From the § Division of Cell Biology, Hospital for Sick
Children, Toronto, Ontario M5G 1X8, Canada, the ¶ Department of
Surgery, Toronto General Hospital, Toronto, Ontario M5G 2C4, Canada,
the Department of Physiology, The Ohio State University,
Columbus, Ohio 43210, and the ¶¶ Department of Medicine,
University of Pennsylvania,
Philadelphia, Pennsylvania 19104-4283
Received for publication, March 6, 2001
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ABSTRACT |
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Recent evidence suggests that
extension of pseudopods during phagocytosis requires localized
insertion of endomembrane vesicles. The nature of these vesicles and
the processes mediating their release and insertion are unknown. COPI
plays an essential role in the budding and traffic of membrane vesicles
in intracellular compartments. We therefore assessed whether COPI is
also involved in phagosome formation. We used ldlF cells, a mutant line
derived from Chinese hamster ovary cells that express a
temperature-sensitive form of Phagocytosis of microbial pathogens by leukocytes is an
essential component of the host defense against infection.
Microorganisms become internalized into a membrane-bound vacuole called
a phagosome, which subsequently matures upon fusion with endosomes and
lysosomes into a powerful microbicidal organelle (1). The phagocytic capacity of neutrophils and macrophages is remarkable; individual cells
can take up multiple and/or very large particles. As a result, the
area of membrane internalized is significant and has in some cases been
estimated to approach or exceed the total initial surface area of the
phagocyte (2). Despite the internalization of a considerable membrane
expanse, no net loss of surface has been detected, and, in fact,
surface gains have been documented electrophysiologically (3) and by
spectroscopic means (4). These observations therefore suggest that
active exocytosis of endomembranes accompanies phagocytosis.
Pharmacological studies have in fact suggested that secretion of
endomembranes is essential for optimal phagocytosis. First, inhibitors
of phospholipase A2 precluded phagocytosis, with a concomitant accumulation of clear vesicles under the site of particle attachment (5). Second, Cox et al. (6) found that inhibition of phosphatidylinositol 3'-kinase by wortmannin, which can inhibit exocytosis, prevented phagocytosis of large particles, with
comparatively minor effects on the ingestion of smaller ones. Third,
botulinum and tetanus toxins, which interfere with SNARE-mediated
membrane fusion, were found to induce partial inhibition of
phagocytosis (7). Finally, it was shown that exocytic vesicles enriched in VAMP3, a v-SNARE that mediates secretion of recycling vesicles (8),
were locally secreted at sites of phagocytosis (9). Jointly, these
studies suggest that extension of pseudopods during particle engulfment
involves the focal exocytosis of endomembrane vesicles.
The mechanisms mediating the formation and traffic of these putative
vesicles are not understood. However, existing information regarding
endomembrane traffic may be applicable to the genesis of phagosomes. In
particular, COPI has been convincingly shown to participate in the
budding and traffic of vesicles between the Golgi complex and the
endoplasmic reticulum (ER)1
(10-12) as well as vesicles involved in recycling and endosome maturation (13, 14). COPI exists as a protein complex in the cytosol
and can assemble on docking sites of membranes, where it promotes the
fission of vesicles (15, 16). We therefore considered the possibility
that COPI may participate in the process of phagosome formation.
To this end, we used brefeldin A (BFA), which inactivates the GTP
exchange factors of adenosine ribosylation factor, to inhibit COPI or
the Chinese hamster ovary (CHO) mutant cell line called ldlF,
originally isolated by Hobbie et al. (10). These cells harbor a temperature-sensitive mutation in the gene encoding Materials, Constructs, and Antibodies--
Fura-2
acetoxymethyl ester, zymosan, FM1-43, rhodamine-123, and
rhodamine-phalloidin were from Molecular Probes, Inc. (Eugene, OR).
Hepes-buffered medium RPMI 1640, 0.8-µm dyed latex beads, BFA, and
cycloheximide were obtained from Sigma. 125I-Diferric human
transferrin (125I-Tfn) was from PerkinElmer Life Sciences,
and [35S]methionine/cysteine was from Amersham Pharmacia
Biotech. pEGFP, the plasmid encoding GFP, was from
CLONTECH. The VAMP3-GFP chimera was previously
described (9). Human IgG was from Baxter Healthcare Corp. (Glendale,
CA). Rabbit anti-GFP and anti-catalase antibodies were from Molecular
Probes, Inc. and Calbiochem (La Jolla, CA), respectively. The rabbit
polyclonal antibodies to Cell Culture and Handling--
The murine macrophage cell lines,
J774 and RAW 264.7, were obtained from the ATCC (Manassas, VA). RAW
macrophages were transfected with Fugene 6, as described by the
manufacturer (Roche Molecular Biochemicals). ldlF cells, bearing a
thermolabile mutation in the Assessment of Phagocytosis and Particle Adherence--
To assess
phagocytosis, SRBC were opsonized with goat or rabbit anti-SRBC IgG
(1:40 for 1 h at 37 °C) and then washed in cold phosphate-buffered saline to remove unbound IgG. The opsonized SRBC
were added to plated phagocytic cells (~10 SRBC/cell) and incubated
for 1 h at 37 °C. Extracellular SRBC were then removed by a
brief (30-s) hypotonic lysis in water, and internalized SRBC, which
resist lysis, were quantified under Nomarski optics. To assess
particle adherence, FcR-CHO cells or FcR-ldl cells were incubated with
opsonized SRBC for 30 min at 4 °C. Nonadherent SRBC were removed by
washing in phosphate-buffered saline, and the number of bound SRBC/cell
was quantified under light microscopy. Zymosan particles and 0.8-µm
latex beads were opsonized with 1 mg/ml human IgG and washed as
described above.
