The Non-Catalytic Carboxyl-Terminal Domain of
ARFGAP1 Regulates Actin Cytoskeleton Reorganization
by Antagonizing the Activation of Rac1
Ka Yu Siu1, Mei Kuen Yu1,2, Xinggang Wu1, Min Zong1, Michael G. Roth3, Hsiao Chang Chan1,2, Sidney
1School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, 2Epithelial Cell Biology Research Center, The Chinese
University of Hong Kong, Shatin, New Territories, Hong Kong, 3Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas,
United States of America
Background: The regulation of the actin cytoskeleton and membrane trafficking is coordinated in mammalian cells. One of
the regulators of membrane traffic, the small GTP-binding protein ARF1, also activates phosphatidylinositol kinases that in
turn affect actin polymerization. ARFGAP1 is a GTPase activating protein (GAP) for ARF1 that is found on Golgi membranes.
We present evidence that ARFGAP1 not only serves as a GAP for ARF1, but also can affect the actin cytoskeleton.
Principal Findings: As cells attach to a culture dish foci of actin appear prior to the cells flattening and spreading. We have
observed that overexpression of a truncated ARFGAP1 that lacks catalytic activity for ARF, called GAP273, caused these foci
to persist for much longer periods than non-transfected cells. This phenomenon was dependent on the level of GAP273
expression. Furthermore, cell spreading after re-plating or cell migration into a previously scraped area was inhibited in cells
transfected with GAP273. Live cell imaging of such cells revealed that actin-rich membrane blebs formed that seldom made
protrusions of actin spikes or membrane ruffles, suggesting that GAP273 interfered with the regulation of actin dynamics
during cell spreading. The over-expression of constitutively active alleles of ARF6 and Rac1 suppressed the effect of GAP273
on actin. In addition, the activation of Rac1 by serum, but not that of RhoA or ARF6, was inhibited in cells over-expressing
GAP273, suggesting that Rac1 is a likely downstream effector of ARFGAP1. The carboxyl terminal 65 residues of ARFGAP1
were sufficient to produce the effects on actin and cell spreading in transfected cells and co-localized with cortical actin foci.
Conclusions: ARFGAP1 functions as an inhibitor upstream of Rac1 in regulating actin cytoskeleton. In addition to its GAP
catalytic domain and Golgi binding domain, it also has an actin regulation domain in the carboxyl-terminal portion of the
Citation: Siu KY, Yu MK, Wu X, Zong M, Roth MG, et al. (2011) The Non-Catalytic Carboxyl-Terminal Domain of ARFGAP1 Regulates Actin Cytoskeleton
Reorganization by Antagonizing the Activation of Rac1. PLoS ONE 6(4): e18458. doi:10.1371/journal.pone.0018458
Editor: Neil Hotchin, University of Birmingham, United Kingdom
Received June 27, 2010; Accepted March 8, 2011; Published April 4, 2011
Copyright: ? 2011 Siu, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grant GM37547 from the NIH to MGR, and the One-line budget of the School of Biomedical Sciences, CUHK, to SY. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Sidney.firstname.lastname@example.org
The small GTPase ARF serves as a key regulator of a number of
cellular processes including vesicle trafficking, signal transduction
and regulation of the actin cytoskeleton . The six isoforms of
ARF in mammals can be categorized into three classes: Class I
(ARF1, 2 and 3), Class II (ARF4 and 5) and Class III (ARF6).
Among them, ARF1, the most extensively studied Class I member,
regulates the formation of coated vesicles on secretory and
endocytic membranes  and also take part in signal transduction
in a number of signaling pathways [3,4,5,6,7,8,9,10,11,12]. ARF6
functions in the endocytic pathway and regulates actin cytoskel-
eton . Because of their diverse and complex cellular functions,
the activity of ARF proteins is highly regulated. Like other small
GTPases, ARFs cycle between an active, GTP-bound form and an
inactive, GDP-bound form. The intrinsic GTPase activity of ARF
is negligible  and the inactivation of ARF requires interactions
with GTPase activating proteins (ARF GAPs). All ARF GAPs have
an ARF GAP catalytic domain of 120 amino acids enriched in
cysteine. To date, there are at least 24 such sequences identified in
the human genome  and the genes containing them are divided
into 10 subfamilies. With the exception of the ADAP subfamily, at
least one member within all other subfamilies have been
demonstrated experimentally to have ARF GAP activity .
Other than the ARF GAP catalytic domain, these genes are vastly
different in terms of their size, the identifiable features that they
contain and their subcellular localization . The existence of
multiple domains in these molecules not only reflects the complex
functions of ARF but also indicates that these ARF-GAPs have
functions in addition to stimulating GTP hydrolysis on ARF.
ARFGAP1 was the first ARF-GAP protein isolated . This
protein contains 415 amino acids, with a GAP catalytic domain
located in the amino terminal 120 residues. ARFGAP1 was shown
to localize primarily on Golgi membranes where it is thought to
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function in the formation of coated vesicles [15,16]. The non-
catalytic sequences of ARFGAP1 are necessary for associating with
membranes. We have identified a region in the middle of the
protein that is responsible for the membrane targeting of the whole
molecule both in vivo and in vitro . This region contains 132
residues starting from amino acid 203 to 334. Subsequently, a lipid-
package sensor domain that allows the protein to sense membrane
curvature was discovered in this region of the molecule .
Gcs1p is the ARF GAP in S. cerevisiae with the greatest
sequence similarity to ARFGAP1 in its non-catalytic sequences.
