Sequential Interaction of Actin-related Proteins 2 and 3 (Arp2/3) Complex with Neural Wiscott-Aldrich Syndrome Protein (N-WASP) and Cortactin during Branched Actin Filament Network Formation*
Takehito Uruno
,
Jiali Liu
,
Yansong Li
,
Nicole Smith
and
Xi Zhan
¶
From the
Department of Experimental Pathology,
Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross,
Rockville, Maryland 20855 and the
Department of
Cell Biology and Anatomy, The George Washington University, Washington D. C.
20037
Received for publication, February 25, 2003
, and in revised form, May 2, 2003.
 |
ABSTRACT
|
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The WASP and cortactin families constitute two distinct classes of Arp2/3
modulators in mammalian cells. Physical and functional interactions among the
Arp2/3 complex, VCA (a functional domain of N-WASP), and cortactin were
examined under conditions that were with or without actin polymerization. In
the absence of actin, cortactin binds significantly weaker to the Arp2/3
complex than VCA. At concentrations of VCA 20-fold lower than cortactin, the
association of cortactin with the Arp2/3 complex was nearly abolished.
Analysis of the cells infected with Shigella demonstrated that N-WASP
located at the tip of the bacterium, whereas cortactin accumulated in the
comet tail. Interestingly, cortactin promotes Arp2/3 complex-mediated actin
polymerization and actin branching in the presence of VCA at a saturating
concentration, and cortactin acquired 20 nM affinity for the Arp2/3
complex during actin polymerization. The interaction of VCA with the Arp2/3
complex was reduced in the presence of both cortactin and actin. Moreover, VCA
reduced its affinity for Arp2/3 complex at branching sites that were
stabilized by phalloidin. These data imply a novel mechanism for the de
novo assembly of a branched actin network that involves a coordinated
sequential interaction of N-WASP and cortactin with the Arp2/3 complex.
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INTRODUCTION
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Dynamic cortical actin assembly is intimately associated with membrane
protrusions, cell crawling, phagocytosis, and intracellular vesicle
trafficking. While assembly of actin bundles or cables requires formin-like
proteins (1,
2), de novo actin
polymerization in the cell cortex occurs primarily as the result of the
function of actin-related proteins 2 and 3
(Arp2/3),1 which form
a stable protein complex with five other unique proteins
(35).
The Arp2/3 complex serves as a nucleation site for actin elongation, a major
rate-limiting step in actin assembly
(68),
and forms distinct Y-shaped actin branches
(9,
10). Branched actin network is
a characteristic of cortical actin filaments found in cell leading edges such
as lamellipodium, membrane ruffles, and membrane vesicles
(11,
12). The formation of branched
actin filaments is presumably the result of actin assembly at an Arp2/3
complex that binds to an existing actin filament
(9,
13). However, the intrinsic
actin nucleation activity of Arp2/3 complex is normally weak
(9) and requires activation by
binding to other cellular factors
(68).
All Arp2/3 activators have been reported to bind to actin as well. The
Arp2/3 activators can be categorized into two distinct classes based on their
actin binding properties. Members of the first group bind to the monomeric
form of actin (G-actin), and include the WASP family proteins (WASP, Ref.
14; N-WASP, Refs.
15 and
16; and SCAR/WAVE, Refs.
17 and
18 in metazoans; Las17 in
yeast, Ref. 19), and ActA in
Listeria (20). These
proteins are able to activate the Arp2/3 complex by recruiting G-actin to the
proximity of Arp2 and Arp3 subunits, resulting in a heterogeneous nucleation
site for actin assembly (7).
Members of the second group bind to filamentous actin (F-actin), and include
the cortactin family proteins (cortactin, Refs.
21 and
22 and HS1, Ref.
23) in metazoans, and
myosins-I
(2426)
and Abp1 (27) in yeast. The
ability of these proteins to promote actin assembly, however, appears to be
modest compared with the WASP-related proteins, and the significance of such
modest activations remains unclear.
Cortactin contains an N-terminal acidic domain that binds to the Arp2/3
complex and a central repeat domain consisting of 6.5 tandem repeats of unique
37 amino acids that binds to F-actin
(21,
28,
29). Its C-terminal region
contains a Src homology 3 (SH3) domain that associates with a variety of
cellular proteins including CortBP1
(30), CBP90
(31), ZO-1
(32), and dynamin-2
(33). The structure of
cortactin is well conserved in higher eukaryotes. Cortactin-like proteins have
been also found in sea urchin, sponge, fruit fly, frog, chicken
(35), mouse
(36), and human
(37), indicating an
established function of cortactin at a very early stage of evolution. In
contrast, no myosin I- or Abp1-like proteins that contain the acidic motif
required for Arp2/3 binding have been found in mammalian cells
(38). The only
cortactin-related protein is HS1, a hematopoietic cell-specific protein, which
also contains a similar N-terminal region, the repeat, and the C-terminal SH3
domains. The major difference between HS1 and cortactin is that HS1 has only
3.5 tandem repeats (23,
34).