Immunofluorescence and F-actin Staining--
To label F-actin,
cells were fixed for up to 3 h with 4% paraformaldehyde and then
permeabilized with 0.1% Triton X-100 and incubated with
rhodamine-phalloidin (0.01 unit/ml phosphate-buffered saline). To stain
for Quantification of Surface Fc Measurement of Free Cytosolic Calcium
([Ca2+]i)--
Cells grown on glass
coverslips were incubated overnight at either 34 or 39 °C and then
loaded with fura-2 by incubation with 10 µM of the
parental acetoxymethyl ester for 30 min. Coverslips were then mounted
in a thermostatted Leiden holder on the stage of a Zeiss IM-35
microscope, equipped with a × 63 oil immersion objective. The
microscope set-up has been previously described in detail (18).
Calibration of fluorescence ratio versus
[Ca2+]i was performed as described (20). All
measurements were at 37 °C.
Scanning and Transmission Electron Microscopy--
For scanning
electron microscopy, cells were fixed in 2% glutaraldehyde and
postfixed with 1% OsO4. Following washing, the cells were
dehydrated in a graded series of ethanol. The samples were then
critical point-dried in a Pelco CPD-2 critical point drying device and
mounted on EM stubs with colloidal silver glue. They were then coated
with evaporated gold/palladium with a Pelco sputter coater model 3 for
50 s at 18 mA. The samples were then examined with a Philips XL 30 scanning electron microscope.
For transmission EM, cells were fixed as for scanning EM. The cells
were washed extensively and then en bloc stained with 1%
aqueous uranyl acetate for 30 min. Following washing, the samples were
dehydrated through a graded series of ethanol and then embedded in Epon
as we have described previously (21). Thin sections were cut and
stained with lead citrate and uranyl acetate and observed with a
Philips CM-12 electron microscope.
SDS-Polyacrylamide Gel Electrophoresis and
Immunoblotting--
Samples were solubilized in Laemmli's sample
buffer (22), resolved by SDS-polyacrylamide gel electrophoresis using
the Protean II minigel system (Bio-Rad), and transferred onto
polyvinylidene difluoride membranes. Membranes were then immersed in
blocking buffer (5% milk and 0.05% Tween 20) overnight at 4 °C.
Blots were incubated with anti- Receptor-mediated Endocytosis and Recycling of
125I-Transferrin--
FcR-ldl cells were either maintained
at 34 °C or incubated at 39 °C for Modulation of
The disappearance of
In contrast to the Golgi complex, incubating cells at 39 °C did not
appear to cause detectable changes to peroxisomes, mitochondria, and
the ER. The punctate distribution of catalase, a marker of peroxisomes,
was indistinguishable in cells incubated at 34 and 39 °C (not
shown), implying that peroxisomes are able to import and retain this
soluble protein in their lumen. Furthermore, rhodamine-123 accumulated
normally in the mitochondria of cells incubated at 39 °C (Fig.
1C), indicating that the mitochondrial membrane potential was unaffected, which implies that the activity of the respiratory chain is normal. In addition, the reticulate morphology of the ER,
revealed by immunostaining of calnexin, was similarly unaltered by
prolonged incubation at the restrictive temperature (not illustrated). These results were consistent with ultrastructural analysis of thin
sections by transmission EM, which showed no alterations in the
structure or distribution of mitochondria or ER in cells treated at
39 °C (100 sections from three different experiments; not
illustrated). Together, these observations imply that treatment of the
cells at the restrictive temperature for Effect of
Consistent with the findings of Daro et al. (13) in ldl
cells, the cells stably transfected with Fc receptors (FcR-ldl) were
found to take up and recycle Tfn, albeit at reduced rates. As shown in
Fig. 2D, the initial rate of uptake was >2-fold lower in
Effect of
Further evidence that this effect was specifically related to loss of
COPI was obtained by treating FcR-CHO and FcR-ldl cells (maintained at
34 °C) with the fungal metabolite BFA. BFA associates with and
blocks the activity of nucleotide exchange factors that regulate
ARF, thereby preventing the association of coatomer subunits with
membranes (23, 24). Treatment with 100 µM BFA for 30-60 min induced rounding of both cell lines (not shown).
The cell rounding observed upon inactivation of COPI was associated
with an apparent decrease in net surface area. This was determined by
confocal microscopy, quantifying the amount of cell-associated FM1-43
(not shown), a dye that becomes fluorescent when it intercalates into
the plasmalemma, thereby providing a measure of cell surface area (6,
25). The decrease in
Several separate lines of evidence argue that rounding of
COPI-deficient cells is not an indication of detrimental effects on
cell function or viability. First, the cells remained impermeant to
vital dyes and to the fluorescent dye FM1-43. Second, the cytosolic free calcium concentration, a sensitive measure of cell integrity and
well being, was unaltered by depletion of Effect of COPI Depletion on Receptor Expression and
Function--
In view of the apparent loss of surface area, the
effects of COPI depletion on phagocytosis could result from alterations in Fc receptor expression, distribution, or function. This possibility was considered in the experiments illustrated in Fig.
3. At the permissive temperature,
Fc
The ability of the receptors to engage their ligand was also assessed,
quantifying the number of opsonized SRBC bound to the surface of the
Fc The Effect of Brefeldin A on Phagocytosis by FcR-ldl and Macrophage
Cells--
Our results indicated that functional COPI was required for
phagocytosis in FcR-ldl cells. These findings appear to be in conflict
with earlier findings of Zhang et al. (26), who reported that in RAW 264.7 macrophages phagocytosis was insensitive to BFA. BFA
is a specific inhibitor of GTP exchange factors of the ARF family of
GTPases, which are essential for COPI function (23, 24, 27). We
therefore tested the effect of BFA on phagocytosis in FcR-ldl cells and
macrophages. The inhibitor induced a rapid dispersal of
The primary difference between the two protocols used to inactivate
COPI is the time involved in the development of the inhibitory effect.
To resolve the apparent inconsistency, we compared the rate of
disappearance of
However, these findings imply that COPI is not directly involved as an
essential component of the phagocytic process. Instead, loss of COPI
function abolishes phagocytosis indirectly, perhaps by gradual
depletion of an essential component required for the late stages of
signal transduction, actin polymerization, and/or pseudopod extension.