Besides its anticipated functions in the Golgi [19,20], Gcs1p is also
required for normal organization of the actin cytoskeleton .
GCS1 interacts genetically with SLA2 and SAC6. Mutants in the
SLA2 gene are defective in actin polarization and endocytosis
[22,23]. SAC6 encodes an actin cross-linking protein, homologous
to human fimbrin. In addition to this genetic evidence for a role
for GCS1 in regulating actin, Gcs1p stimulates actin polymeriza-
tion and inhibits depolymerization in vitro .
The Rho family of small GTPases, consisting of Cdc42, Rac
and Rho, are the key regulators of the actin cytoskeleton .
Each of these small GTPases stimulates the formation of a unique
type of actin morphology, although their activities are often
coordinated. For example, Cdc42 stimulates the production of
actin microspikes or filopodia . Rac1 stimulates the formation
of lamellipodia and membrane ruffles . RhoA stimulates the
formation of stress fibers and focal adhesions . ARF6 has also
been demonstrated to regulate the actin cytoskeleton. Earlier cell
biology studies using GTP-binding mutants of ARF6 has
demonstrated the important function that ARF6 regulates the
reorganization of cortical actin [28,29]. ARF6 appears to exert its
effect on the cortical actin cytoskeleton by acting coordinately with
Rac1 [30,31]. ARF6 activates Rac by interacting with Rac GEFs
or by regulating the availability of lipid rafts required for Rac
attachment at the plasma membrane.
Of note, a subfamily of the ARF GAPs, called ARAP1, 2 and 3,
have been shown to act as GAPs for both the ARF family small
GTPases and the Rho family small GTPases Cdc42 and RhoA
[4,5]. ARAP1 is primarily localized on Golgi membranes, similar
to ARFGAP1, suggesting that it might function in Golgi-derived
vesicle trafficking. The presence of both an ARF GAP and a
RhoGAP domain in ARAP proteins suggests that Class I ARFs
and Rho proteins might interact functionally, as has been
demonstrated for ARF6.
While studying the vesicle trafficking functions of ARFGAP1 we
noticed that in cells overexpressing GAP273, a truncated protein
containing only the carboxyl terminal 273 residues (residues 142–
415) of ARFGAP1, cell spreading and flattening appeared to be
impaired. Because the closest homologue of ARFGAP1 in yeast,
Gcs1p, regulates the actin cytoskeleton as well as membrane
traffic, we investigated the potential effect of ARFGAP1 on actin.
We found that ARFGAP1, besides being localized on Golgi
membranes, is present in the cell periphery associated with actin
foci. We observed that cells over-expressing GFP-GAP273 are
slow to spread when plated on fresh culture dishes. This effect is
mediated by the carboxyl terminal 65 residues of ARFGAP1, and
is related to the function of Rac1, as the over-expression of
GAP273 inhibits the activation of Rac1.
GFP-GAP273 inhibits cell spreading and is enriched in
We have previously generated Chinese Hamster Ovary (CHO)
cell lines over-expressing a fusion protein consisting of the green
fluorescent protein (GFP) at the amino terminus and a 273 amino
acid, non-catalytic domain of ARFGAP1, called GAP273. In
CHO cells stably transfected with GFP-GAP273, the fluorescent
fusion protein was observed primarily on Golgi membranes in cells
that had spread on their glass substrate [14,17,32]. However, we
noticed that cells expressing GFP-GAP273 spread and flattened on
their substrate slower than non-transfected CHO cells and often
had a knobby appearance. These slowly spreading cells contained
concentrated green fluorescence on both perinuclear membranes
at the cis Golgi (Figure 1A, large arrowhead) and in peripheral foci
near the plasma membrane (Figure 1A, small arrowheads) and at
the tips of membrane protrusions (Figure 1C, arrows). The
peripheral structures containing concentrations of GFP-GAP273
appeared to be surrounded by actin (Figure 1B and D). The
presence of these actin-rich membrane ‘‘blebs’’, which we will
refer to as actin foci, decreased as a function of time after plating
the cells and appeared to correlate inversely to cell spreading. As
shown in Figure 2A, the percentage of cells having multiple (at
least 3) actin foci decreased from about 73.3% (Standard
deviation, S.D.=4.5, n=3) at 12 hours after re-plating to 5.7%
(S.D.=3.2%, n=3) at 36 hours in wild type CHO cells
(Figure 2A). In GFP-GAP273 cells, however, 96% (S.D.=1.0%,
n=3) of the cells contained multiple actin foci at 12 hours after re-
plating. 36 hours after re-plating this percentage decreased to
about 68.3% (S.D.=6.3%, n=3) for GFP-GAP273(3.2) cells and
19.3% (S.D.=4.0%, n=3) for GFP-GAP273(3.5) cells (Figure 2A).
In this experiment, at least one hundred cells were analyzed in
each data point. 3.2 and 3.5 cells are two individually isolated
clones from a transfection of GFP-GAP273 into CHO cells. Since
the 3.2 cell line expressed a much higher level of GFP-GAP273
than the 3.5 cell line (Figure 2B), there was a positive correlation
between the level of GFP-GAP273 expression and the frequency
of cells having multiple actin foci. We further observed that the
level of GFP-GAP273 expression inversely correlated with the
ability of the GAP273 cells to spread after re-plating. In non-
transfected CHO cells, the cells were efficiently spread out 12 h
after re-plating. The mean area covered by the cells was
approximately 800 mm2(S.D.=261 mm2, n=50) at 12 h but did
not increase over time (Figure 2C). The relatively large standard
deviation is due to large variation in the size of the CHO cells.
