator of actin
polymerization and depolymerization
(Ramesh et al., 1997
). WIP
overexpression in human B cell lines causes an increase in cellular F-actin
content and induces the formation of subcortical actin patches
(Ramesh et al., 1997
). WIP
microinjection to fibroblasts induces filopodia. This process depends on Cdc42
(Martinez-Quiles et al.,
2001
), a GTPase that (in its active GTP-loaded form) binds to the
hematopoietic-cell-specific WASP
(Aspenstrom et al., 1996
;
Kolluri et al., 1996
;
Symons et al., 1996
) and its
more ubiquitously expressed homolog N-WASP
(Miki et al., 1998a
), causing
a conformational change that allows WASP and N-WASP to interact with the
Arp2/3 complex and initiate actin polymerization
(Kim et al., 2000
;
Miki et al., 1996
;
Rohatgi et al., 2000
). Because
WIP regulates N-WASP-induced actin nucleation and is important for induction
of actin-containing microspikes by bradykinin and Cdc42 in 3T3 fibroblasts
(Martinez-Quiles et al.,
2001
), we set out to analyse its role in PDGF-stimulated actin
cytoskeletal rearrangement in 3T3 cells. We found that WIP is involved in
PDGF-induced the formation of dorsal circular ruffles, and that WIP binding to
actin is essential for this function.
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Materials and Methods |
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3T3 fibroblast culture and PDGF stimulation
NIH 3T3 or Swiss 3T3 murine fibroblasts were transfected with control
plasmid (pcDNA), with pcDNA containing human WIP coding sequence (pcDNA-WIP)
or with pcDNA containing a deletion mutant coding sequence
(pcDNA-WIP43-54) using the standard calcium phosphate protocol. The
vector used as a control or as backbone for cloning was a modified pcDNA3
vector that expresses cloned cDNA as a N-terminal FLAG fusion protein
(Ramesh et al., 1997
).
pcDNA-WIP
43-54 was constructed using the Qickchange®
Site-Directed mutagenesis kit (Stratagene) with the oligo
5'-GCTCTCCTTTCTGATATCGACAGAAGTGCACC-3'.
Transfected cells were selected in the presence of G418 (1 mg ml-1) and maintained in Iscove's medium supplemented with 10% fetal bovine serum (FBS) and G418. Confluent 3T3 cells were split onto glass coverslips (1:10 for 3T3pcDNA and 1:20 for 3T3pcDNA-WIP) in Iscove's 10% FBS containing 1 mg ml-1 G418, grown for 24 hours and then serum-starved for 3-4 days in Iscove's 0.5% FBS with G418. Cells were washed twice with warm Iscove's medium and then stimulated or not with human recombinant PDGF-bb (Intergen) (50 ng ml-1) for 8 minutes at 37°C. In some experiments, cells were pretreated with DMSO as a control or with 12 nM wortmannin for 30 minutes at 37°C and then washed and stimulated with PDGF-bb as described above.
Microinjection
Confluent NIH 3T3 transfected cells were split onto glass coverslips in
Iscove's 10% FBS containing G418 (1 mg ml-1), grown for 24 hours
and then serum-starved for 3-4 days in Iscove's 0.5% FBS with G418.
Microinjection was carried out using an Eppendorf automatic microinjector
(Transjector 5246 and InjectMan Micromanipulator 5179) set at 50-60 hPa
(hectopascals) for 0.3 seconds. Following microinjection, cells were incubated
for 20 minutes prior to stimulation. Dextran cascade blue (Molecular Probes)
was used as a tracer for microinjected cells mixed with affinity-purified
anti-WIP IgG rabbit antibody or control purified rabbit IgG (Cappel) at 500
µg ml-1. At least 150 cells were injected for each reagent. A
total of 200 noninjected cells in the areas surrounding the injected cells
were count for each cover slip.
Immunofluorescence
Stably transfected NIH or Swiss 3T3 fibroblasts, microinjected NIH 3T3
fibroblasts, or primary murine fibroblasts grown and stimulated as described
above were fixed with 4% formalin solution in PBS (Sigma) for 10 minutes at
room temperature, washed twice with PBS and permeabilized with 0.2% Triton
X-100 in PBS at room temperature for 5 minutes. Cells were rinsed twice with
PBS and non-specific binding was blocked with 10% bovine serum albumin (BSA)
in PBS for 20 minutes at room temperature. Rabbit antiserum specific for WIP
was diluted 1/250 in PBS containing 1% BSA and added to the cells for 1 hour
at room temperature. Cells were rinsed twice with PBS and incubated with
Alexa488-conjugated goat anti-rabbit (Molecular Probes; 1/1000) and TRITC
(rhodamine)-conjugated phalloidin (Sigma; 1 µg ml-1) in PBS
containing 1% BSA for 1 hour at room temperature. Cells were rinsed three
times with PBS and air dried, and the coverslips were then mounted with
antifade reagent (Molecular Probes) and visualized with a Nikon Eclipse E800
microscope. Photographs were taken using a CCD-300-RC camera and images were
processed using Adobe Photoshop and Microsoft PowerPoint software. Software
program NIH Image 1.62 was used to quantify fluorescence intensity.
