Phagocytosis requires extension of F-actin-rich
pseudopods and is accompanied by membrane fusion events. Members of the
ARF family of GTPases are essential for many aspects of membrane
trafficking. To test a role for this family of proteins in Fc
receptor-mediated phagocytosis, we utilized the fungal metabolite
brefeldin A (BFA). The addition of 100 µM BFA to a
subclone of RAW 264.7 macrophages disrupted the appearance and function
of the Golgi apparatus as indicated by altered immunofluorescent
distribution of
-COP and reduced efflux of BODIPY
C5-ceramide, a phospholipid that normally accumulates
in the Golgi apparatus. In contrast, BFA had no effect on phagocytosis
of IgG-coated erythrocytes. These results suggested that activation of
BFA-sensitive ARFs is not required for phagocytosis. ARF6 is unique
among members of the ARF family in that its membrane association is
unaffected by BFA. Expression of ARF6 mutants defective in either GTP
hydrolysis (Q67L) or binding (T27N) inhibited phagocytosis of
IgG-coated erythrocytes and attenuated the focal accumulation of
F-actin beneath the test particles. These results indicate a
requirement for ARF6 in Fc
receptor-mediated phagocytosis and suggest that ARF6 is an important mediator of cytoskeletal alterations after Fc
receptor activation.
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INTRODUCTION |
During phagocytosis, foci of F-actin accumulate beneath bound
particles, conforming to the cytoarchitecture of pseudopods that engulf
the particles (for review, see Ref. 1). Macrophages and other
phagocytically competent cells are capable of ingesting many particles
without an apparent reduction in surface area, suggesting that
F-actin-based membrane protrusive events are coupled to membrane
recruitment. The exact relationship between actin assembly and membrane
protrusion and recruitment is not clear.
Members of the ARF family of GTPases have been implicated in numerous
membrane trafficking events in eukaryotic cells (for review, see Refs.
2 and 3). ARF proteins contain consensus amino acid sequences for GTP
binding as well as NH2-terminal glycine-containing consensus sequences required for myristoylation and membrane targeting. ARF1, the best characterized member of this family, cycles between cytosol and membrane depending on its nucleotide status. ARF1 binding
of the activating nucleotide, GTP, results in a conformational change
of the protein and enhanced affinity for membranes. Once membrane-bound, the ARF1 participates in recruitment of coatamer protein that is required for budding and fission of membrane vesicles (4-7).
Many insights into the process of membrane trafficking came from the
use of the fungal metabolite brefeldin A
(BFA),1 a substance that
impairs the association of ARF proteins with membranes and therefore
blocks early steps in the budding of vesicles. Although the exact
mechanism of action of BFA is unknown, it appears to inhibit
membrane-catalyzed guanine nucleotide exchange activity on ARFs
(8-10). More recently, several BFA-sensitive guanine nucleotide exchange factors for ARFs have been identified (11, 12). ARF6 is unique
among the ARFs in that its association with membranes is not sensitive
to BFA (13-15). Immunolocalization studies indicate that ARF6 resides
primarily in the plasma membrane, and studies using various mutant
alleles of ARF6 suggest that ARF6 cycles between an intracellular
compartment and the plasma membrane, depending on its nucleotide
status. Expression of a GTP hydrolysis-deficient ARF6 mutant, Q67L,
leads to accumulation of the protein in the plasma membrane and the
induction of plasma membrane invaginations, whereas expression of the
GTP binding-defective mutant, T27N, results in accumulation of the
protein in an internal tubulovesicular compartment (13, 16, 17). A
recent report suggests that ARF6 is also found in the cytosol (18),
raising the possibility that its function may also be regulated by its
subcellular distribution. Although the function of ARF6 is less well
understood than that of ARF1, recent studies using mutant alleles of
ARF6 suggest a prominent role for ARF6 in endocytosis (19), membrane
recycling (16, 17), and regulated exocytosis (20). In addition,
expression of the Q67 allele of ARF6 results in F-actin-rich plasma
membrane protrusions in HeLa cells (15) and accumulation of F-actin at the cell periphery in Chinese hamster ovary cells (21). In the latter
study, D'Souza-Schorey et al. (21) showed that ARF6 was capable of interacting with POR1, a Rac1-binding protein implicated in
membrane ruffling (22). Interestingly, these workers suggested that
Rac1 and ARF6 functioned on separate signaling pathways.