There is precedent for depletion of membrane components in ldlF cells,
which were initially isolated on the basis of reduced ldl receptor
expression at the restrictive temperature (10). Reduction of these
essential components may conceivably occur by inhibition of the
biosynthetic pathway when COPI is inactivated (11). Nonetheless,
treatment of cells with cycloheximide for 24 h did not preclude
phagocytosis, although protein synthesis was demonstrably arrested (not shown).
We also investigated if the prolonged absence of functional COPI was
equally effective at blocking phagocytosis of smaller particles. RAW
cells were treated with BFA for 8-10 h and then allowed to internalize
either SRBC, which have a diameter of ~4 µm, or 0.8-µm latex
beads coated with IgG, the latter approaching the size of bacterial
pathogens. At this time, phagocytosis of SRBC was blocked by ~65% ± 10% relative to control cells. The uptake of 0.8-µm beads was also
clearly inhibited by depletion of Effect of
We also investigated whether increase in cytoplasmic free
Ca2+ concentration ([Ca2+]i) was
normal in COPI-deficient cells. An increase in [Ca2+]i mediated by activation of phospholipase C
and release of Ca2+ from intracellular stores is one of the
earliest consequences of Fc receptor ligation (30, 31). Although not
essential for phagocytosis, this [Ca2+]i
transient is invariably associated with receptor clustering and
activation in professional phagocytes and nonprofessional engineered
phagocytes (32, 33). In FcR-ldl cells maintained at 34 °C,
[Ca2+]i spikes were similarly triggered by
binding of IgG-opsonized particles (Fig. 5F,
squares). Such spikes were absent in ldl cells lacking Fc
receptors (Fig. 5F, circles). Importantly,
[Ca2+]i transients were elicited by particles in
FcR-ldl cells pretreated at 39 °C (Fig. 5G). Similar
observations were obtained with FcR-CHO cells preincubated at both
temperatures (not illustrated). In contrast,
[Ca2+]i transients were not observed upon the
addition of opsonized particles to the parental wild-type CHO (not
shown) and ldlF cells (Fig. 5F, circles), which
do not express Fc Effect of Ultrastructural Analysis of Phagocytosis in COPI-containing or
-depleted Cells--
We next used transmission EM to compare the
ultrastructure of normal and COPI-deficient cells during the course of
phagocytosis. FcR-ldl cells preincubated at either 34 or 39 °C were
exposed briefly to opsonized SRBC, fixed, and processed for EM. As
described earlier for both professional (38) and engineered phagocytes (18), the early stages of phagocytosis in cells with functional COPI
are characterized by extension of elaborate pseudopods that surround
and ultimately engulf the particles (Fig.
7, A and B). In
these cells, SRBC located well within the cytosol (often near the
nucleus; Fig. 7B) were seen frequently, probably
representing complete (sealed) phagosomes. Such structures were
virtually never observed in COPI-deficient cells (e.g. Fig.
7, C and D). Nevertheless, adherent SRBC were
found routinely on the surface of the COPI-depleted cells (Fig. 7,
C and D). Of note, the pseudopods extending from these were much shorter, if present at all. Often, the SRBC were apposed to flat regions of the plasma membrane. These sites of apposition were sometimes raised above the surface of the cells, corresponding to the "pedestals" noted in Fig. 5. In a significant proportion of cells that were pretreated at 39 °C, clear vesicular structures were found to accumulate beneath the bound SBRC
(small arrowheads in Fig. 7D and
inset). These structures were not found at 34 °C.
Focal Secretion during Phagocytosis in COPI-deficient
Cells--
Pseudopod extension during Fc
VAMP3-GFP was found both in endomembrane vesicles and on the surface of
RAW cells performing phagocytosis (Fig. 8, B-D).
Importantly, as reported in FcR-CHO cells (8), VAMP3-GFP accumulated in the membrane of nascent phagosomes (Fig. 8C). The
accumulated VAMP3-GFP was inserted on the plasma membrane and not
simply concentrated in vesicular form below the surface. This was
confirmed by the observation that the phagosomal VAMP3-GFP was
accessible to anti-GFP antibodies in intact cells (Fig. 8D).
In contrast, VAMP3-GFP was often intracellular in cells treated with
BFA, distributed in tubules or cisternae (Fig. 8E).
Importantly, little accumulation was generally observed at the sites of
aborted phagocytosis where particles attached (Fig. 8, F and
G). These results suggest that COPI deficiency precludes the
mobilization of a VAMP3-expressing endomembrane pool necessary for
pseudopod extension during phagocytosis.
The central observation described in this paper is that
phagocytosis was impaired when COPI function was inhibited. The
requirement for COPI was documented by two independent approaches: the
use of a mutant cell line expressing a thermolabile form of The inhibition of phagocytosis lagged behind the disappearance of
Integrins have been reported to act synergistically with Fc receptors
to promote phagocytosis (35-37). A possible role of integrins was also
suggested by morphological changes undergone by cells following COPI
inactivation. The rounding of the cells was consistent with diminished
integrin activity, inasmuch as integrins are important in cell
spreading (34). However, the expression of
The occurrence of seemingly normal signaling after engagement of the Fc
receptors suggests that the inhibitory effect of COPI depletion is
exerted downstream. Some insight as to the possible site of action was
gained from ultrastructural analysis. Two features are noteworthy.
First, unlike the cells with normal COPI, which extended long and
convoluted pseudopodia, those lacking COPI extended only short
pseudopods or none at all (Fig. 7). Second, clear vesicles were
frequently seen to accumulate under the sites of frustrated phagocytosis in COPI-deficient cells (Fig. 7D). These
observations resemble findings of Cox et al. (6) and
Lennartz et al. (5). In the former study, actin
polymerization but not pseudopod extension was seen when
phosphatidylinositol-3'-kinase was inhibited. In the latter, impairment
of phopholipase A2 also limited the extension of pseudopods
and in addition promoted the accumulation of small electron lucent
vesicles underneath the sites of particle attachment. In both cases, it
was suggested that failure to perform exocytosis was responsible for
the inability of the cells to extend pseudopods. In accordance with
this interpretation, there is mounting evidence that secretion of
endomembranes is involved in pseudopod extension (see Introduction). It
is therefore conceivable that delivery of vesicles required for
pseudopod elongation is impaired by neutralization of COPI and ARFs.