While WASP proteins potentiate strongly the actin nucleation activity of
the Arp2/3 complex, the branched filaments formed by WASP and the Arp2/3
complex are unstable and quickly undergo debranching by a mechanism that is
still not clear (10,
39,
40). On the other hand,
cortactin has a modest activity for actin nucleation, but promotes
significantly the formation of stable actin branches
(22,
23). Despite these
differences, WASP and cortactin bind to the Arp2/3 complex through a similar
structural domain consisting of multiple acidic residues and a single
tryptophan (7,
21). A recent study has
reported that although both N-WASP and cortactin bind to the Arp3 subunit of
the Arp2/3 complex (41),
N-WASP binds to Arp2 and p41 subunits as well
(41,
42). Interestingly, a
cortactin fragment containing only the Arp2/3 binding domain competes poorly
with N-WASP for the Arp2/3 complex even at a concentration 1000-fold higher
than N-WASP (21,
41). Based on this finding, it
has been suggested that a ternary complex of N-WASP, cortactin, and Arp2/3
might exist in the presence of excess amounts of cortactin, and the activities
of WASP and cortactin might be synergistic for actin assembly under this
condition (41). Indeed, both
proteins have been implicated in the same actin-dependent cellular processes
such as membrane ruffling (33,
43), podosome dynamics
(35,
44), vesicle propulsion
(45,
46), and actin comet tail
formation by infectious agents
(16,
4749).
However, there is evidence suggesting that cortactin may interact with the
Arp2/3 complex in a mechanism distinct from N-WASP. For example, both
cortactin and N-WASP are abundant proteins, and their cellular concentrations
are in micromolar ranges (21,
50). It is also known that the
majority of cortactin proteins are intimately associated with the Arp2/3
complex within cells (21).
Thus, the precise role of cortactin and N-WASP and the nature of their
functional relationship in actin assembly remain to be defined.
Here, we report that although the affinity of VCA, a constitutively active
N-WASP peptide, for the Arp2/3 complex is much higher than that of cortactin
in the absence of actin, such affinity is significantly reduced once actin
polymerization is initiated. The release of VCA from the Arp2/3 complex is
further promoted by the presence of cortactin, which apparently has increased
its affinity for the activated Arp2/3 complex. Our data suggest that while
N-WASP interacts primarily with the free form of the Arp2/3 complex, the more
likely target for cortactin is the complex of Arp2/3 and F-actin at a
branching site. Thus, we propose that the rapid formation of actin filaments
requires a sequential event involving an initial activation of the Arp2/3
complex by N-WASP and a subsequent interaction between activated Arp2/3
complex and cortactin at the branching point.
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EXPERIMENTAL PROCEDURES
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ProteinsMurine cortactin tagged with 6x His at its C
terminus was expressed in Escherichia coli and purified as described
previously (21). Human
N-WASP-derived VCA peptide tagged by GST was expressed and purified from
E. coli as described
(16) and further purified by
Mono Q chromatography. For some experiments, GST-VCA was digested with
thrombin at room temperature overnight, and the released tag-free VCA was
further purified by Mono Q chromatography. GST-cort-(180), comprising
the acidic domain and a region before the central repeat domain, and
GST-cort-(1375), containing both the acidic and the repeat domains,
were expressed in E. coli and prepared as described
(21). Bovine Arp2/3 complex
was purified by a three-step procedure involving Q Sepharose ion-exchange,
GST-VCA affinity chromatography and additional Q Sepharose chromatography
(23). The protein in the
flow-through fraction of the final Q Sepharose was concentrated by a Centricon
30 (Amicon), and the buffer of the sample was changed to 1x
Ca2+-free polymerization buffer (50 mM KCl, 2
mM MgCl2, 1 mM EGTA, 0.25 mM ATP,
10 mM imidazole, pH 7.3, 3 mM NaN3, and 0.5
mM dithiothreitol). Protein concentration was determined by the
Bradford method using BSA as the standard.
GST Pull-down AssayTo measure the effect of VCA on the
binding of cortactin to the Arp2/3 complex, 10 nM Arp2/3 complex
was mixed with GST-cort-(180) or GST-cort-(1375), and untagged
VCA at different concentrations in a total 200-µl volume of 1x
polymerization buffer. After a 15-min incubation at 22 °C, the reaction
mixture was supplemented with BSA (1 mg/ml) and transferred to a 1.5-ml vial
containing 20 µl of glutathione-Sepharose beads, and then incubated for 30
min with gentle rotation. The beads were pelleted by centrifugation at 300
x g for 1 min and subsequently subjected to SDS-PAGE followed
by immunoblotting using anti-Arp3 polyclonal antibody
(21). The blot was digitalized
by film scanning and quantified by Scion Image software. The result was
normalized based on five control samples of the Arp2/3 complex with different
amounts on the same gel.
To measure the binding of VCA to the Arp2/3 complex during actin assembly,
polymerization of 1.5 µM actin was initiated in the presence of
10 nM Arp2/3 complex, 100 nM GST-VCA, and cortactin at
concentrations from 0 to 400 nM in 200 µl of 1x
polymerization buffer for 30 min. The reaction mixture was then supplemented
with BSA (1 mg/ml), and mixed with 20 µl of glutathione-Sepharose beads,
and incubated for 30 min with gentle rotation. The beads were sedimented at
300 x g for 1 min, and the supernatant was transferred to a new
1.5-ml tube. 50 µl of the supernatant was mixed with an equal volume of
2x SDS sample buffer and boiled for 5 min. 16 µl of the sample was
subjected to SDS-PAGE (12%) followed by transferring to a nitrocellulose
membrane. Arp2/3 complex was detected by immunoblot analysis using Arp3
antibody.
Actin PolymerizationPolymerization of G-actin (10%
pyrene-labeled, rabbit skeletal muscle actin from Cytoskeleton Inc.) was
performed as described previously
(21) with a modification.