Figure 1. Co-localization of GFP-GAP273 with actin in the cell
periphery. A, C. GFP fluorescence in CHO cells stably expressing GFP-
GAP273 [GAP273(3.2)] cells. B, D. Actin staining of the same cells as in A
and C, respectively. Scale bar = 25 mm.
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Two GFP-GAP273 clones showed delayed cell spreading with
severities dependent on GFP-GAP273 protein expression. Clone
3.2 cells had a mean area covered by the cells of 117 mm2
(S.D.=21 mm2, n=60) at 12 h, and increased to approximately
249 mm2(S.D.=74 mm2, n=60) at 36 h after re-plating. Clone
3.5 cells spread from 245 mm2(12 h, n=60) to 762 mm2(36 h,
n=60). The observation of delayed cell spreading and numerous
actin-rich membrane blebs caused by GFP-GAP273 was also
confirmed when we overexpressed this construct in other cell lines
such as Hela or HEK293 (data not shown).
Cell migration requires extensive reorganization of actin. To
determine if actin reorganization is compromised during migra-
tion in GFP-GAP273 cells, we conducted a cell migration
experiment in which a monolayer of confluent cells was scratched
and the migration of cells into the cleared area recorded as a
function of time. As shown in figure 3, HEK293 cells transiently
transfected with GFP-GAP273 are slow to close the scratched area
(Figure 3A, left panels). Control cells that were transfected with
GFP-GalT or GFP empty vector were able to migrate into and
cover more than half of the scratched area in 24 hours (Figure 3A,
middle and right panels, respectively). To quantify the extent of
cell migration, we measured the gap of the scratched area at
various points and then plotted the average width of the scratched
area as a function of time after scratch. GFP-GAP273 clearly
impeded cell migration in a statically significant manner
(Figure 3B). These results further demonstrate that the reorgani-
zation of actin cytoskeleton during cell spreading and migration, is
impaired by GAP273 overexpression.
To investigate the dynamics of membrane blebs caused by
GFP-GAP273, we performed live cell imaging and recorded the
appearance of such membrane blebs in cells transfected with
GFP-GAP273 (movie S2). As a control, we also recorded images
of cells transfected with GFP-Sec13, which has no known effect
on actin (movie S1). As shown in the movie S1, GFP-Sec13 gives
a largely diffused, cytosolic signal when overexpressed in actively
spreading cells. Active membrane ruffling was present in the cell
edges (boxed areas), suggesting that GFP-Sec13 overexpression
does not inhibit this process. In contrast, in GFP-GAP273
overexpressing cells, the green fluorescent signal was concentrat-
ed in the perinuclear structures that likely represent the cis Golgi
or the intermediate compartment, but diffused cytosolic signal
was also observed. The extent of membrane movements was not
reduced but the structures of membrane protrusions appeared to
be much different from the GFP-Sec13 cells (movie S2).
Spherically-shaped membrane protrusions containing green
fluorescent signal and emerging at the cell edges were observed
throughout the whole recording period (arrows). Active mem-
brane ruffles similar to those found in GFP-sec13 cells were not
obvious. These results confirm our initial observation that
GAP273 must play a role in cell spreading and membrane
ruffling, possibly by affecting the dynamics of actin reorganization
at the cell cortex.
Rac1 and ARF6 can suppress the effect of GAP273 on
The actin cytoskeleton is regulated by members of the Rho
family of small GTPases and by ARF6. Actin foci like the ones that
we observed have been reported previously. They appeared in
cultured cells that over-expressed a dominant negative, inactive
allele of ARF6 (Supplementary Figure S1A) or Rac1 (Figure S1B
and ). Moreover, a catalytically inactive mutant allele of ARF6
exchange factor ARNO, ARNO(E156K) , could also promote
the appearance of actin foci when over-expressed (unpublished
data). Over-expressing constitutively active alleles of ARF6
[ARF6(Q67L)] (Figure 4A, left panels) in GFP-GAP273(3.2) cells
suppressed the formation of actin foci caused by GAP273 and
stimulated cell spreading. In contrast, the inactive allele
ARF6(T27N) promoted or perhaps stabilized the actin foci
(Figure 4A, right panels). Similarly, the GFP-GAP273(3.2) cells
over-expressing Rac1(G12V) were flat, extended and contained
membrane ruffles, whereas the neighboring cells that were not
transfected with the Rac mutant contained actin foci and were less
Figure 2. The ability of ARFGAP1 to stabilize the formation of
cortical actin foci and inhibit cell spreading correlates with the
level of expression of GFP-GAP273. A. Non-transfected CHO, 3.2
and 3.5 cells were plated into fresh culture dishes in medium containing
serum. At the indicated intervals after plating, cells were fixed and
stained with TRITC phalloidin. For each interval, more than 100 cells
were observed and the fraction having 3 or more foci of peripheral actin
is graphed as a function of time after plating. The data shown are from
three independent experiments. Error bars = S.D. B. Relative expression
levels of GFP-GAP273 in 3.2 cells and 3.5 cells was determined by
immunoblotting of lysates from the indicated cells. GFP-GAP273 was
detected by anti- GFP antibody. For a loading control, lysates were
blotted with anti- ER60 antibody, which recognizes a 60 kDa ER resident
protein. C. The mean surface areas occupied by CHO, 3.2 and 3.5 cells
were measured at the indicated intervals after re-plating as described in
Methods. A total of 50, 60, 60 cells from each time point for CHO-K1, 3.2,
3.5, respectively, were measured. Error bar = S.D.
