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J. Biol. Chem., Vol. 277, Issue 40, 37771-37776, October 4, 2002
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,,,,¶, and
From the Department of Cell Biology, Gesellschaft
für Biotechnologische Forschung (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Germany and the § Cell Motility Group,
Cancer Research UK, Lincoln's Inn Fields Laboratories, 44 Lincoln's
Inn Fields, London WC2A 3PX, United Kingdom
Received for publication, April 29, 2002, and in revised form, July 6, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Wiskott-Aldrich syndrome protein
(WASP)/Scar family proteins promote actin polymerization by stimulating
the actin-nucleating activity of the Arp2/3 complex. While Scar/WAVE
proteins are thought to be involved in lamellipodia protrusion, the
hematopoietic WASP has been implicated in various actin-based processes
such as chemotaxis, podosome formation, and phagocytosis. Here
we show that the ubiquitously expressed N-WASP is essential for actin
assembly at the surface of endomembranes induced as a consequence of
increased phosphatidylinositol 4,5-biphosphate (PIP2) levels. This
process resulting in the motility of intracellular vesicles at the tips
of actin comets involved the recruitment of the Src homology 3 (SH3)-SH2 adaptor proteins Nck and Grb2 as well as of WASP interacting
protein (WIP). Reconstitution of vesicle movement in N-WASP-defective
cells by expression of various N-WASP mutant proteins revealed three
independent domains capable of interaction with the vesicle surface, of
which both the WH1 and the polyproline domains contributed
significantly to N-WASP recruitment and/or activation. In contrast,
the direct interaction of N-WASP with the Rho-GTPase Cdc42 was not
required for reconstitution of vesicle motility. Our data reveal a
distinct cellular phenotype for N-WASP loss of function, which adds to accumulating evidence that the proposed link between actin and membrane
dynamics may, at least partially, be reflected by the actin-based
movement of vesicles through the cytoplasm.
The dynamic turnover of actin filaments is required for multiple
processes as diverse as cell migration and cytokinesis. De novo nucleation of actin filaments is most prominently catalyzed by the Arp2/3 complex, whose activity is regulated by various proteins
(1). Potent stimulators of the actin-nucleating activity of the Arp2/3
complex are members of the Wiskott-Aldrich syndrome protein
(WASP)1/Scar family, which have
been implicated in a variety of cellular processes (2). The
hematopoietic WASP and the ubiquitously expressed N-WASP can both bind
directly to the Rho-GTPase Cdc42 through their CRIB
(Cdc42/Rac-interactive-binding) domain (3, 4). Both proteins are
therefore thought to serve key effector functions in the signaling of
this small GTPase to the actin cytoskeleton (5). In vitro
experiments have demonstrated that the auto-inhibitory conformation of
N-WASP can be released upon interaction with both Cdc42 and
phosphatidylinositol 4,5-biphosphate (PIP2) (6, 7). These and
additional interactions e.g. of Src-homology 3 (SH3)-containing proteins like Grb2 (8) with the proline-rich region of
WASP/N-WASP have led to a model (5) for the regulation of these
proteins that awaits further confirmation in vivo. While
cell lines from N-WASP knockout mice have confirmed the requirement of
this protein for the actin-based motility of Shigella
flexneri or Vaccinia virus, the cellular function of N-WASP has
remained unclear (9, 10).
It has been shown previously that N-WASP can localize to the surface of
endo- and lysosomal vesicles in Xenopus oocyte extracts (11), the motility of which is multiplied by activation of protein kinase C with phorbol myristate acetate. Phorbol myristate
acetate treatment in combination with hyperosmolarity has also been
used to stimulate actin-based pinosome rocketing in mast cells (12), a
response that requires annexin 2 (13). In fibroblasts, actin-based vesicle propelling can be visualized for instance upon overexpression of phosphatidylinositol-4-phosphate 5-kinase (PIP5K) (14), which generates PIP2, or of constitutively active ADP-ribosylation factor 6 (15). Interestingly, the endocytic GTPase dynamin has recently been
demonstrated to be recruited to the actin tails induced by PIP5K (16,
17). Because this GTPase can link to N-WASP via proteins such as Grb2
or syndapin, it has been proposed that this type of actin comet
formation may be regulated by N-WASP-mediated activation of the Arp2/3
complex (14).