Briefly, Ca2+-ATP-G-actin in G-actin buffer (5 mM
Tris-HCl, pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP,
and 0.5 mM DTT) was mixed with one-tenth volume of 10x
exchange buffer (2 mM EGTA, 1 mM MgCl2) for 3
min at 22 °C to convert to Mg2+-ATP-G-actin. Polymerization was
initiated by adding 60 µl of Mg2+-ATP-G-actin (7.5
µM) to 240 µl of 1.25x polymerization buffer (62.5
mM KCl, 2.5 mM MgCl2, 12.5 mM
imidazole, pH 7.3, 1.25 mM EGTA, 0.125 mM
CaCl2, 0.625 mM DTT, 0.3125 mM ATP, and 3.75
mM NaN3) containing Arp2/3 complex, GST-VCA, and
cortactin at concentrations as indicated. The kinetics of actin polymerization
was monitored by measuring the increase in pyrene fluorescence detected by an
LS50B spectrophotometer (PerkinElmer Life Sciences) with filters for
excitation at 365 nm and emission at 407 nm.
Fluorescence Microscopy Analysis of Branched Actin
FilamentsThe analysis was carried out essentially as described
previously (10,
13). The specific conditions
of actin polymerization for each experiment were described in the
corresponding legends. Rhodamine-phalloidin (Molecular Probes) was added at
the times indicated to actin polymerization reactions at a molar concentration
equivalent to actin. After 5 min of incubation, the mixture was diluted
400-fold in fresh fluorescence buffer (100 mM KCl, 1 mM
MgCl2, 100 mM DTT, 10 mM imidazole, pH 7.3,
0.5% methylcellulose, 20 µg/ml catalase, 100 µg/ml glucose oxidase, and
3 mg/ml glucose). The diluted samples (2.4 µl) were applied onto cover
slips precoated with 0.1% nitrocellulose in n-amyl acetate, and
examined under an Olympus IX-70 inverted microscope using a x100
objective lens with numerical aperture (NA) of 1.35. Images were captured by a
charge-coupled device camera, and further processed on Adobe Photoshop to
generate monochromatic images. The length of filaments and the number of
branch points were measured by Scion Image software, and the degree of
branching was calculated as the number of branches per micrometer of
filament.
Immunofluorescence Analysis of Shigella-infected MDA-MB-231
CellsBreast cancer MDA-MB-231 epithelial cells expressing GFP
(enhanced green fluorescent protein) alone, GFP-N-WASP, or cortactin-GFP were
prepared by retrovirus-mediated gene transfer according to the protocol
described previously (51).
Infection with Shigella flexneri M90T strain (a kind gift of C.
Egile) was carried out according to the method described
(52). Briefly, overnight
culture of the bacteria was diluted in trypticase soy broth (BD Biosciences)
at 1:100 and grown for 2 h at 37 °C to an OD600 =
0.20.3. Bacteria (100 bacteria/1 cell) were centrifuged at 3200 rpm for
4 min, rinsed twice with the cell growth medium (Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum), and resuspended with the
growth medium (2 ml/well). Cells, seeded on fibronectin-coated cover slips in
a 6-well plate a day before infection, were washed once with the growth
medium, overlaid with the bacterial suspension, and centrifuged at 2000 rpm
for 10 min at 20 °C. Cells were incubated for 1 h at 37 °C to allow
bacterial entry, then washed three times with the growth medium, and incubated
in the medium containing 50 µg/ml of gentamicin for an additional 12
h at 37 °C before inspection.
Cells were fixed for 20 min in PBS containing 3.7% paraformaldehyde,
incubated for 10 min in PBS-50 mM NH4Cl twice to quench
the fixative, and permeabilized in PBS containing 0.5% Triton X-100 for 10
min. The cells were blocked for 1 h with PBS containing 2% BSA, incubated with
anti-GFP polyclonal antibody (Molecular Probes, 1:100), and anti-cortactin
antibody (4F11; Upstate Biotechnology Inc., 1:100) for 1 h in PBS containing
0.2% BSA. After three washes with PBS, the cells were further incubated with
FITC-labeled anti-rabbit IgG antibody (1:50) and rhodamine-labeled anti-mouse
IgG antibody (1:50) in PBS containing 0.2% BSA for 1 h. The cells were then
incubated with diamidine-2-phenyl indole (DAPI) (10 µg/ml) for 5 min and
washed three times with PBS. The stained cover slips were mounted on a glass
slide with 20 µl of Prolong Antifade preservative (Molecular Probes) and
sealed with nail polish. The cells were examined under an Olympus IX-70
inverted microscope using a x60, NA = 1.25 objective lens.
 |
RESULTS
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N-WASP-VCA Inhibits the Association of Cortactin with Arp2/3
Complex in the Absence of ActinIn an effort to understand the
relationship of N-WASP and cortactin during actin assembly, we examined
whether the two types of Arp2/3 activators could bind simultaneously to the
Arp2/3 complex. As a result, the interaction of cortactin and Arp2/3 complex
was examined in the presence of a VCA peptide derived from human N-WASP
(16) by pull-down analysis
with murine cortactin recombinant proteins GST-cort-(180) and
GST-cort-(1375), both of which have a similar affinity for the Arp2/3
complex with a Kd of about 1 µM
(21). As shown in
Fig. 1, GST-cort-(1375)
was able to pull-down 41% of 10 nM Arp2/3 complex at 1.5
µM, and 52% at 5 µM, in the absence of VCA.
However, this ability to pull-down the Arp2/3 complex was dramatically
inhibited by VCA in a dose-dependent manner and nearly abolished by VCA at
concentrations significantly lower than GST-cort-(1375). (200
nM VCA abolished the association of Arp2/3 complex with 1.5
µM cortactin protein, and 600 nM VCA abolished that
with 5 µM cortactin.) Binding of GST-cort-(180) to the
Arp2/3 complex was also inhibited by VCA in a similar manner
(Fig. 1). With either of the
cortactin proteins, VCA inhibited 50% of cortactin binding to the Arp2/3
complex at
50 nM. This result is consistent with the
previously reported relative affinities of VCA and cortactin for the Arp2/3
complex: VCA has a Kd value from 0.1 to 0.2
µM (16,
21,
23), and cortactin has a
Kd from 0.7 to 1.3 µM
(21,
23,
29,
41). Our finding also agrees
with a recent report showing that a similar cortactin peptide encoding the
N-terminal region (180) competed poorly with VCA in GST pull-down
assays (41).