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spread out (Figure 4B, left panels). The inactive allele Rac1(T17N)
promoted or stabilized the formation of actin foci just as did
ARF6(T27N) (Figure 4B, right panels), and at the same time,
inhibited cell spreading. We observed no change in cell spreading,
the appearance of actin foci or the distribution of GFP-GAP273 in
clone 3.2 cells transiently transfected with active or inactive alleles
of ARF1 (unpublished data). The quantification of the results on
cell spreading and actin foci formation shown in Figure 4A and 4B
demonstrates statistically significant differences from data obtained
from three independent experiments (Figure 4C and 4D). In GFP-
GAP273 (3.2) cells, 33% (S.D.=2.1%, n=3) of the cells contain
multiple (at least 3) actin foci 40 hours after re-plating. This
percentage is much lower than that obtained at 36 hour after re-
plating (compare 33% at 40 hours versus 68% at 36 hours, see
Figure 2A). We think the difference may be an effect of adding
fresh growth medium, which promotes cell spreading, to the cells
Rac1(T17N) or ARF6(T27N), 86% (S.D.=3.0%, n=3) and
cells transfectedwith either
66% (S.D.=5.0%, n=3) of the transfected cells have multiple
actin foci, respectively (Figure 4C), suggesting that dominant
negative mutants of Rac1 and ARF6 promoted or stabilized the
actin foci, and concomitantly, inhibited cell spreading. The data
shown here was collected from three independent experiments, in
each of which at least 100 cells were analyzed for each data point.
Cell spreading and membrane ruffling induced by activated alleles
of ARF6 and Rac1 could suppress the formation of actin foci in
GFP-GAP273 (3.2) cells. The areas occupied by ARF6(Q67L)- or
Rac1(G12V)- transfected 3.2 cells were significantly larger than
those occupied by the neighboring non-transfected cells within the
same image (Figure 4D). On average, the Rac1(G12V)-transfected
cells occupied397 mm2
ARF6(Q67L) occupied 241 mm2(S.D.=72 mm2, n=41). Howev-
er, the mean area covered per GFP-GAP273 (3.2) cell was
153 mm2(S.D.=36 mm2, n=123). Therefore, constitutive Rac1 or
ARF6 activity suppressed the inhibitory effect of GAP273 on cell
Figure 3. Cells over-expressing non-catalytic GFP-GAP273 migrate more slowly in an in vitro wound healing assay. Monolayers of
HEK293 cells transiently transfected with GFP-GAP273, GFP-GalT, or pEGFP-C3 empty vector were scratched and the cell were allowed to spread into
the scratched area. The degree of cell migration into scratched area was recorded at the indicated times. A. Images of cell migration over time. B. The
average widths of the scratched area measured at various points are graphed. (n=5, Error bars =S.D.)
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ARFGAP1 functions upstream of Rac1
The observation that the effect of GAP273 on the formation of
actin foci could be suppressed by the constitutive activation of
ARF6 or Rac1 might be explained by the hypothesis that
ARFGAP1 might act as a negative regulator upstream of Rac1
and ARF6 in one or more signaling pathway(s). If so, one would
expect a reduced fraction of GTP-bound Rac1 and/or Arf6 in
GFP-GAP273 cells. To test this hypothesis, we compared the level
of GTP-Rac1, GTP-ARF6, and GTP-RhoA in vivo in wild type
CHO cells and in GFP-GAP273 (3.2) cells as a function of time
after serum stimulation. If ARFGAP1 acted upstream of any of the
small GTP-binding proteins tested, serum activation of that
protein should be diminished in GFP-GAP273 (3.2) cells. In wild
type CHO cells, Rac1 was activated by serum over the course of
20 minutes after serum was added to the cells. However, the serum
activation of Rac1 was attenuated in the GFP-GAP273 (3.2) cells
(Figure 5, top panels). Activation of another Rho family member,
RhoA, by serum was not inhibited in GFP-GAP273 (3.2) cells
compared to CHO cells (Figure 5, middle panels). Addition of
serum did not increase the fraction of ARF6 bound to GTP in
either cell line (Figure 5, bottom panels). Therefore, GAP273
affects actin cytoskeleton most likely via Rac1. The decreased
activation of Rac1 in GFP-GAP273 (3.2) cells has been observed
in three independent experiments. It is clear that the abnormal
actin reorganization in GFP-GAP273 (3.2) cells is, at least in part,
due to failure in Rac1 activation in these cells.
The carboxyl-terminus of ARFGAP1 mediates the effect
To determine the region of GAP273 responsible for the
formation of actin foci, we measured the number of actin foci in
cells transiently expressing various fragments of ARFGAP1 fused
to GFP as a function of time after plating the cells (Figure 6). The
information of this domain mapping experiment (left panel) and a
control immunoblot showing the lack of extensive protein
degradation of the various transfected ARFGAP1 fragment (right
panel) is shown in Figure 6A. The carboxyl-terminal 65 amino
acids of ARFGAP1 were sufficient to inhibit cell spreading and
promote or prolong actin foci in CHO cells (Figure 6B). As
controls, neither GFP-CAT, the amino-terminal one-third of
ARFGAP1 that has GAP catalytic activity on ARF1, nor GFP-
GAP132, the middle third that is sufficient for targeting to Golgi
, stimulated the formation of actin foci. Three independent
experiments were performed for each time points and transfected
constructs. Typically 50 to 100 cells were analyzed in each of the
independent experiments. Indeed, images of cells over-expressing
GFP-GAP65 showed that GAP65 co-localized with actin foci
(Figure 7, arrows). These results suggest that there are two
separable functions in the non-catalytic domain of ARFGAP1,
binding to Golgi membranes and interacting with the actin
In this report, we have demonstrated a novel effect of
ARFGAP1 on the actin cytoskeleton. In cells transfected with
the catalytically inactive, carboxyl terminal domain of ARFGAP1,
GAP273, cell spreading was inhibited and more foci of cortical
actin were observed in the transfected cells. Activation of Rac1 by
serum was inhibited in cells over-expressing GAP273 and we saw
no inhibition of RhoA or ARF6. The over-expression of
constitutively active alleles of ARF6 or Rac1 suppressed the effect
of GAP273 on actin. These observations are consistent with the
hypothesis that ARFGAP1 affects actin cytoskeleton at least in
part by inhibiting the activation of Rac1.