Here we show that N-WASP or the hematopoietic WASP are essential for
PIP5K-induced vesicle rocketing. In addition we demonstrate that the
molecular pathway leading to recruitment and activation of WASP/N-WASP
involves multiple proteins including SH2/SH3 adaptors and WIP.
Expression Constructs--
The murine WIP cDNA was obtained
by reverse transcription-PCR on RNA derived from brain of adult C57BL/6
mice using primers 5'-CTACCCAAGATGCCTGTCCC-3' and
5'-GAAGAGCAGGCAAAGATCACC-3'. The GFP-tagged expression construct was
prepared by cloning of murine WIP cDNA into pEGFP-C3-vector
(CLONTECH). GFP-tagged full-length N-WASP and a
subset of mutants have been described (9) (see Fig. 4A).
Additional mutants used in this study corresponding to the residues of
murine N-WASP again indicated in the table in Fig. 4A were
amplified by PCR and fused into pEGFP-C1-vector (CLONTECH). Expression was verified by Western
blotting using a polyclonal antiserum raised against enhanced green
fluorescent protein. The cDNAs for human WASP, which were cloned
into pEGFP-C1-vector (CLONTECH) and the murine,
Myc-tagged PIP5KI Cells, Transfections, and
Immunolabelings--
N-WASPdel/del and their respective
parental fibroblast cell lines were grown as described (9) and
transfected using FuGENE6 (Roche Molecular Biochemicals) for 12-24 h
according to the manufacturer's instructions.
For immunolabelings, cells grown on acid-washed glass coverslips were
fixed with formaldehyde (4%) for 20 min in phosphate-buffered saline
and extracted with a mixture of formaldehyde (4%) and Triton X-100
(0.1%) for 1 min. Indirect immunofluorescence was carried out
essentially as described (22). Polyclonal N-WASP antiserum was kindly
provided by Hiroaki Miki (Tokyo, Japan). As secondary reagent we used
Alexa-Fluor 488-conjugated goat anti-mouse antibodies, which were
routinely mixed with Alexa 594-phalloidin (both Molecular Probes) to
stain filamentous actin.
Microscopy--
Live cells were observed with an open, heated
chamber (Warner Instruments) mounted on an inverted microscope
(Axiovert 135TV, Zeiss) equipped with computer-controlled shutters
(Optilas) in the transmitted and epifluorescence light paths to
minimize radiation damage to the cells. Images of fixed and live
samples were acquired with a back-illuminated, cooled
charge-coupled-device camera (Princeton Instruments) driven by IPLab
software and processed on Macintosh G4 computers using IPLab, Scion
Image 1.62c and PhotoShop 5.0.2 software.
Quantification--
Vesicle speed was measured in
N-WASPflox/flox fibroblasts co-transfected with PIP5K and
GFP-actin by tracking the paths of 102 individual vesicles in seven
cells using the Dynamic Imaging Analysis System (DIAS, Solltech Inc.).
Reconstitution efficiency of actin tail formation in
N-WASPdel/del fibroblasts was assessed by co-expression of
different N-WASP or WASP constructs with PIP5K and counting of the
number of fluorescent cells showing actin tails. Efficiency of actin
tail formation in N-WASPflox/flox cells was determined as
in N-WASPdel/del cells except that PIP5K was
co-expressed with GFP-actin to identify transfected cells. More than
100 cells from at least three independent experiments were photographed
and analyzed for each construct. Statistical analyses were carried out
using Microsoft Excel 2001 and Minitab 10.5 software.
Vesicle Rocketing Is Abolished in N-WASP-defective
Cells--
Transfection of N-WASPflox/flox precursor cell
lines expressing functional N-WASP (9) with PIP5K caused the formation
of multiple actin tails in 89 ± 2% of the transfected cells (see
Fig. 4B) characterized by strong accumulation of endogenous
N-WASP (Fig. 1A). However, no
single actin tail was detectable in >300 N-WASP-defective cells
(N-WASPdel/del) (9) upon expression of PIP5K (Fig.
1B). In contrast, co-expression of this enzyme with
full-length N-WASP fused to GFP restored the formation of actin tails
in 88 ± 2% of transfected N-WASPdel/del cells (see
also Fig. 4B), now being tipped by ectopically expressed N-WASP (Fig. 1C).