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FIG. 1. VCA inhibits the association of the Arp2/3 complex with cortactin in the
absence of actin. GST-cort-(1375) at a final concentration of 1.5
µM (filled circles) or 5 µM (open
circles), or 10 µM GST-cort-(180) (filled
squares) was incubated with 10 nM Arp2/3 complex in the
absence or presence of VCA at the indicated concentrations in 1x
polymerization buffer (50 mM KCl, 2 mM
MgCl2,10 mM imidazole, pH 7.3, 1 mM EGTA, 0.1
mM CaCl2, 0.5 mM DTT, 0.25 mM ATP,
and 3 mM NaN3). After incubation for 15 min at 22
°C, the reaction mixture was supplemented with 1 mg/ml BSA, and
GST-cortactin proteins and associated fractions were recovered by incubating
with glutathione-Sepharose beads for 30 min followed by brief centrifugation.
The presence of Arp2/3 complex in the beads fraction was detected by
immunoblotting using anti-Arp3 antibody. The bands corresponding to Arp3 were
digitalized by film scanning, and the density of each band was quantified by
Scion Image software and normalized based on Arp2/3 complex samples of five
different concentrations loaded on the same gel. The percentage of the
normalized values for each sample in relation to the total amount of Arp2/3 in
the reaction was plotted as a function of VCA concentration.
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Cortactin and N-WASP Localize in Distinct Areas in the Comet Tail
Induced by S. flexneriThe above data suggest that binding of
cortactin and VCA to Arp2/3 complex is mutually exclusive, making it less
likely that they form a ternary complex with the Arp2/3 complex as previously
suggested (41). To further
confirm this notion, we examined the distribution of cortactin and N-WASP in
the actin comet tail formed by the infectious agent S. flexneri in
the cytoplasm of epithelial cells expressing human N-WASP fused with GFP
(GFP-N-WASP). It has been shown that Shigella protein IcsA at the
bacterial surface is able to interact with and activate N-WASP, which in turn
activates Arp2/3 complex-mediated actin assembly to induce comet tails
(16). The bacteria were
stained with DAPI, a DNA binding dye (blue); and GFP-N-WASP and cortactin were
stained with anti-GFP (green) and cortactin (red) antibodies, respectively.
Although both GFP-N-WASP and cortactin were found in the comet tails, two
proteins displayed distinct localization profiles in relation to bacteria
(Fig. 2). N-WASP was
predominantly localized at either one or both polar ends of the bacterial
surface (Fig. 2, arrowheads). This observation is consistent with previous reports
(16,
48). In contrast, cortactin
was primarily associated with the entire tail
(Fig. 2, arrows),
where Arp2/3 complex is known to be abundant
(16). Colocalization of
GFP-N-WASP and cortactin was found, if any, only in a narrow region where the
bacterial head and comet tail meet. Thus, it appears that N-WASP is closely
associated with the initiation site for actin assembly while cortactin is
stably associated with Arp2/3 complex and the more established actin network.
Similar distribution pattern of the two proteins was also reported for the
actin comet tail induced by vaccinia virus
(53).

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FIG. 2. Distinct localization of N-WASP and cortactin in the comet tails induced
by S. flexneri. MDA-MB-231 epithelial cells expressing GFP-N-WASP
were infected with S. flexneri M90T strain, and the localization of
GFP-N-WASP (green) and cortactin (red) in the comet tail
induced by the bacteria was analyzed by immunofluorescent staining using
anti-GFP and anti-cortactin antibodies, respectively. Host and bacterial
nuclei (blue) were stained with DAPI. Panels A to C
show examples of GFP-N-WASP (arrowheads) associated at both ends of a
bacterium with either a double or a single cortactin tail (arrows).
Panels D and E show examples of GFP-N-WASP (green,
arrowheads) found at the posterior tip of a bacterium with a single
cortactin tail (red, arrow).
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Cortactin Is Able to Promote Actin Polymerization in the Presence of
VCA at Saturating ConcentrationsTo further explore the specific
role of VCA and cortactin in actin polymerization mediated by the Arp2/3
complex, we examined Arp2/3 complex-mediated actin assembly in the presence of
both cortactin and GST-VCA. At low concentrations of the Arp2/3 complex
(
10 nM), which showed very little intrinsic nucleation
activity, cortactin had no significant effect on the activation of the complex
even at 500 nM until GST-VCA was added (data not shown), suggesting
a synergistic function between cortactin and VCA
(21,
22). To understand better the
nature of this synergistic function, we analyzed the ability of cortactin to
promote actin polymerization in the presence of 1 µM GST-VCA.
Since GST-VCA has a 100 nM affinity for the Arp2/3 complex, and 1
µM VCA was able to abolish binding of 10 µM
cortactin to the Arp2/3 complex (Fig.
1), it was assumed that 1 µM GST-VCA would have
saturated the binding sites on 8 nM Arp2/3 complex available for
cortactin. Surprisingly, under this condition cortactin was still able to
provoke a strong actin assembly even at concentrations as low as 5
nM (Fig.
3A). A dose dependence analysis with various
concentrations of GST-VCA further demonstrated that cortactin induces
consistently a half-maximal stimulation at
20 nM
(Fig. 3B and Refs.
21 and
23), indicating that this
value reflects a biochemical property of cortactin independent of GST-VCA. The
similar result was also obtained with untagged VCA (data not shown).