These results are surprising because the ARFGAP1 has never
been postulated to function in the cell periphery involving in actin
regulation. However, in view of the signaling capacity of ARF1 at
the plasma membrane, it is possible that ARFGAP1, a molecule
whose functions are intimately linked to ARF1, can regulate actin
reorganization at the cell periphery. Other ARF GAPs have been
extensively documented to regulate actin cytoskeleton. Among
them, the best studied are GIT1, 2, and ASAP1. GIT1 and 2 bind
to a number of molecules, through which they affect actin
remodeling, cell spreading, focal adhesion turnover [34,35,36,37].
ASAP1hasalso been shown
Our results are consistent with the hypothesis proposed by
others that small GTPases of the ARF and Rho families function
coordinately in vivo both at the cell periphery and at the Golgi
[4,42]. Here, we have demonstrated that ARFGAP1 also acts in a
bifunctional manner. It is a GAP protein for ARF1 and also
inhibits the activation of Rac1, although at present we do not
know how direct this effect may be. We do not believe that the
effects of GAP273 on actin are due to an effect on ARF1. We
observe no change in the kinetics of glycoprotein processing or
transport in CHO cells stably over-expressing this protein, no
change in the morphology of the Golgi in these cells. Transient
expression of active or inactive alleles of ARF1 also does not
change the actin phenotype of cells expressing GAP273 (unpub-
lished data). Much higher over-expression of GAP273 is required
to inhibit transport in the early secretory pathway .
Furthermore, ARFGAP1 has no detectable GAP activity towards
ARF6 in vitro (unpublished data) and we observed no change in
cellular pools of GTP-bound ARF6. If GAP273 were acting to
inhibit GAP activity on ARF6, we would expect that the
phenotype caused by over-expression of GAP273 would be
mimicked by over expressing the constitutively active ARF6 allele,
but we observed the opposite. Although it has been established
that ARF6 functions to regulate cortical actin, our results show
that the effect of ARFGAP1 is not directly mediated by ARF6. We
speculate that ARFGAP1 may exert its effect on actin reorgani-
zation via an interaction with POR1/Arfaptin. POR1/Arfaptin
was initially shown to interact with both Rac1-GDP and ARF6-
GTP and regulate cytoskeleton rearrangement . Subsequently
it was shown to bind to ARF1, 5 and 6, but did not affect the GEF
or GAP activities towards the ARFs .
There is a growing literature on the presence of, and
requirements for, actin and actin-binding proteins in vesicles
produced at the Golgi where ARFGAP1 is thought to function
[42,45,46,47,48,49]. ARF1 can directly  and indirectly 
stimulate the production of phosphatidylinositol(4,5)bisphosphate
on Golgi membranes, a lipid that is bound by several proteins that
regulate actin polymerization as well as by proteins that regulate
coated vesicle production. Thus, ARF1 has activities that could
lead towards changes in the actin cytoskeleton. There is evidence
that ARFGAP1 acts as an effector of ARF by interacting with
vesicle cargo as well as coat proteins [52,53]. Our observations
suggest that an additional effector function of ARFGAP1 may be
to link vesicle production to the local regulation of actin. An
activated allele of Rac1 has been shown to inhibit membrane
traffic to the apical, but not the basolateral surface in polarized
epithelial cells . This effect of Rac1 may not be limited to
polarized epithelia, as fibroblasts maintain membrane traffic
pathways analogous to those in polarized epithelia . Another
small GTPase of the same family, Cdc42, has been shown to
regulate dyneine recruitment to COPI vesicles on Golgi .
to affectthese processes
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However, the function of actin in coated vesicles is not completely
understood. It has been noted previously that in addition to
providing attachment sites for motors  or directly moving
vesicles from the donor membrane through polymerization
[57,58], actin might also provide a barrier to vesicle formation
. Thus, whatever role actin plays in vesicle production, the
Figure 4. ARF6 and Rac1 can suppress the reorganization of the actin cytoskeleton by GAP273. A. Transient over-expression of
ARF6(Q67L) in GFP-GAP273(3.2) promotes cell spreading (left panels). Actin foci are also absent from the cell. Transient over-expression of
ARF6(T27N) in GFP-GAP273(3.2) cells (right panels) promotes or stabilizes the formation of actin foci. Images of GFP-GAP273 fluorescence of the same
cells are shown in the bottom panels. The GFP-GAP273 fluorescence co-localizes with ARF6 staining in the cell transfected with ARF6(T27N). B.