Recycling endosomes are likely to be one of the endomembrane
pools affected by COPI. These membranes have been linked to
phagocytosis by two observations: (a) impairment of Rab11, a
GTPase that regulates fusion of recycling endosomes (38, 39),
attenuated phagocytosis (40) and (b) exocytic vesicles
containing VAMP3 were locally inserted into phagocytic cups (9).
Interestingly, treatment with BFA and genetic ablation of COPI lead to
defects in transferrin receptor recycling, implicating COPI in the
recycling pathway (Fig. 2D and Refs. 13, 41, and 42).
These findings prompted us to analyze the effects of COPI inactivation
on the distribution and mobilization of VAMP3-containing vesicles. We
found that treatment with BFA drastically reduced the amount of
VAMP3-GFP integrated into the plasma membrane of RAW cells. Of
particular significance, the localized secretion of
VAMP3-GFP-containing vesicles at the sites of phagocytosis observed in
untreated cells was largely eliminated by BFA (Fig. 8, F and
G). We concluded that COPI is required to maintain a pool of
endomembrane vesicles that need to insert into the plasma membrane to
promote the extension of pseudopods around the phagocytic particle.
In summary, our findings and those of others suggest that COPI and ARF
are important for the homeostasis of the endocytic recycling
compartment. Recycling endosomes, in turn, seem to provide at least
part of the membrane required for pseudopod extension. Last, as in the
case of wortmannin-treated cells, our observations dissociate the
polymerization of actin from the extension of pseudopods during
phagocytosis, suggesting that multiple events are required for
successful pseudopodial elongation.
COP. To confer phagocytic ability to
ldlF cells, they were stably transfected with Fc receptors type IIA
(Fc
RIIA). In the presence of functional COPI, Fc
RIIA-transfected
ldlF cells effectively internalized opsonized particles. In contrast,
phagocytosis was virtually eliminated after incubation at the
restrictive temperature. Similar results were obtained impairing COPI
function in macrophages using brefeldin A. Notably, loss of COPI
function preceded complete inhibition of phagocytosis, suggesting that
COPI is indirectly required for phagocytosis. Despite their inability
to internalize particles, COPI-deficient cells nevertheless expressed
normal levels of Fc
RIIA, and signal transduction appeared unimpeded. The opsonized particles adhered normally to COPI-deficient cells and
were often found on actin-rich pedestals, but they were not internalized due to the inability of the cells to extend pseudopods. The failure to extend pseudopods was attributed to the inability of COPI-deficient cells to mobilize endomembrane vesicles, including a
VAMP3-containing compartment, in response to the phagocytic stimulus.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
COP, a
component of COPI (10, 11). When incubated at the permissive temperature (34 °C), such cells express functional
COP, while incubation at the restrictive temperature (
39 °C) results in destabilization and rapid degradation of the mutant
COP, thereby inactivating COPI (10, 11). While extremely useful for the study of
COPI function, ldlF cells are not phagocytic. In order to analyze the
role of COPI in phagocytosis, ldlF cells were stably transfected with
Fc receptors (Fc
RIIA). Such heterologous transfection of opsonin
receptors was shown earlier to confer phagocytic capacity to nonmyeloid
cells, including CHO cells (17-19). We found that, like CHO cells,
ldlF cells transfected with Fc
RIIA (called FcR-ldl hereafter)
effectively internalized IgG-opsonized particles when grown at the
permissive temperature. However, following down-regulation of COPI,
phagocytosis was progressively and drastically inhibited. Evidence is
provided that COPI is indirectly required for phagocytosis by
maintaining a VAMP3-containing endomembrane pool that appears to be
required for pseudopod extension during Fc
R-mediated phagocytosis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
COP,
-mannosidase II, and calnexin were
generous gifts from Drs. M. Krieger (Massachusetts Institute of
Technology, Cambridge, MA), K. Moremen (Emory University, Atlanta, GA),
and David Williams (University of Toronto), respectively. Mouse
anti-phosphotyrosine antibody mixture, containing equivalent amounts of
monoclonal antibodies PY-7E1, PY-1B2, and PY-20 was from
Zymed Laboratories Inc. (San Francisco, CA). Mouse
anti-Fc
RIIA monoclonal antibody IV.3 and the
anti-
1-integrin monoclonal antibody, 7E2, were
from Medarex (Annandale, NJ) and from the Developmental Hybridoma
Studies Bank (Iowa City, IA), respectively. Cy3-conjugated anti-mouse
and anti-rabbit IgG were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Sheep erythrocytes (SRBC) and goat and
rabbit anti-SRBC antibodies were from ICN/Cappel (Aurora, OH).
COP, were the generous gift of Dr. M. Krieger. Wild-type CHO and ldlF cells were stably transfected with
Fc
RIIA cDNA using calcium phosphate, yielding FcR-CHO and
FcR-ldl cells, respectively. All cell lines were grown in Dulbecco's
modified Eagle's medium with 10% fetal bovine serum and 5%
penicillin-streptomycin (Life Technologies, Inc.) and maintained under
5% CO2. Unless otherwise indicated, FcR-CHO cells were
grown at 37 °C, and FcR-ldl cells were grown at 34 °C. For
down-regulation of COPI, FcR-ldl cells were placed at the restrictive
temperature of 39 °C for the specified periods. Alternatively, cells
were incubated with 100 µM BFA for the indicated periods,
followed by phagocytosis in the continuous presence of BFA.
-mannosidase II, calnexin, catalase, and phosphotyrosines, fixed
and permeabilized cells were incubated with a 1:100, 1:1000, 1:500, or
1:100 dilution of the respective antibodies for 1 h and
subsequently incubated for 1 h with fluorochrome-conjugated secondary antibodies used at 1:1000. To stain for GFP, fixed, nonpermeabilized VAMP3-GFP-transfected cells were incubated with a
1:600 dilution of rabbit anti-GFP antibody followed by Cy3-conjugated anti-rabbit at 1:1000. FcR-ldl (not permeabilized) cells were stained
with a 1:50 dilution of anti-Fc
RIIA and undiluted
anti-
1-integrin antibodies to quantitate surface
expression of Fc
RIIA and
1-integrin, respectively.