Therefore, it appears that cortactin promotes actin polymerization in a unique
mechanism that apparently requires neither the formation of a complex with
N-WASP nor a competition with N-WASP for Arp2/3 binding. Previous studies have
demonstrated that cortactin stimulates actin polymerization in a Arp2/3
binding-dependent manner (21,
22), indicting that 20
nM concentration for a half-maximum stimulation likely reflects the
affinity of cortactin for the Arp2/3 complex during actin assembly.

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FIG. 3. Cortactin stimulates actin nucleation by the Arp2/3 complex in the
presence of GST-VCA at a saturating concentration. A, kinetics of
actin polymerization in the presence of 1 µM GST-VCA.
Polymerization of 1.5 µM pyrene-labeled actin was recorded over
time in the presence of 1 µM GST-VCA with or without 8
nM Arp2/3 complex and cortactin at 0, 5, or 100 nM,
respectively. B, dose dependence of the stimulation of VCA-Arp2/3
complex-mediated actin polymerization by cortactin. Actin polymerization was
performed under the same condition as described for A, except a
broader range (from 1 to 500 nM) of cortactin concentrations was
used. The time required to reach half-maximal polymerization (t
) was calculated from each polymerization curve and is
presented as a function of cortactin concentration.
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Cortactin Induces the Release of VCA from the Arp2/3 Complex
through the Formation of Branched Actin FilamentsThe above
observations led us to hypothesize that the relative affinities of VCA and
cortactin for the Arp2/3 complex might have been changed during the course of
actin polymerization. To test this possibility, we first examined the
interaction of VCA and the Arp2/3 complex by pull-down analysis under the
conditions where actin polymerization was involved. Actin polymerization was
initiated in the presence of GST-VCA and the Arp2/3 complex with or without
cortactin. After 30 min when actin polymerization was completed, the reaction
mixture was incubated with glutathione beads to pull-down GST-VCA and the
associated Arp2/3 complex. The Arp2/3 complex remaining in the supernatant was
analyzed by immunoblotting of the Arp3 subunit
(Fig. 4A). In the
absence of cortactin GST-VCA was able to pull-down more than 90% of the Arp2/3
complex with or without adding actin (Fig.
4A, lane 3;
Fig. 4B, at 0
nM cortactin). In the presence of cortactin and actin, the ability
of GST-VCA to pull-down Arp2/3 was altered. Under this condition, the
interaction between VCA and the Arp2/3 complex was remarkably reduced in a
cortactin dose-dependent manner, and the reduction reached a half-maximum at
25 nM cortactin, the value that is consistent with its affinity for
the Arp2/3 complex as estimated from the actin polymerization assay. In a
control experiment without actin, cortactin showed no detectable effect on the
ability of VCA to pull-down the Arp2/3 complex, consistent with the view that
cortactin binds weakly to the actin-free form of the Arp2/3 complex.

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FIG. 4. Cortactin induces dissociation of Arp2/3 complex from VCA under the
condition for actin polymerization. A, analysis of VCA-associated
Arp2/3 complex during actin polymerization. GST-VCA (100 nM) and
Arp2/3 complex (10 nM) was preincubated with or without cortactin
at the indicated concentrations for 3 min. G-actin (1.5 µM) was
added to the mixture to initiate actin polymerization in a final volume of 200
µlof1x polymerization buffer at 22 °C. In the control experiment
(-actin), the G-actin buffer alone was added. After 30 min of polymerization,
the reaction mixture was supplemented with BSA (1 mg/ml) and mixed with 20
µl of glutathione beads. The beads were pelleted by centrifugation. The
supernatant fractions were subjected to SDS-PAGE followed by immunoblotting
using anti-Arp3 antibody. The bands corresponding to Arp3 were digitalized by
film scanning. Lane 1, half the amount of the sample on lane
2; lane 2, 0 nM GST-VCA and cortactin; lane
3, 100 nM GST-VCA, 0 nM cortactin; lanes
49, 100 nM GST-VCA plus 10, 25, 50, 100, 200, and 400
nM cortactin, respectively. B, the quantification of the
release of Arp2/3 complex from VCA. The digitalized image of the blot as shown
in A was quantified using Scion Image. The Arp2/3 complex associated
with GST-VCA was estimated by subtracting that in the supernatant from the
total amount in the reaction and plotted as a function of cortactin
concentration.
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VCA Is Unable to Inhibit the Branching Activity of
CortactinTo further confirm the changes in the relative affinities
of cortactin and VCA for the Arp2/3 complex during the course of actin
polymerization, we examined the effect of VCA on cortactin-induced actin
branch formation. Previous studies have demonstrated that VCA itself is unable
to stabilize actin branches
(10,
54). Therefore, one would
expect that branched actin could become less stable should cortactin be
replaced by VCA from the Arp2/3 complex. Thus, actin polymerization was
assembled first in the presence of cortactin and the Arp2/3 complex and
GST-VCA to a steady state, and additional GST-VCA was added afterward to the
reaction to compete for Arp2/3 binding. The actin branching was then compared
with that formed without adding further GST-VCA. Consistent with previous
reports (22,
23), actin filaments assembled
in the presence of 50 nM cortactin, 100 nM GST-VCA, and
10 nM Arp2/3 complex after 60 min displayed extensive actin
branching (Fig. 5A,
panel a) to a similar extent as that that occurred at 3 min
(Fig. 5A, panels
e and f). In contrast, the reaction carried out in the absence
of cortactin yielded longer but unbranched actin filaments after 60 min
(Fig. 5A, panel
b). However, further addition of 1 µM GST-VCA to the
reaction at 15 min after actin polymerization initiation, when actin
polymerization had been completed (data not shown), did not reduce
significantly cortactin-mediated actin branching even after 45 min of
incubation (Fig.