In addition, co-expression of PIP5K with GFP-actin in
N-WASPflox/flox cells followed by video microscopy showed
that the formation of PIP5K-induced actin tails reflected the rapid
rocketing motility of intracellular membranes, which are thought to
include endocytic and/or Golgi-derived vesicles (14, 17). As reported
before (14, 16), this type of actin-based motility was random in direction and caused the formation of multiple protrusions. Actin comets in N-WASPflox/flox cells had an average speed of
10 ± 2 µm min PIP5K Comets Are Tipped by SH3-SH2 Adaptors and WIP--
To
gain more insight into the molecular pathways leading to recruitment of
N-WASP onto the surface of these actin filament-nucleating endomembranes, we co-expressed PIP5K with various GFP constructs and/or
analyzed PIP5K-induced actin tails by immunolabeling. We found a strong
phosphotyrosine signal at the tips of actin tails and incorporation of
the Arp2/3 complex into these tails (not shown) as demonstrated
previously (14). In addition, the SH3-SH2 adaptor proteins Nck1 (not
shown), Nck2 (Fig. 1D), and Grb2 (Fig. 1F) as
well as WIP (Fig. 1E) were recruited to vesicle surfaces. WIP was also detected along actin tails, albeit at reduced levels relative to the tips. These data suggest that the recruitment of
N-WASP, which is essential for actin-based vesicle rocketing, may be
accomplished by a pathway similar to that occurring at the surface of
Vaccinia virus (21, 23). Interestingly, pinosomes induced by
constitutively active ADP ribosylation factor 6 recruit N-WASP and
Grb2, but not Nck adaptor proteins (15). However, the reason for this
difference is currently unclear.
Nck, WIP, and Grb2 Are Recruited to Vesicles in N-WASP-defective
Cells--
Vaccinia virus usurps host cell receptor tyrosine kinase
signaling pathways, which involve the recruitment and activation of
N-WASP and the Arp2/3 complex via Nck and WIP (18, 19). While both
proteins are also recruited to the surface of S. flexneri, neither Nck nor WIP seem to be required for the actin-based motility of
this pathogen. In contrast to Vaccinia virus, the recruitment of WIP to
the Shigella surface is mediated by N-WASP and the
positioning of Nck might involve both WIP and N-WASP (19). These
observations have demonstrated that recruitment of WIP and Nck can
occur both upstream and downstream of N-WASP, depending on the
biological system investigated (23). Therefore we tested if Nck and WIP are recruited to PIP5K-induced vesicle surfaces in the absence of
functional N-WASP.
Co-expression of GFP-tagged Nck or WIP with PIP5K in
N-WASPdel/del fibroblasts and subsequent video microscopy
revealed that in the absence of functional N-WASP, both Nck (Fig.
2A) and WIP (Fig. 2D) are recruited to PIP2-induced vesicles, which under
these conditions were passively floating in the cytoplasm due to the lack of actin-based motility. To test if the recruitment of Nck to the
surface of these vesicles depends on its direct interaction with WIP
and/or with a phosphotyrosine signal, we made use of specific point
mutations within Nck known to eliminate ligand binding of the
respective SH2 or SH3 domains (24). Nck binds to WIP through its second
SH3 domain (25). Therefore we tested if a GFP-tagged Nck1 mutant
incapable of ligand interaction through its second SH3 domain
(Nck1-W143K) could still be recruited in N-WASP-defective fibroblasts.
Nck1-W143K targeted to these vesicles similar to wild-type Nck1 (Fig.
2B), whereas a Nck1 mutant harboring an inactive SH2 domain
(Nck1-R308K) (24) did not (Fig. 2C). These data indicate
that the recruitment of Nck occurs independently of its interaction
with both N-WASP and WIP but requires interaction with phosphotyrosine
residues of unknown origin via its SH2 domain. The conclusion that WIP
can be recruited to vesicles without interaction with N-WASP (see Fig.
2D) is further corroborated by the observations that a
WIP mutant lacking the WBD, WIP
Nck is not the only SH3-SH2 adaptor protein located at these
endomembranes because Grb2 was also targeted both to the tips of actin
tails induced by PIP5K expression in N-WASPflox/flox
cells (Fig. 1F) and to the surfaces of non-motile vesicles
in the absence of functional N-WASP (Fig. 2F). This suggests
a potential role for Grb2 in N-WASP recruitment and/or activation on
vesicle membranes, which fits the observations that this adaptor
protein can enhance N-WASP-mediated Arp2/3 activation in
vitro (8) and promote the actin-based motility of Vaccinia virus
(21).