Fluorescence was analyzed using either a Leica model TCS4D or a Zeiss
LSM 510 laser confocal microscope. Composites of confocal images were
assembled and labeled using Photoshop (Adobe, Mountain View, CA) and
Microsoft Powerpoint software. Quantification of immunofluorescence was
performed using Scion Image (Scion Image, MA).
RIIA and
1-Integrin--
Surface
1-integrin was
quantified using fixed, nonpermeabilized cells attached to glass
coverslips by confocal microscopy. This was accomplished by acquiring
confocal optical slices at a constant interval and integrating the
total cellular fluorescence by stacking the collected slices.
Fluorescence intensity of the reconstructed images was quantified using
Scion Image. For flow cytometry, the cells were immunostained as above
and then scraped off the coverslips in ice-cold divalent cation-free
phosphate-buffered saline. After washing, the cells were analyzed as in
Ref. 7.
COP antibody (1:8000) and
anti-tubulin antibody (1:500) for 1 h at room temperature. The
blots were then washed three times for 10 min each in antibody buffer
(50 mM Tris/HCl, 150 mM NaCl, 0.05% Tween 20, 0.04% Nonidet P-40, pH 7.5) and next incubated with
peroxidase-conjugated anti-rabbit IgG (1:5000) for 1 h. Membranes
were washed and developed using enhanced chemiluminescence (Amersham
Pharmacia Biotech).
12 h and then serum-starved
for 1 h. The rate of receptor-mediated endocytosis of
125I-Tfn was then measured by incubating cells with 0.4 µCi/ml of 125I-Tfn for either 10 or 30 min. Extracellular
125I-Tfn was then washed and stripped with
phosphate-buffered saline solution at pH 3.0. To measure the rate of
125I-Tfn recycling, cells were incubated for 15 or 30 min
in the absence of 125I-Tfn after a pulse of 30 min. All
steps were performed at 37 °C. The cells were then lysed, and the
amount of internalized 125I-Tfn was quantified with a
counter.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
COP Expression in FcR-ldl Cells--
To study the
role of COPI in phagocytosis, ldlF cells were stably transfected with
Fc
RIIA, yielding FcR-ldl cells. To verify that the mutation
characteristic of ldlF cells persisted in FcR-ldl cells, the
COP
content of both cell types was assessed by immunoblotting following
incubation at the permissive (34 °C) or restrictive (39 °C)
temperature. For comparison, wild-type CHO stably transfected with
Fc
RIIA (FcR-CHO cells) were also analyzed. Tubulin, which is
constitutively expressed in these cells in a temperature-independent manner, was used as a reference. When grown at 34 °C, both ldlF and
FcR-ldl express
COP, although at levels that are 2-4 times lower
than that found in FcR-CHO cells (Fig.
1A). A similar differential expression of
COP at 34 °C was reported earlier between ldlF and
wild-type CHO cells (12). Incubation at the restrictive temperature for
18 h resulted in the complete disappearance of
COP in ldlF and
FcR-ldl cells but had no detectable effect on the expression of this
protein in FcR-CHO cells (Fig. 1A), as found earlier for
wild-type CHO cells (12).
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Fig. 1.
Characterization of
Fc RIIA-transfected ldlF fibroblasts.
A, immunoblotting of
COP (top row)
and tubulin (bottom row) in extracts of CHO cells
transfected with human Fc
RIIA (FcR-CHO) and in ldlF cells that were
either untransfected or transfected stably with the human Fc
RIIA
receptor (FcR-ldl). The cells were either maintained at the permissive
temperature (34 °C) or preincubated for
18 h at the restrictive
temperature (39 °C), as indicated. B, effect of
COP
depletion on the morphology of the Golgi complex. FcR-CHO cells
(i and iii) and FcR-ldl cells (ii and
iv) were pretreated at 34 °C (i and
ii) or 39 °C (iii and iv) as above
and immunostained with antibodies to
-mannosidase II. C,
mitochondrial membrane potential is intact in
COP-containing
(i) or -depleted (ii) FcR-ldl cells. The presence
of an internally negative mitochondrial membrane potential was detected
by the accumulation of rhodamine-123. Bar, 10 µm.
COP was also apparent by analyzing the
functional consequences of incubation at 39 °C. As illustrated in
Fig. 1B, i, the Golgi complex normally displays a
tight juxtanuclear structure composed of cisternae and vesicles.
Maintenance of this structure is known to depend on the continued
function of COPI, which in turn requires the presence of
COP (13,
14). Degradation of
COP in FcR-ldl cells was associated with
dispersal of the juxtanuclear complex, resulting in a diffuse punctate
staining of
mannosidase II-reactive vesicles (Fig.
1B, iv). Dispersal of the Golgi complex was due
to disappearance of
COP and not to the temperature shift itself,
since FcR-CHO cells were found to preserve their juxtanuclear Golgi
cisternae after overnight incubation at 39 °C (Fig. 1B,
iii).
15 h does not induce
wholesale, nonspecific disorganization of the cellular ultrastructure
and that only organelles dependent on COPI for their homeostasis, such
as the Golgi apparatus, undergo visible alterations, as shown
previously (11, 13, 14).
COP Depletion on Phagocytosis--
Previously, it had
been shown that ldlF cells stably transfected with Fc
RIIA were able
to internalize IgG-opsonized particles (17). Therefore, we next
investigated whether COPI is required for phagocytosis. FcR-ldl cells
were maintained at either 34 or 39 °C and then exposed to
IgG-opsonized SRBC to initiate phagocytosis. As illustrated in Fig.
2A and quantified in Fig.