Confocal microscopy was performed on a Bio-Rad MRC600. The CM program was used throughout. For double-labeling visualization, K1 and K2 filters were used. Software programs NIH Image 1.57 and Photoshop were used to analyse and construct images.
Electron microscopy
WIP-transfected and vector-transfected NIH 3T3 fibroblasts were grown and
plated on coverslips as described above and starved for 4 days in Iscove's
medium 0.5% FBS with G418. Starved cells were stimulated for 8 minutes with 50
ng ml-1 PDGF and fixed with PHEM buffer (60 mM PIPES, 25 mM HEPES,
10 mM MgCl2 and 10 mM EGTA) containing 0.75% Triton X-100, 1 µM
phallacidin and 0.05% glutaraldehyde for 2 minutes. Permeabilized cells were
washed in PHEM buffer without fixative and then fixed with 1% glutaraldehyde
in PHEM buffer for 10 minutes. The cytoskeletons were extensively washed into
water, rapidly frozen on a helium-cooled copper block, freeze-dried in a
Cressington CFE-50 apparatus at 90°C and rotary coated with 1.4 nm
of platinum and 4 nm carbon without rotation. The cells were examined in a
JEOL 1200 EX electron microscope using a 100 kV accelerating voltage.
Actin-binding assay
The expression and purification of the recombinant proteins
glutathione-S-transferase/WIP (GST-WIP) 1-127 and GST-WIP
1-12743-54, which lacks the actin-binding domain, were performed as
previously described (Martinez-Quiles et
al., 2001
). The ability of these fusion proteins to bind G-actin
was tested using a GST pull-down assay as previously described
(Martinez-Quiles et al.,
2001
).
Video microscopy
Stably transfected NIH 3T3 fibroblasts were grown on glass coverslips,
serum-starved for 4 days in the presence of 0.5% FBS, washed twice with warm
Iscove's medium and then stimulated with PDGF-bb (50 ng ml-1) at
37°C using a warm stage. Frames were taken at 2-second intervals starting
at 5 minutes using NIH Image 1.62 software at 400x magnification on a
Nikon Eclipse TE200 microscope with a CCD-300-RC charge-coupled device camera.
Images were processed using Microsoft PowerPoint software.
Derivation of murine primary fibroblasts
Lung pieces from wild-type or WIP-/- mice were washed with PBS,
minced and cultured in Iscove's medium supplemented with 10% FBS, penicillin
and streptomycin (50 U ml-1) for several days. After removal of
unattached debris, adherent cells were trypsinized and maintained in culture.
Trypsinized cells were lysed in SDS gel-loading buffer and analysed by western
blot with rabbit anti-WIP serum as previously described
(Anton et al., 1998).
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Results |
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Induction of ruffles by PDGF depends on PI3K-mediated activation of Rac
(Wennstrom et al., 1994a).
However, PDGF also activates other signaling pathways that include
phospholipase C-
and Ras-GTPase-activating protein
(Olivera and Spiegel, 1993
).
WIP overexpression might enhance PI3K-dependent ruffling or, alternatively,
might synergize with a PI3K-independent pathway to induce dorsal ruffles. To
distinguish between these two possibilities, we examined the effect of the
PI3K inhibitor wortmannin on the PDGF response of control and WIP-transfected
cell lines. Pretreatment with wortmannin completely inhibited PDGF-induced
membrane ruffling in both control and WIP-transfected NIH 3T3 cells
(Fig. 1A, right). This suggests
that WIP exerts its effect on PI3K-dependent ruffle formation.
The actin networks in the cortical cytoplasm of PDGF-stimulated NIH 3T3 cells were examined electron microscopically after detergent extraction of the membrane. As observed by light microscopy, large ruffles within the cortex were readily visible as bands of dense actin lattice in WIP-transfected cells (Fig. 1B, left, inset ruffles at low magnification). By contrast, PDGF-stimulation of vector transfected cells shows only small ruffles at the cell edge (Fig. 1B, right, inset low magnification).