5A, panel c compared with panel a). A
quantitative assay with different amounts of GST-VCA added after actin
polymerization at 15 min was also performed
(Fig. 5B). No
significant effect on cortactin-mediated actin branching was observed in the
range of GST-VCA concentrations from 100 nM to 1 µM.
Thus, the data demonstrate that VCA is incapable of replacing cortactin
associated with Arp2/3 complex on the branched actin filaments that have been
already established.

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FIG. 5. VCA is unable to inhibit actin branching activity of cortactin.
A, actin (1.5 µM) was polymerized in the presence of
100 nM GST-VCA and 10 nM Arp2/3 complex with (a
and c) or without (b and d) 50 nM
cortactin in a total 300-µl volume of reaction. After 15 min of
polymerization, 45 µl of the reaction was transferred to a new tube
containing either 5 µl of 1x polymerization buffer (a and
b) or 5 µl of 10 µM GST-VCA (c and
d, the final concentration was 1.1 µM), and incubated
for additional 45 min. Actin branching was then examined by fluorescence
microscopy. Panels e and f are the samples for actin
branching at 3 min after polymerization initiation in the presence
(e) or absence (f) of 50 nM cortactin.
Bar in panel c indicates a 5-µm length. B, actin
polymerization was performed as above and GST-VCA at a series of
concentrations was added after 15 min of polymerization. The degree of actin
branching was calculated by counting the number of branching points from actin
filaments of 200 µm or longer in length and expressed as branch per µm
length filament. The value of actin branching was then plotted as a function
of GST-VCA concentration.
|
|
VCA Reduces Its Affinity for the Arp2/3 Complex at Branched
Actin FilamentsCortactin inhibited maximally only 50% of the
binding of the Arp2/3 complex to GST-VCA
(Fig. 4). The remaining bound
Arp2/3 complex could be in the F-actin-free form to which cortactin binds
weakly compared with GST-VCA. These Arp2/3 complexes in the free form might be
either released from F-actin as a result of a debranching process
(54) or those that did not
participate in actin nucleation. To verify that VCA may only bind to the free
form of Arp2/3 complex and may acquire a relatively lower affinity than
cortactin for Arp2/3 complex once it is associated with F-actin, we examined
the interaction of GST-VCA with the Arp2/3 complex at branched actins in the
absence of cortactin. Previous studies have shown that branched actin
filaments initiated by the Arp2/3 complex can be also stabilized by fixation
with phalloidin (10,
54). Thus, we analyzed the
interaction of VCA with the Arp2/3 complex in the presence of phalloidin that
was added at either 3 or 60 min after nucleation of actin assembly. Actin
filaments formed under each condition displayed different degrees of actin
branching. At 3 min, many actin filaments remained branched, indicating that a
significant portion of the Arp2/3 complex was still at actin branches
(Fig. 5A, panel
f). On the other hand, the most branched actin filaments had undergone
debranching at 60 min (Fig.
5A, panel b), and the majority of the Arp2/3
complex was assumed to be in the free form. When GST-VCA was precipitated from
the actin assembly reaction fixed at 3 min with glutathione beads, only 60% of
the Arp2/3 complex was able to be pulled down
(Fig. 6, A and
B). In contrast, nearly 90% of the Arp2/3 complex was
pulled-down by GST-VCA at 60 min. The efficiency of pull-down at 60 min was
the same as that observed without phalloidin and cortactin
(Fig. 4) where most branched
actin filaments have been debranched. Thus, the ability of GST-VCA to interact
with the Arp2/3 complex is apparently inversely correlated with the degree of
actin branching. In addition, the pull-down of the Arp2/3 complex by GST-VCA
reached a maximum level at concentrations as low as 250 nM
(Fig. 6B) at either 3
or 60 min, suggesting that the remaining Arp2/3 complex, which was likely
associated with branched F-actin, was unable to be accessed by GST-VCA even at
high concentrations. Indeed, when the similar assay was performed in the
presence of cortactin that was added after phalloidin fixation, cortactin was
no longer able to inhibit the interaction between GST-VCA and the Arp2/3
complex (Fig. 6C),
indicating that the Arp2/3 complex associated with GST-VCA was in the free
form for which cortactin had a much lower affinity than VCA
(Fig. 1). Taken together, these
results suggest that VCA had reduced its affinity for the Arp2/3 complex on
the branched F-actin.

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FIG. 6. GST-VCA is unable to associate with Arp2/3 complex in branched actin
filaments. A, effect of actin branching on the association of VCA
with Arp2/3 complex. GST-VCA (100 nM) and Arp2/3 complex (10
nM) were preincubated for 3 min at 22 °C. G-actin (1.5
µM) was added to the mixture to initiate polymerization in a
final volume of 200 µl of 1x polymerization buffer. After 3 or 60
min, 1.5 µM phalloidin was added to the reaction, incubated for
3 min, and additional GST-VCA was added at final concentrations of 200, 400,
700, 1100, and 2100 nM, respectively. The amount of Arp2/3 complex
that was not associated with GST-VCA was determined by immunoblotting of Arp3
present in the supernatant after GST-VCA precipitation. B,
quantification of the effect of actin branching on the association of Arp2/3
complex with VCA. Arp2/3 complex association as analyzed in A was
calculated based on the difference between the density of Arp3 band in the
supernatant and that in the control sample without GST-VCA. The percentage of
the association was estimated by comparison to that in the control sample and
further plotted as a function of the concentration of GST-VCA. C,
effect of actin branching on the cortactin-mediated inhibition of VCA binding
to the Arp2/3 complex. Actin polymerization was performed as described above.