We showed recently that Rac1 and Cdc42 are required for Fc
R-mediated
phagocytosis (23). Given the likelihood that extensive membrane
remodeling is required for phagocytosis of multiple particles and the
findings that phagocytosis requires actin assembly at the plasma
membrane (24, 25), we wondered whether one or more members of the ARF
family participated in the process of particle engulfment. We tested
the sensitivity of phagocytosis to BFA and probed the role of ARF6 in
Fc
R-mediated phagocytosis.
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EXPERIMENTAL PROCEDURES |
Cells and Reagents--
RAW LacR/FMLPR.2 cells, a clone derived
from the RAW 246.7 cell line (American Type Culture Collection) was
maintained in RPMI medium containing 10% fetal bovine serum, 100 units/ml penicillin G, and 100 µg/ml streptomycin at 37 °C in a
CO2 incubator. Rabbit anti-ARF6 serum was derived as
described (15). Rabbit serum against
-COP was from J. Lippincott-Schwartz. Rabbit anti-sheep erythrocyte IgG was from
Diamedix (Miami, FL). Rhodamine-phalloidin and BODIPY
C5-ceramide were from Molecular Probes (Eugene, OR). Fluorescein isothiocyanate-, AMCA-, and rhodamine-conjugated
anti-rabbit IgG were from Jackson ImmunoResearch (West Grove, PA).
Superfect transfection reagent was from QIAGEN (Santa Clarita, CA).
Efflux of BODIPY FL C5-Ceramide--
RAW
LacR/FMLPR.2 cells were plated overnight in 96-well plates and were
incubated with 1 µM BODIPY C5-ceramide
(Molecular Probes, Eugene, OR) in a buffer containing 125 mM NaCl, 5 mM KCl, 1 mM
KH2PO4, 5 mM glucose, 10 mM NaHCO3, 1 mM MgCl2,
1 mM CaCl2, 20 mM HEPES, pH 7.4, and 1 mg/ml defatted bovine serum albumin for 30 min at 4 °C. The
cells were washed four times and incubated in RPMI containing 10%
fetal bovine serum with or without 100 µM BFA at
37 °C. At varying time intervals, cells were washed, fixed in 3.7%
formaldehyde, and the cell-associated remaining fluorescence was
measured in a fluorescence plate reader (Cytofluor II, Millipore;
excitation, 485 nm; emission, 530). To normalize for the cell number
present in the excitation beam, 1 µM ethidium bromide in
the presence of 0.2% Triton X-100 was added, and fluorescence (excitation, 530 nm; emission, 590) was recorded. Data are reported as
the ratio of BODIPY C5-ceramide/ethidium bromide
fluorescence intensities.
Fluorescence Microscopy and Immunoblotting--
Fluorescence
microscopy using a confocal scanning system (Zeiss) was performed as
described (24). For visualization of submembranous accumulations of
F-actin ("phagocytic cups"), transfected cells were washed once and
incubated with 2 × 107 EIgG at 37 °C for 7 min
before fixation. Bound EIgG were stained with fluorescein
isothiocyanate-conjugated anti-rabbit IgG. Cells were permeabilized at
this stage and stained for F-actin with rhodamine phalloidin and for
ARF6 expression with rabbit anti-ARF6 followed by AMCA- conjugated
anti-rabbit IgG. Immunoblotting was performed as described previously
(26).
Phagocytosis Assays--
All transfections were done using
Superfect according to the manufacturer's recommendations. RAW
LacR/FMLPR.2 cells were transfected overnight with the indicated
constructs subcloned into pXS (13, 27) and replated onto 13-mm round
coverslips in RPMI medium containing 10% fetal bovine serum.
Experiments were carried out 3-5 h after replating. 2 × 107 EIgG were added for 30 min at 37 °C followed by
washing and hypotonic lysis. Bound, uningested EIgG were detected by
staining with fluorescein isothiocyanate anti-rabbit IgG for 30 min at
4 °C. Cells were fixed in 3.7% formaldehyde, permeabilized, and
stained for expression of ARF6 proteins using anti-ARF6 serum. After
the addition of rhodamine anti-rabbit IgG, to detect both the presence
of ARF6 proteins and EIgG, cells were examined by fluorescence
microscopy. Ingested EIgG were identified as fluorescein
isothiocyanate-negative rhodamine-positive phagocytic vacuoles. 50 cells expressing the indicated ARF constructs and 50 nonexpressing
control cells on the same coverslip were scored for internalized or
bound EIgG. The phagocytic index was calculated as the average number
of EIgG ingested per cell, and the association index was calculated as the sum of bound plus ingested EIgG per cell. Data represent an average
of five to seven experiments performed in duplicate. A one-tailed
Student's t test was performed on the phagocytosis data.