N-WASP Is Targeted to Vesicle Membranes by Multiple
Pathways--
Co-expression of PIP5K with a variety of N-WASP mutants
(9) (Fig. 4A) in N-WASP-defective cells and subsequent
analysis by video microscopy enabled us to assess the domains required either for reconstitution of motility and/or for recruitment (Figs. 3 and 4). Full-length N-WASP
reconstituted actin tail formation in N-WASP-defective cells as shown
in Fig. 1C. Video microscopy of N-WASPdel/del
cells expressing GFP-N-WASP confirmed that a significant number of
fluorescent spots visualized in the GFP channel were highly motile
(Fig. 3 and supplemental video 2 at
http://www.jbc.org) corresponding to vesicles,
which had regained actin-based motility by recruiting GFP-N-WASP onto
their surfaces. Co-expression of a GFP-tagged mutant protein lacking
the WA domain with PIP5K did not restore this type of motility.
Instead, this mutant just localized to non-motile vesicles (Fig. 3 and
supplemental video 2 at http://www.jbc.org), which proves the requirement for N-WASP to interact with and activate the Arp2/3 complex to restore vesicle motility (14). Fig.
4A summarizes the results
obtained upon co-expression of PIP5K with WASP, WAVE2, or various
N-WASP mutants in N-WASPdel/del cells. In support of the
observation that WIP can target to vesicles in the absence of
functional N-WASP, we found recruitment of the WH1
domain (Fig. 4A), probably mediated by
interaction with WIP (19), supportive of a recruitment mechanism
reminiscent of the sequence of events occurring at the surface of
Vaccinia virus (23).
However, two regions of N-WASP were recruited to PIP5K-induced vesicles
in addition to the WH1 domain, showing that besides the
potential Nck/WIP/N-WASP cascade, there are alternative mechanisms to
recruit N-WASP in this system. First, a construct harboring the basic
and CRIB domains (B-GBD) and incapable of targeting to Vaccinia (19)
was recruited to vesicles (Fig. 4A). More detailed analyses
revealed that the region required for binding is located within the
N-terminal half of B-GBD because residues 156-226 were still recruited
to vesicle surfaces, but a construct comprising residues 226-274 (Fig.
4A) was not. Whether the targeting to vesicles of residues
156-226 is mediated by direct interaction with PIP2, Cdc42, or both is
currently being investigated. In an earlier study, we have refined one
recruitment domain of N-WASP to the Shigella surface to the
latter residues 226-274 (9). The conclusion that the same
domain can by itself not target to vesicles was corroborated by the
observation that the construct
Secondly, a construct corresponding to the polyproline domain (residues
268-393) showed strong recruitment to vesicle surfaces, although a
similar construct has again been demonstrated to be absent from the
Vaccinia virus (19). Interestingly, recruitment of both WASP and N-WASP
to the T-cell-antigen-presenting cell contact site has also been shown
to be mediated by the polyproline domain (26), but as opposed to
antigen-presenting cells, this domain is not essential for recruitment
of N-WASP to vesicles (see the
Taken together, although three independent regions were able to target
to vesicle surfaces, none of them was essential for N-WASP-based
reconstitution of vesicle motility (see Fig. 4A), although
they contributed to actin tail formation to variable degrees (Fig.
4B and see below). In addition, although the targeting of
N-WASP to the surfaces of both Shigella and Vaccinia virus stimulates a type of actin-based motility highly reminiscent of the
actin "tailing" observed on endosomal vesicles, the mechanism of
recruitment to these vesicles still differs from the one evolved by
either pathogen.