To determine the dynamics of ruffle formation, we videotaped serum-starved control and WIP-transfected fibroblasts starting 5 minutes after addition of PDGF and continuing for 5-10 minutes. Fig. 1C and see movie online (http://jcs.biologists.org/supplemental) show that control cells developed few ruffles on the cell surface that, in phase-contrast microscopy, are visualized as black curves (arrows in Fig. 1C upper frames; see movie online). WIP-transfected fibroblasts showed more ruffles that were more motile than the ruffles of control cells (arrows in Fig. 1C lower frames; see movie online). WIP-transfected cells form 1.8 times more dorsal ruffles than pcDNA-transfected cells (11±2 versus 6±1 ruffles per field). Video microscopy confirmed that the time course of ruffle formation was similar in both cells lines (data not shown). These data suggest that overexpression of WIP enhances ruffle formation in response to PDGF stimulation.
WIP partially redistributes to membrane ruffles after PDGF
stimulation
We next investigated whether WIP localizes to ruffles in PDGF-stimulated
fibroblasts. Immunofluorescence analysis using rabbit anti-WIP serum revealed
that endogenous WIP in quiescent control fibroblasts is primarily located in
the cytoplasm with a punctate distribution that has a higher density in the
perinuclear area (Fig. 2A, top
left). PDGF stimulation induced redistribution of a small amount of WIP
towards the membrane ruffle area (Fig.
2A, white arrows) while the strong staining in the perinuclear
area persisted. Redistribution of WIP to membrane ruffles was more prominent
in WIP-transfected cells (Fig.
2B, white arrows).
|
To determine the localization of WIP in ruffles, we performed confocal microcopy. WIP-transfected fibroblasts stimulated with PDGF were double stained for WIP and actin, and a total of 11 stacks from the substrate to the highest level with detectable fluorescence signal were recorded. Weak but specific anti-WIP staining was observed in the middle sections, with maximal intensity in stack 6 (Fig. 3, Section 6), but not in the lowermost or uppermost sections (Fig. 3, Sections 1 and 11). This signal was specific because no signal was detected using preimmune rabbit serum (data not shown).
|
The above results raised the possibility that WIP associates with the actin cytoskeleton in ruffles. We were, however, unable to detect an increase of WIP in the Triton-insoluble cytoskeletal fraction after PDGF stimulation using western blot analysis (data not shown). This might have been because only a small proportion of WIP localized to ruffles as assessed by immunofluorescence.
Microinjection of anti-WIP antibody decreases ruffle formation
induced by PDGF
To determine the role of WIP in PDGF induction of ruffles, we examined the
effect of microinjection of anti-WIP antibody on the cellular response to
PDGF. The antibody we used has previously been shown to inhibit filopodium
induction by bradykinin (Martinez-Quiles
et al., 2001). NIH 3T3pcDNA fibroblasts were injected with rabbit
anti-WIP antibody. The proportion of anti-WIP-injected cells with ruffles was
54%, compared with 78% for uninjected cells and 72% for cells injected with
control IgG (Fig. 4). Similar
inhibition levels were obtained after injection of anti-WIP IgG in
WIP-transfected fibroblasts (data not shown). These results suggest that WIP
plays an important role in ruffle formation induced by PDGF.
|
PDGF-induced ruffle formation is impaired in WIP-deficient
fibroblasts
To establish a definitive role for WIP in ruffle formation by PDGF, we
examined the effect of PDGF on lung fibroblasts derived from WIP-deficient
mice. First, we confirmed, by western blot, WIP expression in the fibroblast
cell lines derived from wild-type (+/+) mice and lack of WIP expression in
fibroblasts derived from knockout (/) mice
(Fig. 5A). Later, we treated
serum-starved primary murine lung fibroblasts derived from wild-type
(WIP+/+) or WIP-/- mice
(Anton et al., 2002) with PDGF
and stained for F-actin with TRITC-phalloidin. Actin distribution in
serum-starved fibroblasts from WIP-/- mice was similar to control
fibroblasts from WIP+/+ mice
(Fig. 5B, left). Most of the
actin accumulated at the periphery of the cell (cortical actin), as previously
observed for starved 3T3 fibroblasts. PDGF treatment of WIP+/+
cells caused the formation of many circular or dorsal ruffles heavily enriched
in actin, peaking at 8 minutes and mostly disappearing by 15 minutes
(Fig. 5B, top middle and top
right, respectively). By contrast, ruffle formation was virtually absent in
WIP-/- fibroblasts after 8 minutes of PDGF treatment
(Fig. 5B, bottom middle). A
very few WIP-/- cells exhibited fewer ruffles 15 minutes after PDGF
treatment, at a time point when the ruffles had disappeared from most
WIP+/+ fibroblasts (Fig.