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RESULTS |
Brefeldin A Disrupts the Golgi Apparatus (GA) in RAW LacR/FMLPR.2
Cells but Has No Effect on Fc
R-mediated Phagocytosis--
To test
the effects of BFA on phagocytosis in RAW LacR/FMLPR.2 cells, we first
verified that this fungal toxin disrupted the GA. We examined the
distribution of
-COP, a coat protein that accumulates in the GA in a
BFA-sensitive fashion (28-30). Similar to results in other cell types,
the addition of BFA resulted in the redistribution of
-COP (Fig.
1), consistent with disruption of the
integrity of the GA. To confirm that the addition of BFA resulted in
functional disruption of the GA, we determined the effect of the toxin
on the efflux of BODIPY C5-ceramide, a fluorescent lipid
analog that accumulates in GA (31, 32). The presence of BFA resulted in
a 73% inhibition in efflux rate (15 min to 4 h) of BODIPY
C5-ceramide (Fig. 2). Thus,
the effects of BFA on the GA in this macrophage-like cell line were
similar to those observed in other cell types. Despite disruption of
the GA, concentrations up to 100 µM BFA had no effect on
the rate or extent of phagocytosis of EIgG (Fig.
3). These results are consistent with a
lack of requirement for an intact GA in Fc
R-mediated phagocytosis
and suggest that BFA-sensitive ARFs are dispensable for
phagocytosis.

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Fig. 1.
BFA disrupts the GA in RAW LacR/FMLPR.2
cells. Adherent RAW LacR/FMLPR.2 cells were incubated in the
absence (panel A) or presence (panel
B) of 100 µM BFA for 30 min at 37 °C, and
fixed and stained for the presence of -COP as described under
"Experimental Procedures." Note focal accumulation of -COP in
untreated cells. Bar = 10 µm.
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Fig. 2.
BFA inhibits efflux of BODIPY FL
C5-ceramide in RAW LacR/FMLPR.2 cells. Adherent RAW
LacR/FMLPR.2 cells were incubated with 1 µM BODIPY
C5-ceramide for 30 min at 4 °C, washed, and incubated
further for various times at 37 °C in the presence ( )
or absence ( ) of 100 µM BFA. Cell-associated
fluorescence was measured and normalized to cell number using ethidium
bromide as described under "Experimental Procedures." Data are
expressed as the mean ± S.E., n = 3.
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Fig. 3.
BFA does not inhibit Fc R-mediated
phagocytosis in RAW LacR/FMLPR.2 cells. Adherent RAW LacR/FMLPR.2
cells were incubated in the presence ( ) or absence ( ) of 100 µM BFA for 30 min at 37 °C before and during the
addition of EIgG. Phagocytic indices were performed as described under
"Experimental Procedures." Data are expressed as the mean ± S.E., n = 3.
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Expression of GTP Hydrolysis-defective or GTP Binding-defective
Alleles of ARF6 Inhibits Fc
R-mediated Phagocytosis--
Although
the experiments described above suggested that BFA-sensitive ARFs do
not play a role in phagocytosis of EIgG, they did not test the
requirement for ARF6 in phagocytosis. To test if the BFA-insensitive
ARF6 plays a role in phagocytosis, we transiently expressed wild type
(WT), GTP hydrolysis-deficient (Q67L), or GTP binding-deficient (T27N)
mutant ARF6 proteins in RAW LacR/FMLPR.2 cells. These cells were chosen
because they consistently demonstrated enhanced transfection efficiency
compared with the parental RAW 264.7 strain (not shown). Expression of
ARF6 protein was assessed by immunofluorescence (not shown) and
immunoblotting. Endogenous ARF6 protein was detected in
mock-transfected cells, and higher levels of ARF6 were detected in
transfectants (Fig. 4). Expression of WT
or Q67L was always greater that that of T27N, although on a per cell
basis, it was comparable because transfection of RAW LacR/FMLPR.2 cells
with T27N led to 2.8-fold fewer T27N-expressing cells compared with
transfection of this cell line with Q67L. Expression of all three
constructs led to markedly enhanced staining for ARF6 throughout the
cytoplasm and, in the case of WT and Q67L, the plasma membrane; similar
results were obtained using anti-hemagglutinin monoclonal antibodies
with hemagglutinin-tagged versions of these proteins (not shown). To
test if ARF6 is required for Fc
R-mediated phagocytosis, we performed
phagocytosis assays on RAW LacR/FMLPR.2 cell transfectants. Expression
of various ARF6 constructs had a moderate effect on the total number of
EIgG associated with the macrophages (ranging from a 24% reduction in
cells overexpressing WT ARF6 to a 47% reduction in cells expressing
ARF6 Q67L; Fig. 5A). Although
expression of WT ARF6 led to a proportionate decrease in ingestion,
expression of either Q67L or T27N led to a disproportionate decrease in
phagocytosis (Fig. 5B). When the efficiency of ingestion was
expressed as the percentage of total cell-associated (i.e. bound and ingested) EIgG ingested (Fig. 5C), expression of
Q67L and T27N led to a 71% and 93% inhibition, respectively, in the phagocytosis of EIgG. Expression of WT ARF6 did not affect
significantly the percent of total cell-associated EIgG ingested
compared with controls (p = 0.17). The inhibitory
effects of the T27N mutant required membrane association because a
nonmyristoylated double mutant (ARF6 G2A/T27N) did not inhibit
phagocytosis (113 ± 7.5% of control, n = 4).