Transient over-expression of Rac1(G12V) (top left) promotes cell spreading and induces membrane ruffling in GFP-GAP273(3.2) cells but suppresses
the localization of peripheral concentrations of GFP-GAP273 at the actin foci (compare the cell transfected with Rac1(G12V) with the surrounding
non-transfected cells, bottom left). Transient over-expression of Rac1(T17N) (top right) in GFP-GAP273(3.2) cells inhibits cell spreading and prolongs
the appearance of membrane projections containing GFP-GAP273. The GFP image (bottom right) shows that surrounding non-transfected cells are
more spread and contain little, if any, peripheral concentrations of GFP-GAP273. For cells transfected with Rac1(G12V) or ARF6(Q67L), the cells had
been re-plated on glass cover slips for about 20 hours before fixation and immunofluorescence labeling. For cells transfected with Rac1(T17N) or
ARF6(T27N), the cells had been re-plated for 40 hours. Scale bar = 25 mm. C. Quantification of the effect of Rac1(T17N) and ARF6(T27N) on the
formation of actin foci. The percentage of cells transiently over-expressing Rac1(T17N) or ARF6(T27N) in GFP-GAP273(3.2) cells was measured. In each
of the three independent experiments, one hundred cells were counted. Error bar = S.D. D. Quantification of the effect of Rac1(G12V) and
ARF6(Q67L) on cell spreading. The area covered by GFP-GAP273(3.2) cells transiently over-expressed with Rac1(G12V) or ARF6(Q67L) was measured.
The mean area covered were obtained from 41, 60 and 123 cells, for ARF6(Q67L), Rac1(G12V) or non-transfected GFP-GAP273(3.2), respectively. Error
bar = S.D.
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local polymerization of actin and vesicle coats must be
coordinated, and ARFGAP1 is a candidate for a component of
Taken together, these results strongly indicate that the non-
catalytic carboxyl terminus of ARFGAP1 is involved in regulating
the function of actin cytoskeleton. This effect is likely mediated by
inhibiting the activation of Rac1, a crucial regulator of actin
Materials and Methods
Cell cultures and transfections
Chinese hamster ovary CHO-K1 cells were grown in DMEM
supplemented with 10 mM Hepes, pH 7.2, 0.35 mM L-proline,
5% fetal bovine serum (FBS), 50 U ml21penicillin and 50 U ml21
streptomycin. Hela and HEK293 cells were grown in DMEM
supplemented with 10% FBS. All three cell lines were obtained
from the ATCC (Manassas, VA). Green fluorescent fusion proteins
were made by inserting various fragments of ARFGAP1 cDNA
into the expression plasmid pEGFP-C3 (Clontech). Transient
transfections were carried out using 1 mg of plasmid DNA in a 6
well plate, together with transfection reagent Fugene 6 (Roche) or
with polyethylenimine .
Plasmid pCMV3.1 containing cDNA of ARF6 (kindly provided
by Julie Donaldson, NIH), myc-tagged-Rac1 mutant alleles
(provided by Bill Singer and Paul Sternweis, U.T. Southwestern),
or GFP fused in frame to various fragments of ARFGAP1 were
transiently transfected into CHO cells. CHO cells stably
expressing GFP-GAP273 have been previously described .
Rabbit IgG against Rac1 was purchased from Millipore (Billerica,
MA). Rabbit antiserum against ARF6 was obtained from Julie
Donaldson (NIH). Mouse monoclonal antibody, 9E10, specific for a
c-myc epitope was obtained from Yoav Henis (Tel Aviv University).
Rabbit antiserum to ARFGAP1 was prepared as described  and
IgG was isolated by affinity chromatography on GST-GAP273
protein. Rabbit anti-GFP antibody was purchased from Santa Cruz
obtained from Paul Kim (University of Cincinnati).
Immunofluorescence and GFP imaging
Cells transfected with various GFP fusion proteins weregrown on
11 mm diameter cover glasses. To detect GFP fusion proteins, cells
werewashedwith PBS3 timesandthen fixedin3.7%formaldehyde
for 15 minutes at room temperature. Formaldehyde was removed
and the cells were incubated with serum free DMEM for 5 minutes
at room temperature, rinsed in water and mounted on a glass slide.
For co-localization studies with actin, CHO cells expressing
GFP-GAP273 were grown on cover glasses for the 16 h and then
fixed in formaldehyde and permeabilized in 150 mM NaCl,
1 mM EDTA, 50 mM Tris HCl, pH 8.0, containing 1% bovine
skin gelatin (NET/Gel) and 0.1% Triton X-100 for 10 minutes.
For experiments involving the detection of actin, the samples were
incubated with 0.5 ml of PBS plus TRITC-Phalloidin for 1 hour
at room temperature.
In co-localization studies with ARF6 or Rac1, cells transiently
transfected with DNA plasmids carrying cDNA for either protein
were fixed and permeabilized as described and the samples were
incubated with PBS plus 0.5% BSA for 15 minutes. Then rabbit
polyclonal antiserum recognizing ARF6, or mouse monoclonal
antibody 9E10 (to detect Myc-tagged Rac1) was applied to the
samples. Co-localization with actin was achieved by adding
TRITC-phalloidin in the incubation with primary antibody. Goat
anti-rabbit IgG (for ARF6) and goat anti-mouse IgG (for Rac1)
secondary antibodies (Invitrogen) conjugated with Alexa fluor-
ophores were used to generate fluorescence signals. Images were
collected with either a Biorad MRC 600 or a Carl Zeiss LSM5
confocal microscope at 6306 magnifications, or an Olympus
FV1000 at 6006 magnifications. Double-labeled samples were
Figure 5. GAP273 inhibits the activation of Rac1 by serum. A. Activation of Rac1 is inhibited in CHO cells stably expressing GFP-GAP273, but
not in wild type CHO cells. The result shown is a representative of three independent experiments. B. GFP-GAP273 did not inhibit the activation of
RhoA. C. Serum caused little change in binding of GTP by ARF6 in either cell type and GFP-GAP273 did not affect ARF6 activation. The percent of total
GTP-binding protein that was bound to GTP in each sample is listed below the image of the immunoblots.