5B, bottom right). These results are highly indicative that WIP
plays an important role in ruffle formation induced by PDGF.
|
WIP binding to actin is essential for ruffle formation by PDGF
WIP has three functional domains: an N-terminal verprolin-homology domain
(VH; residues 1-116) that binds actin; the central proline-rich region that
interacts with Src-homology domain 3 (SH3); and a C-terminal domain that binds
WASP and N-WASP. To test the hypothesis that WIP binding to actin is important
for its effect on PDGF-induced ruffle formation, we examined the consequences
of overexpressing a WIP mutant that lacks the ability to bind actin on PDGF
ruffling.
We constructed a WIP deletion mutant that lacks a 12 amino acid sequence,
including the putative actin-binding motif45KLKK48 in
the VH domain (WIP43-54). Because expression of recombinant full length
(FL) GST-WIP protein is poor
(Martinez-Quiles et al.,
2001
), we compared the capacity of GST-WIP 1-127
43-54 with
that of GST-WIP 1-127 to bind actin in a pull-down assay. As previously shown,
WIP1-127 bound G-actin (Martinez-Quiles et
al., 2001
). By contrast, there was no detectable binding of
G-actin to GST-WIP 1-127
43-54 (Fig.
6A), indicating that the 12 deleted amino acids are crucial for
actin binding. WIP
43-54 retained the ability to bind Nck and N-WASP in
the yeast two hybrid system (S.P.S. and R.S.G., unpublished).
|
We then examined the PDGF response of Swiss 3T3 fibroblasts transfected
with EL WIP43-54 mutant in pcDNA vector. Transfection with
WIP
43-54 by itself did not alter the morphology or actin distribution
of the cells. Following PDGF treatment, there was a complete absence of
circular ruffles in WIP
43-54-transfected cells at all time points
examined (0-15 minutes) (Fig.
6B). However, PDGF did induce actin clusters in
WIP
43-54-transfected fibroblasts. These results suggest that
pcDNA-WIP
43-54 acts as a dominant negative inhibitor of ruffle
formation by PDGF. More importantly, they also suggest that WIP interaction
with actin is crucial for PDGF ruffle formation.
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Discussion |
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After the long period of serum starvation that was necessary to obtain
maximum quiescence of WIP-transfected cells, PDGF stimulation resulted mainly
in the formation of dorsal and circular membrane ruffles rather than classical
lamellipodia (Figs 1,
2,
6). Circular ruffle formation
has been described in response to growth factors such as PDGF
(Plattner et al., 1999) and
hepatocyte growth factor [HCF (Warn et
al., 1993
)]. HCF-stimulated Madin-Darby canine kidney (MDCK) cells
in confluent islands show very similar structures
(Warn et al., 1993
) to those
described here. Circular ruffle formation in MDCK cells and fibroblasts
correlates with pinocytic activity
(Veithen et al., 1996
;
Warn et al., 1993
), a process
that occurs prior to cell movement. The decreased formation of circular
ruffles in anti-WIP-IgG-injected cells and in WIP-/- fibroblasts
suggests a potential role for WIP in extracellular compound intake and cell
movement, and will be a focus of future analysis. In addition to circular
ruffles, PDGF treatment induced finger-like actin-containing projections and
loss of stress fibers in WIP-transfected cells. This result is consistent with
previous reports that PDGF activates Cdc42 and N-WASP to mediate filopodium
formation and actin stress fiber disassembly
(Jimenez et al., 2000
), and
that WIP plays a role in Cdc42-mediated filopodium formation
(Martinez-Quiles et al.,
2001
). Moreover, a recent report described the involvement of a
novel WIP-related (WIRE) protein in the formation of filopodia and
lamellipodia following PDGF treatment
(Aspenstrom, 2002
).
Activation of PI3K and of the downstream effector Rac is required for
PDGF-stimulated membrane ruffling (Chung et
al., 1994; Wennstrom et al.,
1994a
). The effect of WIP overexpression on PDGF-induced ruffle
formation depended on PI3K because ruffle formation in PDGF-treated
WIP-overexpressing fibroblasts was inhibited by wortmannin, a fungal
metabolite that inhibits the catalytic subunit of PI3K
(Thelen et al., 1994
).