The WH1 and Polyproline Domains Contribute to Actin Tail Formation
Efficiency--
In addition to qualitative analysis of recruitment and
the mere capability of various N-WASP constructs to restore vesicle motility upon co-expression with PIP5K, we have assessed the efficiency of restoring actin tail formation with selected constructs, the summary
of which is presented in Fig. 4B. This analysis was done by
determining the percentage of cells that had developed actin tails upon
co-expression of PIP5K with various N-WASP constructs. Our analysis
revealed that 88% of N-WASP-defective cells co-expressing GFP-tagged
full-length N-WASP and PIP5K developed actin tails, which was virtually
identical to the tail formation efficiency in
N-WASPflox/flox cells (89 ± 2% of
PIP5K-transfectants, Fig. 4B). In contrast, the percentage
of cells with actin tails was reduced by 24% (to 69 ± 4%) and
by 34% (to 59 ± 6%) when GFP-tagged
Significantly, deletion of both the WH1 and the polyproline domains
(
As opposed to
The C-terminal part of the GBD corresponding approximately to residues
220-274 is known to interact with the cofilin homology domain (29), an
interaction that is thought to contribute to the stabilization of the
inactive "closed" state of N-WASP (7). Interestingly, while B-GBD
(residues 156-274) recruited to vesicle surfaces, fusion of the exact
same domain to the WA domain was sufficient to abolish recruitment (see
Fig. 4 and above), presumably because the mutant stayed "locked" in
its inactive conformation. This construct ( WASP, but Not WAVE2, Can Restore Vesicle Motility--
Vesicle
motility could be restored by expression of GFP-tagged WASP (Figs. 3
and 4) to the same extent (84 ± 5%) (Fig. 4B) as
full-length N-WASP, suggesting that in hematopoietic cells, N-WASP, and
WASP may be able to compensate for each other in driving this type of
endosome motility. This proves the lack of WASP expression in
N-WASPdel/del cells, which is in line with the fact that we
were unable to detect WASP expression by Western blotting in both
parental and N-WASP-defective cell lines (supplemental material at
http://www.jbc.org). These observations also rule
out that the filopodia formed in N-WASPdel/del cells (9)
are due to compensatory WASP expression. As opposed to WASP,
overexpression of WAVE2 with PIP5K did not restore actin tail formation
in N-WASPdel/del cells (Fig. 4A) and GFP-tagged
WAVE2 did not localize to PIP2-induced vesicles in these cells or
co-localize with N-WASP at the tips of actin tails in precursor cells
(not shown).
In summary, our results demonstrate a distinct cellular phenotype for
the loss of functional N-WASP. However, at present we do not know
whether this phenotype is responsible for the early embryonic lethality
of N-WASP
It has been postulated that the molecular machinery that underlies
membrane ruffling also drives (macro)pinosome rocketing (12). In
contrast to N-WASP, WAVE2 is absent from the surfaces of PIP2-induced
vesicles but is recruited to the tips of
lamellipodia as previously
described for WAVE1 (34). These observations indicate clear differences
in the pathways leading to actin filament assembly at the plasma
membrane as compared with that at certain endomembranes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
' expression construct were kindly provided
by Laura Machesky (Birmingham, UK) (14). GFP-tagged Nck1 and -2, Grb2,
the WASP binding domain (WBD) of human WIP and WIP
WBD as well as
-actin have been described (18-21). The expression construct for
GFP-WAVE2 was kindly provided by Giorgio Scita (Milan, Italy). The Nck1
point mutations, Nck1-W143K and Nck1-R308K were introduced into
CB6-GFP-Nck1 using the QuikChange* Site-directed Mutagenesis Kit
(Stratagene). The fidelity of all constructs was verified by sequencing.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Actin tail formation induced by PIP5K
requires N-WASP and involves SH3-SH2 adaptors and WIP.
N-WASPflox/flox (A, D-F) and
N-WASPdel/del fibroblasts (B, C) were
transfected with PIP5K or a mixture of PIP5K and GFP-tagged N-WASP
(C), Nck (D), WIP (E), or Grb2
(F), fixed and stained with phalloidin to visualize
filamentous actin (B and red in C-F)
or with phalloidin (red) and N-WASP antiserum
(green in A). Ectopically expressed GFP
constructs are shown in green in C-F.
PIP5K-expression in N-WASPflox/flox cells induces the
formation of actin tails tipped by endogenous N-WASP (A).
Tail formation is absent in N-WASP-defective cells (B) but
restored by co-expression of PIP5K with GFP-tagged N-WASP
(C). Nck, WIP, and Grb2 localize to the tips of
PIP5K-induced actin tails (D-F). In addition to the tips,
WIP is also present in the tails, which explains why the tail in
C appears yellow in the merged image.
Bar in A is valid for A and
C and equals 5 µm; bar in B equals
10 µm; bar in D corresponds to 2 µm and is
valid for D-F.