ARFGAP1 Inhibits Rac1
PLoS ONE | www.plosone.org7 April 2011 | Volume 6 | Issue 4 | e18458
excited with either a single 488 nm wavelength for recording
images of GFP or Alexa 488 labels or with a 568 nm wavelength
for imaging TRITC-phalloidin or Alexa 568 labels.
For real-time imaging of live cells expressing GFP-GAP273 or
GFP-Sec13, 24 hours before transfection, 46105Hela cells were
seeded into each well of a 6-well plate. Transfection was done the
next day with 3 mg DNA (GFP-GAP273 or GFP-Sec13) to 9 mg
PEI in a total volume of 200 ml PBS. The transfected cells were
then trypsinized 6 hours later and plated to a 35 mm petridish,
having a 14 mm microwell attached with a 0.16–0.19 mm thick
coverslip (MatTek). After 16 hours of incubation, GFP signals
from the transfected cells was monitored by real time confocal
imaging technique from an Olympus FV1000 confocal microscope
system at 6006magnifications. Images were taken in a 30 seconds
interval for a total period of 15 minutes.
Measuring actin foci and cell spreading
The experiments shown in Figures 2 examined CHO cell lines
stably expressing different levels of GFP-GAP273 . The
experiments shown in Figure 6 examined the effects of transient
Figure 6. Various deletion mutants of ARFGAP1 were tested for their ability to prolong the appearance of cortical actin foci. A.
Schematic diagram and information of various deletion mutants of ARFGAP1 tagged with GFP (left panel). Immunoblotting using anti-GFP antibody
showed that the level of protein expression for these constructs was similar and no extensive degradation of the fusion proteins was observed. (right
panel). B. Cells transiently transfected with the indicated GFP fusion constructs were analyzed for the presence of multiple (3 or more) actin foci after
re-plating. The constructs having a stabilizing effect on actin foci are labeled in red to aid visualization. Those with no difference with wildtype CHO
cells are labeled in green. 50–100 transfected cells were analyzed in each data point. GFP-GAP65 containing the carboxyl-terminal 65 amino acids of
ARFGAP1 was able to increase the number of cells containing actin foci similar to GFP-GAP273. Three independent experiments were performed.
Error bar = S.D.
ARFGAP1 Inhibits Rac1
PLoS ONE | www.plosone.org8 April 2011 | Volume 6 | Issue 4 | e18458
expression of truncated GFP-GAP proteins in CHO cells. To
measure actin foci, for both sets of experiments CHO cells were
removed from 6-well plastic culture dishes and approximately
26104cells were re-plated onto 11 mm cover glasses. At the
indicated intervals after plating, the cells were fixed and stained
with TRITC-phalloidin to visualize actin. Cells with three or more
actin foci were counted and expressed as a percentage of the total
number of cells analyzed. In every case, more than one hundred
cells were analyzed for each time point. Three independent
experiments were performed to obtain statistical data.
Cell spreading was measured by the area occupied by individual
cells. The area of a cell was traced with the outline of the cell
image using NIH Image J v1.43. The area under this outline was
quantified and converted from pixels to area unit (mm2) based on
the magnification and the aid of scale bar.
In vitro wound healing assay
24 hours before transfection, 86105HEK293 cells were seeded
into each well of a 6-well plate and incubated in 5% CO2
incubator at 37uC overnight. Before further experiment, each well
was examined to ensure that a monolayer of cells was completely
covering the well surface. Transfection was done with 3 mg DNA
(GFP-GAP273) to 9 mg PEI in a total volume of 200 ml PBS. A
scratch was then made across the cell monolayer with a 10 ml
pipette tip and the medium was changed to remove any cell debris
left behind. Brightfield images of the scratch were taken after 0, 12
and 24 hours, respectively.
Preparation of the Rac-binding GST-PBD reagent
E. coli carrying the plasmid pGEX-PBD, which encodes GST
fused to the CRIB domain of p65PAK, that binds to Rac and
CDC42 proteins , were grown to OD600between 0.6–0.8 at
37uC in 200 ml of Luria broth containing 50 mg/ml carbenicillin.
Protein expression of GST-PBD was induced with 0.3 mM of
IPTG for 3 hours at 37uC. The bacteria were harvested by
centrifugation and resuspended in lysis buffer containing 20 mM
HEPES, pH 7.5, 120 mM NaCl, 10% glycerol, 2 mM EDTA,
1 mM PMSF, and protease inhibitor cocktail CompleteH (Roche
Diagnostics, IN) at 4uC. The bacterial cells were broken open by
sonication. Cell debris was pelleted at 30,000 g for 30 minutes at
4uC. The supernatant was taken and NP-40 was added to 0.5% of
the final volume. 0.3 ml bed volume of glutathione-sepharose
beads was added to the supernatant and the mixture was
incubated at 4uC with gentle agitation for 1 hour. Then, the
beads were washed with PBD lysis buffer plus 0.5% NP-40 for five
times and lysis buffer (without NP-40) for 3 more times. The
purified GST-PBD bound to glutathione beads could be frozen as
a 50% slurry in PBD lysis buffer at 280uC.