After 3 or 60 min of initiation, the actin filaments were fixed by adding 1.5
µM phalloidin and incubation for 3 min. Cortactin was then added
to the reaction mixtures at the indicated concentrations. After an additional
15 min of incubation, the reaction mixtures were subjected to GST-VCA
precipitation, and the supernatants were subjected to SDS-PAGE followed by
immunoblotting using anti-Arp3 antibody. The amount of the Arp2/3 complex
associated with GST-VCA was estimated as described above.
|
|
 |
DISCUSSION
|
---|
In this study, we provide evidence for dramatic changes in the relative
affinities of N-WASP and cortactin for the Arp2/3 complex during the course of
actin assembly. Although N-WASP shows predominance for the free form of the
Arp2/3 complex, it appears that cortactin has a much higher affinity for the
Arp2/3 complex once it is incorporated into actin filaments. First, cortactin
promotes significantly Arp2/3-mediated actin nucleation even in the presence
of VCA at a concentration 200-fold higher than cortactin that would have
occupied all the binding sites of the Arp2/3 complex for cortactin. Based on
the analysis of actin nucleation and actin branching
(21,
23) (also in this study), we
estimated the affinity of cortactin for the Arp2/3 complex at
20
nM, which is significantly lower than 0.71.3
µM for the free form of Arp2/3 as reported previously
(21,
23,
29,
41). Second, the interaction
between VCA and the Arp2/3 complex becomes much weaker in the presence of
cortactin and actin. In fact, by analysis of binding to the Arp2/3 complex in
the presence of phalloidin or cortactin, VCA appears to bind to only those
Arp2/3 complexes that are in the free form released from debranching. Finally,
VCA is not able to displace cortactin from the Arp2/3 complex at branching
sites of actin filaments.
Reduced affinity of WASP for the Arp2/3 complex after actin nucleation is
initiated has been postulated because of a presumed requirement for a rapid
motility driven by actin polymerization
(16,
55). It has been reported that
the Arp2/3 complex requires hydrolysis of ATP for actin nucleation
(55,
56), and VCA prefers ATP-bound
Arp2/3 complex with a 140 nM affinity to the ADP-bound form with a
0.7 µM affinity
(55). Thus, the function of
VCA may be to place an actin monomer in contact with an Arp2/3 complex and
stimulate ATP hydrolysis. Hydrolysis of ATP to an ADP-Pi state
would then cause a conformational change that allows Arp2, Arp3, and an actin
monomer to associate and form a nucleus, and subsequent release of VCA from
the Arp2/3 complex. In this study, we have directly observed the reduced
binding of VCA to the Arp2/3 complex, which was only apparent when actin
polymerization took place in the presence of cortactin. Although the
measurement was carried out when actin polymerization was completed, fixation
of actin polymerization by phalloidin at different times has mimicked the
function of cortactin to stabilize actin branching and demonstrated that the
change in the affinity of VCA for the Arp2/3 complex occurs at an early phase
of actin assembly. Since phalloidin is known to stabilize actin-associated
ADP-Pi (57), an
intermediate form from the transition from ATP to ADP, our finding supports
the view that hydrolysis of ATP hydrolysis contributes to the change in the
affinity of VCA for the Arp2/3 complex. However, given a dramatic increase in
the affinity of cortactin from 1 µM to 20 nM, which
is even stronger than VCA for ATP-bound Arp2/3 complex, cortactin could play
an important role in the dissociation of VCA from the Arp2/3 complex in a
manner independent of ATP hydrolysis.
The enhanced affinity of cortactin for Arp2/3 complex during actin assembly
could be a result of a substantial conformational change in the Arp2/3 complex
(58,
59). However, this structural
change could not be simply due to VCA binding because it only occurs when
actin is present. Indeed, GST-cort-(180) fusion protein, which has the
same affinity for the free form of the Arp2/3 complex as intact cortactin
(21), is not able to induce
actin polymerization or branching, nor inhibit the activity of an intact
cortactin (data not shown). Thus, the Arp2/3 complex itself, whether it is in
the activated form or not, is not sufficient to account for the increase in
its affinity for cortactin during actin polymerization. It is more likely that
the acquired high affinity of cortactin for Arp2/3 complex reflects its
interaction with both Arp2/3 complex and actin filament at the branching site.
In fact, cortactin is known as a potent F-actin binding protein with an
affinity from 0.2 to 0.4 nM
(21,
28). Our recent measurements
using freshly polymerized actin fixed with phalloidin estimated a
Kd value even below 100 nM
(23). The alignment of
activated Arp2/3 complex with a daughter filament may further enhance the
affinity of cortactin. Additional evidence supporting this possibility is a
previous observation that cortactin absolutely requires its binding to F-actin
for its ability to promote actin nucleation and actin branching
(21,
22).
Because of its strong dependence on F-actin binding, we propose a model for
a possible interaction between cortactin and Arp2/3 complex at a nucleation
site as shown in Fig. 7. In
this model, N-WASP activates the Arp2/3 complex by a transient interaction,
which results in changes in the configuration of the Arp2/3 complex and
increase in the association of the Arp2/3 complex with an existing actin
filament (60). Once the
branching point is established, N-WASP may be released from the complex as a
result of either ATP hydrolysis or replacement by cortactin, which has
acquired a much higher affinity for the branching point. Since both
WASP-related proteins and cortactin are abundantly present in most cells
(21), a high affinity of the
WASP family proteins for the Arp2/3 complex, 100200 nM with
N-WASP (16,
21) and 400 nM with
WAVE (61,
62), would secure an instant
activation of the free form of the Arp2/3 complex in a de novo actin
assembly. Similarly, the high affinity of cortactin for the activated Arp2/3
complex would be necessary to facilitate the release of WASP from the
branching site of actin filaments and to increase stabilized nucleation sites
for a rapid growth of actin filaments. Consequently, the sequential reaction
will result in a robust polymerization of branched actin network upon
stimulation. Supporting evidence for this novel mechanism for actin
polymerization is the observation that cortactin, which itself may not
directly activate the free form of the Arp2/3 complex as N-WASP does, can
potently stimulate both actin nucleation and actin branching
(Fig. 3).