2C, FcR-ldl cells maintained at the permissive temperature
internalize SRBC effectively; phagocytosis occurred in upwards of 50%
of the cells. This compares favorably with the phagocytosis efficiency
of FcR-CHO cells (~30%; Fig. 2C). The greater phagocytic
ability of FcR-ldl cells is most likely attributable to clonal
differences. Depletion of
COP by overnight incubation at 39 °C
profoundly reduced the ability of FcR-ldl cells to perform
phagocytosis. SRBC were observed in only 4 ± 3% of the
COP-depleted cells. By contrast, FcR-CHO cells internalized SRBC
slightly more effectively following incubation at 39 °C
than when maintained at 34 °C. These observations imply that the
effect noted in FcR-ldl cells is not due to the temperature per
se but instead that the presence of COPI is essential for optimal
phagocytosis.
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Fig. 2.
Effect of COP
depletion on cell morphology, and on phagocytic and endocytic
ability. FcR-CHO and FcR-ldl cells were either maintained at
34 °C or preincubated for
18 h at 39 °C and then exposed to
opsonized SRBC to induce phagocytosis. A and B,
representative light micrographs of FcR-ldl cells pretreated at 34 and
39 °C, respectively. C, summary of the quantification of
phagocytic efficiency in FcR-CHO (solid bars) and
FcR-ldl cells (cross-hatched bars) preincubated at the
indicated temperature. Data are means ± S.E. of 150 determinations from six experiments. D, FcR-ldl cells were
incubated at 34 °C (1, 3, 5, and
7) or 39 °C (2, 4, 6,
and 8) and then allowed to internalize 125I-Tfn
for 10 min (1 and 2) or 30 min (3-8)
at 37 °C. After washing and stripping adherent noninternalized
125I-Tfn, samples were either immediately lysed
(1-4) or chased with Dulbecco's modified Eagle's medium
supplemented with serum for 15 min (5 and 6) or
30 min (7 and 8), followed by lysis. Data shown
are representative of two independent experiments.
COP-depleted cells, although the extent of uptake after 30 min was
only ~30% lower. Also in agreement with Daro et al. (13), we found that the slower rate of Tfn uptake was compensated by a
reduced rate of recycling (Fig. 2D, lanes
5-8), accounting for the nearly normal Tfn content at
steady state. The reduced recycling of transferrin is consistent with
the proposed role of COPI in traffic along the endocytic pathway.
COP Depletion on Cell Morphology--
During the
course of the phagocytosis experiments described above, a striking
morphological difference between cells incubated in different
conditions became apparent. When incubated at the restrictive
temperature, FcR-ldl cells were found to become more rounded (Fig. 2,
compare A and B). As before, the effect was
attributable to the depletion of COPI and not to the incubation at
39 °C, since FcR-CHO cells maintained their spread morphology at
this temperature (not shown).
COP-depleted cells was reproducible and
statistically significant, but since fluorescence is not a simple
function of the area, the change in plasmalemmal surface was not
quantified precisely.
COP (see below). Third,
and as reported earlier for ldlF cells (13, 14), FcR-ldl cells treated
overnight at 39 °C displayed endocytosis and recycling of
transferrin (Fig. 2D). Fourth, the rate of incorporation of [35S]methionine/cysteine into newly synthesized proteins
was not significantly changed (not shown). Fifth, peroxisomes, the ER, and mitochondria in
COP depleted cells resembled those of control FcR-ldl cells, and the mitochondrial potential appeared unaffected. Sixth, cells responded to the addition of stimuli with increased tyrosine phosphorylation and actin assembly (see below). Finally, the effects of incubation at 39 °C were reversible. These findings imply that within the time period examined, suppression of COPI function does not cause generalized detrimental effects on the cell.
RIIA receptors were clearly observable by immunostaining on the
plasmalemma of FcR-ldl cells (Fig. 3A). Neither the
distribution (Fig. 3B) nor the amount of receptors appeared
to be altered following incubation at the restrictive temperature.
Quantitation of receptors by flow cytometry indicated that similar
levels of Fc
RIIA were exposed after incubation at 34 or 39 °C for
18 h in both FcR-CHO and FcR-ldl cells (Fig. 3E).
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Fig. 3.
Effect of COP
depletion on Fc receptor expression and opsonized particle
binding. FcR-ldl cells were either maintained at 34 °C
(A and C) or preincubated for 18 h at
39 °C (B and D). A and
B, immunofluorescence labeling of Fc
RIIA receptors.
C and D, scanning electron micrographs of FcR-ldl
cells with bound SRBC. Bar, 10 µm. E,
quantification of SRBC binding (solid bars) and
Fc
RIIA expression (cross-hatched bars) in FcR-CHO cells
and FcR-ldl cells preincubated at either 34 or 39 °C. Data are
means ± S.E. of six experiments of each kind.
RIIA-transfected cells. As illustrated by the scanning electron
micrographs in Fig. 3, C and D, and summarized in
Fig. 3E, SRBC bound readily to FcR-ldl cells both before and after depletion of
COP by overnight incubation at 39 °C.
Comparable results were obtained with FcR-CHO cells (Fig.
3E).
-mannosidase
II from a perinuclear distribution (Fig.
4A) to a diffuse intracellular
appearance (Fig. 4B), resembling that found in
COP-depleted cells. Despite the demonstrated effect of BFA on COPI
function, phagocytosis was virtually unaffected when measured 2 h
after the addition of the drug (Fig. 4C), consistent with
earlier observations (26).
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Fig. 4.
Time course of inhibition of phagocytosis by
BFA and COP depletion. A and
B, morphology of the Golgi complex, as detected with
antibodies to
-mannosidase II, in FcR-ldl cells maintained at
34 °C without (A) or incubated with 100 µM
BFA for 2 h (B). C, quantification of
phagocytosis by FcR-ldl cells treated with or without BFA for 2 h.
D, FcR-ldl cells were incubated for the indicated period of
time at 34 °C in the presence of BFA (squares) or in its
absence at 39 °C (circles). The
COP content of the
cells incubated at 39 °C was quantified by immunoblotting and
scanning of radiograms (open symbols). The
phagocytic efficiency (solid squares and
circles) was quantified microscopically using SRBC.
E, phagocytosis of 0.8-µm latex beads and SRBC by RAW
macrophages incubated with 100 µM BFA for 8-10 h and
normalized to the respective control. Data are means ± S.E. of at
least three experiments of each kind.