Preparation of the Rho-binding GST-RBD reagent
A 100 ml culture of bacteria carrying the plasmid pGEX-RBD
was grown to OD 0.6–1.0 at 37uC, and GST-RBD protein 
expression was induced by 0.3 mM IPTG at room temperature for
3 hours. The bacteria were harvested by centrifugation and
resuspended in RBD lysis buffer containing 50 mM Tris, pH 7.4,
50 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, and
protease inhibitor cocktail CompleteH at 4uC. The bacterial cells
were broken open by sonication. Cell debris was pelleted at
22,000 g for 30 minutes at 4uC. The supernatant was incubated
with 0.5 ml bed volume of glutathione-sepharose beads for 1 hour
at 4uC. Then, the beads were washed with RBD lysis buffer three
times and with lysis buffer without Triton X-100 for an additional
three times. The purified GST-RBD bound to glutathione beads
could be frozen as a 50% slurry in RBD lysis buffer (without
Triton X-100) at 280uC.
Preparation of the ARF-binding GST-GGA3VHS-GATreagent
GST-GGA3VHS-GATwas prepared from bacteria carrying the
plasmid pGEX-GGA3VHS-GAT, using procedure described in
Puertollano et al. .
Determination of the fraction of GTP-bound small
GTPase in vivo
CHO K1 cells or GFP-GAP273 cells at 70% confluency in a
100 mm plate were starved with serum overnight in DMEM
supplemented with 10 mM Hepes, pH 7.2, 35 mM L-proline,
50 U ml21penicillin and 50 U ml21streptomycin. The next day
the cells were treated with the same medium supplemented with
15% fetal bovine serum for intervals between 0 and 30 min at
37uC. The cells were then placed on ice, quickly washed with PBS
once and lysed with 0.55 ml of ice-cold buffer A (50 mM Tris,
pH 7.6, 500 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1%
Triton X-100, 10 mM MgCl2, and protease inhibitor cocktail
CompleteH). The lysates were scraped from the plates and
collected into microfuge tubes. Insoluble material was removed
by centrifugation at 24,000 g for 10 min at 4uC. 0.5 ml of the
supernatants was transferred to another microfuge tube containing
40–50 mg of glutathione beads loaded with GST-PBD (for the
isolation of Rac1-GTP), GST-RBD (for RhoA-GTP), or GST-
GGA3VHS-GAT (for ARF6-GTP or ARF1-GTP). The mixtures
were incubated at 4uC for 30 minutes with gentle agitation. The
remaining 50 ml of supernatants were mixed with equal volume of
2X SDS sample buffer and would be used to measure the total
amount of Rac1, RhoA or ARF6 present in the lysate. After the
incubation, the beads were washed with ice-cold buffer B (50 mM
Tris, pH 7.6, 150 mM NaCl, 1% Triton X-100, 10 mM MgCl2,
and protease inhibitor CompleteH) four times. After the last wash,
30 ml of 2X SDS sample buffer was added to the beads, which
typically had a volume of 15 ml. Finally, the samples were
subjected to SDS-PAGE and immunoblot analysis using enhanced
chemiluminescence. Films were scanned with a Molecular
Dynamics laser scanning densitometer or a Bio-Rad GS-800
densitometer and quantified with ImageQuant software (Molec-
Over-expression of ARF6(Q67L) (top left) promotes cell spreading
in CHO cells and eliminates actin foci. Over-expression of
ARF6(T27N) (top right and bottom panels) induces the formation
of numerous actin foci. ARF6(T27N) (bottom left) co-localizes with
actin foci (bottom right). B. Over-expression of Rac1(G12V) (top
Actin foci when ARF6 or Rac1 are inactivated. A.
Figure 7. GFP-GAP65 co-localized with actin foci. GFP-GAP65
fluorescence is shown in the left panel and actin staining of the same
cells is shown in the right panel, as indicated by the arrows. Scale bar =
ARFGAP1 Inhibits Rac1
PLoS ONE | www.plosone.org9April 2011 | Volume 6 | Issue 4 | e18458
left) promotes cell spreading and membrane ruffling. Over-
expression of Rac1(T17N) (bottom left) promotes the formation
of and co-localizes with actin foci (bottom right). The expression of
ARF6 and Rac1 was detected by rabbit antiserum specific against
ARF6 and mouse monoclonal antibody (9E10) against the myc-tag
on Rac1. Antibodies were detected with secondary antibodies
conjugated with Alexa 488. Actin staining was detected by
TRITC-conjugated phalloidin. Scale bar = 25 mm.
were recorded at 30-second intervals for 15 minutes. Membrane
ruffling activity is very obvious throughout the recording period
(boxed areas). There are no observable spherical membrane
structures at the cell periphery.
HEK293 cells overexpressing GFP-Sec13. Images
were recorded at 30-second intervals for 15 minutes. Spherical
HEK293 cells overexpressing GFP-GAP273. Images
membrane structures containing green fluorescence signal were
rapidly appearing and retracting at the cell periphery (arrows).
These protrusions seldom generated actin microspikes. Active
membrane ruffling was not obvious in these cells.
We would like to thank Julie Donaldson for plasmids encoding mutants of
ARF6, Juan Bonifacino for the plasmid encoding GST-GGA3VHS-GAT
and Bill Singer and Paul Sternweis for plasmids pGEX-PBD and pGEX-
RBD. We thank Maria Kosfiszer for expert technical assistance.
Conceived and designed the experiments: KYS SY MGR HCC.
Performed the experiments: KYS MKY XW MZ SY. Wrote the paper:
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