1 (n = 102). No such
motility was observed in N-WASP-defective cells co-transfected with
PIP5K and GFP-actin (supplemental video 1 at
http://www.jbc.org). As with PIP5K expression,
N-WASPflox/flox fibroblasts treated with 10 nM
phorbol myristate acetate (12, 13) also developed multiple actin tails,
whereas N-WASPdel/del cells did not (not shown), suggesting
a more general requirement for N-WASP in actin-based endosome movement.
WBD (19), is also
recruited to the tips of PIP5K-induced actin tails in precursor
cells (Fig 2E) and to non-motile vesicles in
N-WASP-defective cells (not shown). Taken together, these results
suggest that at the surface of intracellular membranes stimulated to
nucleate actin filaments by PIP5K activity Nck and WIP are placed
upstream in a pathway leading to N-WASP recruitment as seen in the
Vaccinia system but not in Shigella (23). The observation
that Vaccinia virus surfaces failed to label for Nck or WIP in the
absence of N-WASP (10) substantiated the conclusion that these proteins
can only be recruited to the virus as a trimolecular complex (23).
Although Nck and WIP are clearly targeted to PIP5K-induced vesicle
membranes in N-WASPdel/del cells, we do not exclude that in
the presence of N-WASP a direct interaction of this protein with WIP or
Nck stabilizes their recruitment owing to the formation of intact
trimolecular complexes.
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Fig. 2.
Nck, WIP, and Grb2 can target to
vesicles independently of N-WASP. N-WASPdel/del
(A-D, F) or N-WASPflox/flox cells
(E) were co-transfected with PIP5K and GFP-tagged Nck or WIP
expression constructs as indicated. Video microscopy reveals that
GFP-tagged Nck (A), WIP (D), and Grb2
(F) are recruited to vesicle surfaces in
N-WASPdel/del cells. A Nck mutant lacking the capability to
interact with WIP can still target to vesicles (B), whereas
a mutant incapable of interacting with phosphotyrosine residues cannot
(C). A WIP construct lacking the WASP binding domain is also
recruited to the tips of actin tails in N-WASPflox/flox
cells (E, arrowhead). The merged image in
E shows the GFP construct in green and
phalloidin-stained F-actin in red. Arrows in
A-C, D, and F point to the surfaces
of vesicles that have (A, B, D, and
F) or have not (C) accumulated the GFP-tagged
constructs as indicated. Bar equals 2 µm.
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Fig. 3.
Vesicle rocketing is restored by ectopic
N-WASP or WASP and requires the WA domain.
N-WASPdel/del fibroblasts were co-transfected with PIP5K
and GFP-tagged N-WASP, N-WASP lacking the WA domain (GFP- WA), or
WASP as indicated and analyzed by video microscopy. Time is given in
minutes and seconds. The translocation of vesicles in cells expressing
GFP-tagged N-WASP or WASP is indicated by white arrows,
whereas non-motile vesicles carrying GFP-tagged N-WASPWA are marked
by white arrowheads. The path of translocation of the
vesicle decorated with N-WASP is additionally indicated by a
black line. Bar equals 2 µm.
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Fig. 4.
The WH1 and polyproline, but not the CRIB,
and basic domains contribute to tail formation efficiency.
N-WASPdel/del fibroblasts were co-transfected with PIP5K
and various GFP-tagged N-WASP constructs, WASP or WAVE2. Recruitment to
vesicle surface and reconstitution of vesicle motility were analyzed by
video microscopy or upon fixation and counterstaining with phalloidin
(A). B shows a quantification of PIP5K-induced
actin tail formation in N-WASPflox/flox cells co-expressing
GFP-actin as compared with N-WASPdel/del cells
co-expressing the GFP-tagged constructs as indicated. Values in
B are means ± standard deviations from at least three
independent experiments in which >100 transfected cells were examined
for each condition. WH1 and polyproline constructs were compared
with full-length N-WASP using a paired t test confirming a
statistically significant difference (p 
0.0000 and
p 0.0035, respectively).
(WH1-CRIB) (residues 226-501) was
not able to restore vesicle movement, although it can mediate actin
tail formation on Shigella (9).
polyPro mutant in Fig.
4A).
WH1- and
polyproline-N-WASP were used, respectively (Fig. 4B).