COP with the onset of inhibition of phagocytosis.
The results are summarized in Fig. 4D. Significantly, the
course of disappearance of
COP preceded the complete
ablation of phagocytosis. After 6 h at 39 °C, when
COP was
no longer detectable, phagocytosis was diminished but still clearly
observable (approximately half-maximal; see Fig. 4D).
Similarly, while no significant effects were noted at the early stages
of BFA incubation, phagocytosis was inhibited by nearly 35% after
3 h and by 100% after 20 h (Fig. 4D). BFA also
inhibited phagocytosis in professional phagocytes; particle ingestion
was inhibited by 50% and by >90% in J774.1 and RAW cells incubated
with the ARF antagonist for 6 and 12 h, respectively (not shown).
Importantly, these data argue that a significant attenuation of
phagocytosis can be observed at early time periods (4-8 h) after COPI
deficiency and further suggest that our observations are not due to
generalized, nonspecific detrimental effects on cell function due to
prolonged absence of COPI.
COP, although the inhibition was
significantly smaller (40 ± 9%; Fig. 4E).
COP Depletion on Receptor Signaling--
The
preceding results indicate that the inhibition of phagocytosis induced
by depletion of COPI cannot be attributed to a decrease in the number
of Fc
RIIA receptors or in their ability to ligate SRBC. Instead, the
effect of COPI is probably exerted at a later stage, affecting signal
transduction or downstream effectors. These possibilities were analyzed
next. Clustering of Fc
receptors upon interaction with IgG-opsonized
particles leads to the activation of tyrosine kinases and to
accumulation of F-actin around nascent phagosomes (18, 28, 29). The
occurrence of these steps was tested in COPI-deficient cells. As shown
in Fig. 5, A and B,
accumulation of tyrosine-phosphorylated proteins was detected at sites
of particle attachment to FcR-ldl cells pretreated at 34 °C (not
shown) and 39 °C. Moreover, activation of downstream effectors was
also readily observable. Accumulation of F-actin in the phagosomal cup,
which is evident in the phagocytosis-competent FcR-ldl cells kept at
34 °C (Fig. 5D), was in fact more pronounced in cells
shifted to 39 °C (Fig. 5E). In fact, scanning EM revealed that SRBC were often found tightly attached to "pedestals" that protruded from the
COP-depleted FcR-ldl cells (Fig. 5C).
F-actin was concentrated at the junction between the opsonized
particles and these pedestals (Fig. 5E).
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Fig. 5.
Effect of COP
depletion on early signaling events. FcR-ldl cells were
preincubated at 34 or 39 °C as described. A and
B, phosphotyrosine accumulation at sites of particle
attachment in COPI-deficient cells. C, scanning electron
micrograph illustrating the pedestals (arrowheads) that form
at sites of bound SRBC in FcR-ldl cells preincubated at 39 °C.
D and E, fluorescence micrographs of FcR-ldl
cells maintained at 34 °C (D) or pretreated at 39 °C
(E) and stained for actin after incubation with zymosan. The
arrows point to actin cups. Size bars
in C-E, 10 µm. F and G, cytoplasmic
free [Ca2+]i determinations in ldlF
(circles) and FcR-ldl cells (squares) that had
been maintained at 34 °C (F) and in FcR-ldl cells
preincubated at 39 °C for 18 h to deplete
COP
(G). Where indicated, opsonized zymosan (OPZ) was
added to initiate phagocytosis. The changes in
[Ca2+]i from two separate FcR-ldl cells
(open and solid squares) are shown in
each panel. Results are representative of five separate
experiments with at least eight cells per group.
RIIA. These findings imply that COPI is not
essential for this early response and confirm the notion that
incubation at 39 °C does not have generalized detrimental effects on cells.
COP Depletion on Integrin Expression--
Integrins
are important in cell spreading (34) and have been reported to act
synergistically with Fc receptors to promote phagocytosis (35-37).
Since COPI-deficient cells were morphologically altered
(i.e. rounded), we hypothesized that integrin function was
compromised in these cells. We therefore assessed the presence and
distribution of
1-integrins. We found that focal
contacts were formed in FcR-ldl cells incubated at both 34 and 39 °C
(Fig. 6, A and C).
Actin stress fibers were often seen to abut these focal adhesions under
both conditions, although the length of the stress fibers was reduced
in
COP-deficient cells (Fig. 6, B and D). The
total content of
-integrins in unstimulated FcR-ldl cells was
compared at 34 or 39 °C using flow cytometry of suspended cells and
also by quantitative fluorescence microscopy of adherent cells. No
significant difference was found using the former method, and only a
small decline was observed microscopically (Fig. 6E). Similarly, the F-actin content was unaffected by incubation at 39 °C.
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Fig. 6.
Effect of COPI depletion on integrins.
FcR-ldl cells maintained at 34 °C (A and B) or
at 39 °C for 18 h (C and D) were stained
for 1-integrin (A and C) and for
actin (B and D). Focal adhesion complexes that
co-localize with actin fibers are indicated with arrows.
E,
1-integrins (black and
striped bars) and F-actin (white
bars) were quantified in FcR-ldl cells maintained at the
permissive (34 °C) and restrictive (39 °C) temperatures.
1-Integrin content was measured by
fluorescence-activated cell sorting analysis of suspended cells
(black bars) or by quantitative confocal
microscopy of adherent cells (striped bars) and
F-actin stained with rhodamine-phalloidin by plate microfluorometry.
Data are means ± S.E. of at least three experiments each.
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Fig. 7.
Transmission electron microscopy of FcR-ldl
cells during phagocytosis. FcR-ldl cells were either maintained at
34 °C (A and B) or preincubated for 18 h
at 39 °C (C and D) and then allowed to bind
opsonized SRBC. Cells were then fixed and processed for transmission
EM. The large arrowheads in A-C point
to pseudopods near nascent phagosomes. The small
arrowheads in D indicate the presence of small
clear vesicles beneath adherent SRBC. Notice the absence of these
vesicles in B. The inset in B and
D shows the area within the dotted
line magnified by a factor of 4. SRBC that are fully
internalized within phagosomes are indicated by P.