These results indicate that functional ARF6 is required for
Fc
R-mediated phagocytosis.

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Fig. 4.
Expression of ARF6 proteins in RAW
LacR/FMLPR.2 cells. RAW LacR/FMLPR.2 cells transiently transfected
with the indicated constructs were subjected to detergent lysis,
SDS-PAGE, and immunoblotting using either anti-actin monoclonal
antibody or anti-ARF6 antiserum as indicated.
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Fig. 5.
Inhibition of Fc R-mediated phagocytosis by
ARF6 Q67L or T27N. RAW LacR/FMLPR.2 cells transfected with the
indicated constructs were incubated with EIgG for 30 min at 37 °C.
The association index (panel A), phagocytic index
(panel B), and percent EIgG ingested
(panel C) were determined for RAW LacR/FMLPR.2
cells expressing the indicated constructs or nonexpressing controls as
described under "Experimental Procedures." Data are expressed as
the mean ± S.E., n = 5-7. Differences between
the percent EIgG ingested by either Q67L or T27N and controls were
statistically significant (p < 0.0005).
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ARF6 Q67L or T27N Inhibits Submembranous Accumulations of F-actin
Beneath Attached EIgG in RAW LacR/FMLPR.2 Cells--
To test whether
inhibition of phagocytosis by ARF6 mutants correlated with alterations
in Fc
R-directed actin assembly, we tested the effects of expressing
various ARF6 constructs on phagocytic cup formation. 7 min after the
onset of phagocytosis, well formed phagocytic cups were evident in
WT-expressing cells (Fig. 6). In cells
expressing ARF6 Q67L or T27N, however, focal accumulation of F-actin
beneath most attached EIgG was diminished (Fig. 6, right
panel), despite the presence of surface-bound EIgG (Fig. 6,
left panel). In those instances where focal accumulations of F-actin were apparent, they were poorly formed or attenuated.

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Fig. 6.
Phagocytic cup formation is inhibited by ARF6
Q67L or T27N. RAW LacR/FMLPR.2 cells transfected with the
indicated constructs were incubated with EIgG for 7 min at 37 °C and
were fixed and stained for the presence of EIgG (left) and
for F-actin (right). Expression of the indicated constructs
was confirmed by staining with anti-ARF6 serum followed by
AMCA-conjugated anti-rabbit IgG (not shown). Bar = 10 µm.
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DISCUSSION |
The data presented here demonstrate a requirement for ARF6 in
Fc
R-mediated phagocytosis. Because expression of either GTP-binding or GTPase-deficient alleles of ARF6 inhibited phagocytosis, it is
possible that cycling of ARF6 between GDP- and GTP-bound forms is
required for the ingestion process. Alternatively, because expression
of Q67L produced alterations in the distribution of F-actin even in the
absence of added EIgG, it is possible that expression of this allele
results in a phenotype that does not support stimulated actin assembly
at the plasma membrane. This implies that GTP hydrolysis by ARF6 may
not actually be a prerequisite for phagocytosis, a possibility that
would be difficult to verify. In either case, the requirement for ARF6
in the accumulation of submembranous F-actin induced by IgG-opsonized
particles suggests that ARF6 plays a role in the regulation of the
cytoskeletal changes that underlie Fc
R-mediated phagocytosis.