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FIG. 7. A model for the formation of de novo branched actin filaments mediated
by Arp2/3 complex, N-WASP, and cortactin. In de novo actin
assembly, N-WASP acquires a high affinity for the free form of Arp2/3 complex
upon stimulation by membrane-associated signaling molecules such as Cdc42 and
phosphatidylinositol 4,5-bisphosphate. The association with N-WASP leads to
activation of the Arp2/3 complex, facilitation of its binding to an existing
actin filament, and initiation of new actin assembly. At this stage, the
activated Arp2/3 complex becomes less accessible to N-WASP but is more prone
to the association with cortactin, which binds to both the Arp2/3 complex and
the nascent actin filament with an affinity 50-fold higher than for the free
form of Arp2/3 complex. The complex of cortactin/Arp2/3/F-actin at the
branching site is stable and serves as a better nucleation site for assembly
of branched actin filaments.
|
|
Our conclusion is significantly different from a recent report
(41) that VCA and cortactin
may bind simultaneously to the Arp2/3 complex via different subunits and form
a ternary complex with Arp2/3 complex. The apparent discrepancy is at least
partially due to different conditions used in their experimental procedure,
which was performed with a cortactin fragment containing only the Arp2/3
binding domain and lacking the activity for either actin nucleation or
branching. Although the report has described a competition between VCA and the
cortactin fragment and the authors indicated the presence of a common binding
site at the Arp3 subunit for both types of protein, no clear conclusion was
drawn because of a further finding that the cortactin N-terminal peptide could
not compete with the VCA binding sites at Arp2 and p41 sites of the free form
of the Arp2/3 complex (41).
While our study using fully functional cortactin proteins does not rule out
the possibility that N-WASP might still associate with p41 and Arp2 subunits
in the presence of cortactin, this possibility is very unlikely under the
condition when actin polymerization takes place, given the fact that their
relative affinities have been dramatically changed. Consistent with our
conclusion, cortactin and N-WASP do not appear to be co-localized in the
Shigella-induced actin comet tail that is composed of newly formed
branched actin network driven by constitutively activated N-WASP and Arp2/3
complex (16,
63). However, cortactin could
be colocalized more closely with WASP family proteins in other cellular
systems. For example, cortactin is a primary substrate of the Src protein
tyrosine kinases (35,
64) and binds to several
membrane-associated proteins including dynamin-2, ZO-1, and CortBP1 via its
SH3 domain (30,
32,
33,
65). Thus, cortactin is likely
to function in proximity to the plasma and other intracellular membranes as
well. Indeed, cortactin is abundantly present in lamellipodium, membrane
ruffles, and endosomes (21,
35,
46,
66). By interacting with
different components on the membrane, cortactin and N-WASP likely play an
important role in the organization of actin network by fine-tuning the size
and branches of actin filaments to power movements of particular types of
cellular organelles.
It is also possible that WASP-like proteins may facilitate the association
of cortactin with the Arp2/3 complex during actin assembly. In fact, it has
been reported that cortactin could bind to N-WASP directly though the
cortactin SH3 domain (44). A
recent study has also described an interaction between cortactin and WIP, a
WASP interacting protein (67).
Thus, WASP could also affect cortactin through indirect interaction. While the
actin branching activity of cortactin as analyzed in vitro does not
appear to require the function of its SH3 domain (data not shown) and the VCA
fragment used in this study does not contain an apparent SH3 binding domain, a
transient interaction with WASP proteins could contribute to recruitment of
cortactin to the sites for cortical actin assembly in cells. Since many
cellular proteins have also been found to bind to the cortactin SH3 domain,
distinguishing specific roles of these proteins in the function of
cortactin/WASP/Arp2/3-mediated cortical actin assembly may eventually provide
a detailed mechanism for regulating and directing actin assembly in cells.
 |
FOOTNOTES
|
---|
* This work was supported by National Institutes of Health Research Grants
RO1 HL52753-09, RO1 CA91984-01, Department of Defense Grant DAMD17-01-1-0125,
and American Heart Association Established Investigator Grant 0040135N. The
costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
¶
To whom correspondence should be addressed. Tel.: 301-738-0568; Fax:
301-738-0879; E-mail:
zhanx{at}usa.redcross.org.
1 The abbreviations used are: Arp2/3, actin-related proteins 2 and 3; BSA,
bovine serum albumin; DAPI, diamidine-2-phyenyl indole; DTT, dithiothreitol;
F-actin, filamentous actin; FITC, fluorescence isothiocyanate; GFP, green
fluorescent protein; GST, glutathione S-transferase; PBS,
phosphate-buffered saline; SH3, Src homology 3; VCA, verprolin-cofilin-acidic
motif; N-WASP, neural Wiskott-Aldrich Syndrome protein. 
 |
ACKNOWLEDGMENTS
|
---|
We thank C. Egile and M. B. Goldberg (Harvard Medical School) for technical
help with Shigella infection. We also thank Jeff Winkles for the
critical reading; and Jian-Jiang Hao, Jianwei Zhu and Kang Zhou for helpful
discussions with this work.
 |
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