Micrographs are representative of 100 sections from three different
experiments. Size bar, 1 µm.
R-mediated phagocytosis is
thought to depend on focal exocytosis of endomembranes (3, 6). In fact,
focal exocytosis of VAMP3-containing secretory vesicles was shown
recently to occur in phagocytic cups (9). Because prolonged depletion
of COPI appears to limit the extension of pseudopods around opsonized
particles, we postulated that inhibition of phagocytosis may result
from the impaired delivery of endomembrane vesicles to the site of
phagosome formation. To test this notion, the distribution and
mobilization of VAMP3 was analyzed in RAW macrophages by transient
transfection of a VAMP3-GFP chimeric construct. The predicted
transmembrane topology of the chimera is shown in Fig.
8A. The construct retains the
cytosolic domain of VAMP3, which dictates its intracellular targeting,
while the GFP module is in the lumen of endosomes and appears on the
exofacial side of the plasma membrane upon exocytosis. As a result, the occurrence of exocytosis of VAMP3-containing vesicles can be detected by staining intact (nonpermeabilized) cells with anti-GFP antibodies as
described (9).
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Fig. 8.
Distribution of VAMP3-GFP during
phagocytosis. A, diagrammatic illustration of the
VAMP3-GFP chimera (top) and of its putative orientation in
endosomes and in the plasmalemma (bottom). The VAMP3 module
is cytoplasmic, while GFP is luminal in vesicles and extracellular when
secreted. B-E, confocal images of RAW macrophages
transfected with VAMP3-GFP that were either untreated (B-D)
or treated with 100 µM BFA (E-G). Cells were
then allowed to bind opsonized zymosan particles. Fixed,
nonpermeabilized cells were then immunostained for GFP (D
and G). B and E, single optical slices
of GFP fluorescence from VAMP3-GFP. C and F,
three-dimensional reconstructions of GFP fluorescence from VAMP3-GFP.
D and G, three-dimensional projections of
exofacial VAMP3-GFP detected by antibodies to GFP. The
arrows indicate bound zymosan particles. The
inset in G is an intensified image of the
main panel to reveal the location of the cells
and zymosan particles, which have modest background fluorescence not
visible at normal laser exposure (cf. main
panels in D and G).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
COP and the inhibition of ARF with BFA. It is noteworthy that within the time
frame studied, COPI inactivation did not produce nonspecific deleterious effects on cell viability or function. Thus, COPI-deficient cells accumulated Tfn, had seemingly normal mitochondrial potential, and synthesized proteins at nearly normal rates, and the morphology of
the mitochondria, ER, and peroxisomes was unaltered. More importantly, the cells retained the ability to bind SRBC, triggered cytosolic calcium changes, and induced the focal assembly of F-actin at sites of
frustrated phagocytosis. We believe, instead, that COPI plays a
specific and discrete role in the events leading to phagocytosis. A
recent study demonstrated that interference with ARF6 function depressed phagocytosis (26). However, it is unlikely that our results
are related to ARF6, because this isoform of ARF has not been linked to
COPI function and, in addition, the exchange factors that control ARF6
are thought to be BFA-insensitive (39).
COP or the inactivation of ARF nucleotide exchange factors. Near
normal phagocytosis was seen for up to 2 h after the addition of
BFA, which enters the cells and interferes with ARF function rapidly,
and almost 50% of the phagocytic rate remained immediately after
COP was virtually eliminated. For these reasons, we felt that COPI
was necessary for the maintenance and/or localization of a critical
pool of elements of the phagocytic machinery, instead of playing a
direct role in phagocytosis. We initially considered whether the
plasmalemmal Fc receptors were present and responsive. Direct
assessment by flow cytometry confirmed the presence of nearly normal
quantities of surface receptors, and their ability to signal upon
cross-linking was verified by three different observations: (a) the accumulation of phosphotyrosine in the phagocytic
cup (Fig. 5, A and B); (b) the
initiation of calcium spikes (Fig. 5, F and G),
and (c) the induction of F-actin polymerization (Fig. 5,
D and E). Hence, neither absence nor inactivation
of Fc receptors can account for the inhibition of phagocytosis.
1-integrins quantified by flow cytometry or confocal
microscopy was affected only modestly, if at all, by
COP depletion,
and focal adhesions were clearly visible in adherent cells grown at
39 °C (Fig. 6, A and C). Actin stress fibers
were seen to abut these focal adhesions, and the total content of
F-actin of unstimulated FcR-ldl cells was essentially identical whether
grown at 34 or 39 °C (Fig. 6, B and D).
Therefore, gross alterations in integrin function are unlikely to
account for the inhibition of phagocytosis associated with COPI depletion.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Monty Krieger for
providing the ldlF cell line and the rabbit anti-COP antibody and to
Drs. X. Peng and W. Trimble for providing the VAMP3-GFP construct.
![]() |
FOOTNOTES |
---|
* This work was supported by the Medical Research Council (MRC), the Arthritis Society, and National Sanatorium Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
** Recipient of a postdoctoral fellowship from the MRC. Present address: Dept. of Pediatric Surgery, Childrens Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213.
Recipient of a graduate studentship from the Natural Sciences
and Engineering Research Council of Canada.
An MRC Distinguished Scientist, an International
Scholar of the Howard Hughes Medical Institute, and the current holder
of the Pitblado Chair in Cell Biology. To whom correspondence should be
addressed: Division of Cell Biology, Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-5727; Fax: 416-813-5028; E-mail: sga@sickkids.on.ca.
Published, JBC Papers in Press, March 15, 2001, DOI 10.1074/jbc.M102009200
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ABBREVIATIONS |
---|
The abbreviations used are:
ER, endoplasmic
reticulum;
BFA, brefeldin A;
CHO, Chinese hamster ovary;
EM, electron
microscopy;
FcRIIA, Fc receptors type IIA;
GFP, green fluorescent
protein;
Tfn, transferrin;
ARF, ADP-ribosylation factor.
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