These data suggest that both WH1- and polyproline-dependent
interactions of N-WASP, e.g. with WIP and SH3
domain-containing proteins, contribute to recruitment and/or activation
of N-WASP to stimulate actin-based motility at endosomal membranes. The
decrease in actin tail formation efficiency upon expression of the
mutant lacking the polyproline domain may not only be a result of the
lack of interaction with SH3 ligands because the direct interaction of
this domain with profilin I has previously been shown to be important
for Shigella motility (27). The contribution of the
polyproline domain of N-WASP for recruitment of the profilin-actin
complex and the general role of profilin for actin-mediated vesicle
movement is under current investigation.
WH1polyPro) completely abolished recruitment to vesicles and
reconstitution of actin tail formation (Fig. 4). These data demonstrate
that the presence of either one of these two domains is essential but
not necessarily sufficient (see WH1-CRIB) for reconstitution of
vesicle motility (see also quantifications in Fig. 4B).
WH1- or polyproline-N-WASP, a mutant lacking the
basic and the CRIB domains (B-CRIB) required for PIP2 and Cdc42
binding, respectively (6, 7), restored actin tail formation with the
same efficiency as full-length N-WASP (86 ± 2% and 88 ± 2%, respectively), although a construct corresponding to the deleted
domain (B-CRIB) was by itself able to recruit to vesicle surfaces (see
above). Additionally, expression of a point mutant of N-WASP (H208D)
shown previously to be incapable of binding to GTP-Cdc42 (9, 28)
restored the formation of actin tails with an efficiency (79 ± 5%) similar to B-CRIB and full-length (Fig. 4B). Hence,
we conclude that direct interaction of N-WASP with PIP2 via the basic
domain or with Cdc42 does not contribute to its recruitment to and/or
activation at these endomembranes.
WH1polyPro) as well as
an N-terminally truncated version of it (mini-N-WASP), which
corresponds to a mutant that can be "unlocked" from its inactive
state by PIP2 and Cdc42 in vitro (7), did not localize to
vesicles (Fig. 4A), although for the latter construct,
reconstitution of actin tail formation on the surface of
Shigella proved its functionality in vivo (9). Finally, the fact that (WH1-CRIB) did not reconstitute vesicle rocketing led us to speculate that the binding of N-WASP to polyproline ligands cannot by itself release the intramolecular interaction.
/ mice, although it would certainly be
compatible with the broad abnormalities observed in these embryos (9,
10). Several observations point toward a link between membrane and
actin dynamics (30, 31), as exemplified recently by the description of
the association of dynamin with PIP5K-induced actin tails or those induced by Listeria monocytogenes (16, 17). The finding that N-WASP is essential for PIP2-induced vesicle motility adds further significance to the interactions of this protein with intersectins or
syndapins, which have already implicated coupling of actin nucleation
to endocytic processes (32). The involvement of N-WASP in the
insulin-stimulated translocation of GLUT4-vesicles to the plasma
membranes of adipocytes might reflect just one specific example for the
engagement of actin polymerization in vesicle traffic (33).
| |
ACKNOWLEDGEMENTS |
|---|
We thank Giorgio Scita for kindly sharing unpublished reagents, Laura Machesky for providing WASP and PIP5K expression constructs, H. Miki for N-WASP antiserum, and Petra Hagendorff for excellent technical assistance.
| |
FOOTNOTES |
|---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains a supplemental
figure and two supplemental videos.
The atomic coordinates and the structure factors (code AJ437262) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ Supported by the Deutsche Forschungsgemeinschaft (WE 2047/5-2) and the Fonds der Chemischen Industrie.
Supported by an EMBO long-term fellowship. To whom
correspondence should be addressed. Tel.: 49-531-6181-442; Fax:
49-531-6181-444; E-mail: kro@gbf.de.
Published, JBC Papers in Press, July 29, 2002, DOI 10.1074/jbc.M204145200
2 H. Nakagawa and J. V. Small, personal communication.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: WASP, Wiskott-Aldrich syndrome protein; WIP, WASP interacting protein; CRIB, Cdc42/Rac-interactive-binding; PIP2, phosphatidylinositol 4,5-biphosphate; PIP5K, phosphatidylinositol-4-phosphate 5-kinase; GFP, green fluorescent protein; WBD, WASP binding domain.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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