Pretreatment of WIP-transfected cells with wortmannin completely inhibited
PDGF-induced membrane ruffling, suggesting that WIP participates in the
PI3K/Rac-mediated ruffle formation. Moreover, PDGF stimulation of
WIP-transfected cells pretreated with wortmannin induced an increase in stress
fiber formation. This result suggests a secondary role for WIP in stress fiber
formation that is revealed when Rac activation by PI3K is blocked.
Several lines of evidence suggested that WIP plays a role in the
physiological induction of ruffles by PDGF. Microinjection of anti-WIP
antibody inhibited PDGF ruffle formation
(Fig. 4). More importantly,
PDGF-induced ruffle formation was severely impaired in WIP-deficient
fibroblasts derived from WIP-/- mice
(Fig. 5B). Finally, a WIP
deletion mutant that fails to bind actin (WIP43-54) acted as a dominant
negative inhibitor of ruffle formation by PDGF
(Fig. 6). This last result
strongly suggests that the role of WIP in ruffle formation depends, at least
in part, on its ability to bind actin. In this regard, WIP has been shown to
stabilize actin filaments in vitro
(Martinez-Quiles et al.,
2001
). Binding of WIP to F-actin might also explain the
localization of WIP to ruffles in cells treated with PDGF (Figs
2,
3).
WIP might exert an effect on PDGF-induced ruffle formation at levels other
than actin binding. WIP binds to SH3-domain-containing adaptor proteins such
as Nck-1 (Anton et al., 1998),
which also binds to phosphorylated PDGFR
(Chen et al., 2000
). A recent
report excludes a role for Nck-1 in ruffle formation following PDGF
stimulation but shows that Nck-2 is involved
(Chen et al., 2000
). Given the
high amino acid identity between Nck-1 and Nck-2 (68%), their similar domain
structure, their common partners and their co-expression in 3T3 fibroblasts
(Braverman and Quilliam, 1999
;
Chen et al., 1998
;
Chen et al., 2000
), the
possibility that WIP binds to Nck-2 and that Nck-2 is involved in the effect
of WIP on ruffle formation should be tested.
WIP also binds to the SH3 domain of cortactin (N.M.Q. and R.S.G.,
unpublished). Cortactin binds actin via its N-terminal domain, activates
Arp2/3-dependent actin polymerization and localizes to ruffles. A
cortactin-WIP complex might promote ruffle formation by both inducing F-actin
formation and stabilizing actin filaments. WIP binding to N-WASP seems
unlikely to play an important role in ruffle formation, because
N-WASP-deficient fibroblasts still form lamellipodia after PDGF stimulation
(Snapper et al., 2001). Actin
polymerization might also be promoted by WIP binding to the SH3 domain of
myosin, because it has been shown that myosin-I-induced actin polymerization
in yeast is regulated through interactions with both Las17p, a homolog of
mammalian WASP, and verprolin, a homolog of WIP
(Mochida et al., 2002
).
PDGF is thought transiently to promote, through Rac activation, the
assembly of an actin-based signal transduction unit at sites of actin
remodeling that results in the movement of a range of proteins [including
protein kinase A, Abl and WAVE2, the principal WAVE family member expressed in
fibroblasts (Miki et al.,
2000)] to sites of cytoskeletal reorganization
(Westphal et al., 2000
). The
tyrosine kinase c-Abl not only binds to Nck
(Miyoshi-Akiyama et al.,
2001
), a WIP partner, but also contains an SH3 domain that could
bind directly to the proline-rich WIP. WAVE belongs to the WASP family of
proteins that includes WASP and N-WASP, and stimulates actin polymerization
mediated by the Arp2/3 complex (Miki et
al., 2000
). Because WIP binds directly to WASP and N-WASP, it
would be interesting to determine whether it also binds to WAVE. It is
tempting to hypothesize that WIP-mediated enhanced membrane ruffling might
result from synergy between WAVE's ability to activate actin polymerization
and WIP's ability to stabilize nascent actin filaments. Further work is needed
to test this hypothesis.
![]() |
Acknowledgments |
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![]() |
Footnotes |
---|
There is a recent manuscript describing the presence of WIP in lamellipodia
at the cell periphery and showing a role for WIP on membrane protusions
(Kinley et al., 2003).
Present address: Division of Infectious Diseases, Department of Medicine,
UTHSCSA, South Texas Centers for Biology in Medicine,15355 Lambda Drive, Texas
Research Park, San Antonio, TX 78245, USA
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