The addition of aluminum fluoride to HeLa cells overexpressing WT ARF6
or expression of ARF6 Q67L alone leads to extension of F-actin-rich
membrane protrusions (15). In Chinese hamster ovary cells, expression
of ARF6 Q67L leads to a redistribution of cortical F-actin to the
periphery, which was inhibited by truncation mutants of POR1 (21), a
protein that also interacts with Rac1 and mediates growth
factor-induced membrane ruffles (22). We have shown recently a
requirement for Rac1 and Cdc42 in phagocytosis and phagocytic cup
formation (23). Although the exact relationship among members of the
Rho family and ARF6 remain to be clarified, collectively these data
suggest that ARF6 and members of the Rho family of GTPases are
important effectors of cytoskeletal changes that accompany phagocytosis
and other plasma membrane-based cytoskeletal alterations. Whether ARF6
and Rac1 function in the same or independent pathways during
phagocytosis is currently under investigation in our laboratory.
Recent studies indicate that ARF6 participates in a novel recycling
pathway between the plasma membrane and a juxtanuclear compartment. In
HeLa cells, the intracellular ARF6-containing compartment appears
tubular, is distinct from transferrin-postive endosomes, and is not
readily accessible to water-soluble pinocytic tracers; however, it is
accessible to plasma membrane proteins that lack clathrin/AP-2
cytoplasmic targeting sequences, such as major histocompatibility
complex class I proteins and the interleukin-2
receptor (Tac) (16).
In electron micrographic studies of Chinese hamster ovary cells,
however, there was partial colocalization of ARF6 with transferrin
receptors, indicating that this compartment may partially overlap with
a recycling endosomal compartment (17). Although our study did not
address whether ARF6 in macrophages function in a recycling pathway,
the results in HeLa and Chinese hamster ovary cells suggest that ARF6
may have functions in addition to regulating the cytoskeleton. For
example, it is possible that GTP "cycling" by ARF6 participates in
the delivery of components necessary for remodeling of the cell surface
during phagocytosis. The Fc
receptors themselves are unlikely to be
one of these components because, unlike the major histocompatibility
complex class I and Tac, they contain residues that confer localization
to coated pits (33). Furthermore, expression of Q67L and T27N
inhibited, but did not abolish, binding of EIgG. In mouse macrophages,
phagocytosis proceeds in direct proportion to particle binding even
when Fc
R surface expression is reduced by more than 90% (34). The
disproportionate reduction in phagocytosis compared with binding of
EIgG in cells expressing either Q67L or T27N suggests a more profound
defect in the phagocytic apparatus. It is possible that during
phagocytosis, ARF6 mediates the delivery to the plasma membrane of
additional protein effectors or membrane components required for
phagocytosis, although the nature of these effectors/components remains
to be defined.
From the above discussion, it is not clear whether it is possible to
distinguish how ARF6 functions in membrane trafficking and cytoskeletal
remodeling. Because the two events may be coupled in vivo,
especially during events such as pseudopod extension and membrane
ruffling, ARF6 may, in fact, function in the coupling of these events.
The lack of an inhibitory effect of BFA on phagocytosis suggests that
other ARFs and the GA itself may not be critical for phagocytosis and
pseudopod extension. However, we cannot discount the participation of
the TGN in phagocytosis because BFA may not necessarily affect this
component of the GA, and studies using antiserum against TGN-38, a
marker of the TGN, did not reveal uniform alteration in the
distribution of TGN-38 in macrophages incubated with BFA (not
shown).
The lack of inhibition of phagocytosis using ARF6 G2A/T27N, a
nonmyristoylated mutant of AR6 T27N, indicates that myristoylation and/or membrane association of ARF6 T27N is required for effective inhibition of phagocytosis by this GTP binding-deficient allele of
ARF6. This is consistent with findings that myristoylation of ARF1 is
required for guanine nucleotide exchange activity catalyzed by ARNO
(35) and an unknown component of a retinal extract (36). It is possible
that myristoylation is also required for efficient binding of ARF6 to
its exchange factors. Therefore, nonmyristoylated alleles of ARF6, such
as ARF6 G2A/T27N, may be incapable of interacting with ARF6 exchange
factor(s) and cannot compete with endogenous ARF6 for their binding.
Thus, they could not act in a "dominant-negative" fashion to
inhibit ARF6 function.