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INTRODUCTION |
Macrophages are monocyte-derived cells that upon activation
acquire phagocytotic activity for pathogens, capability for antigen presentation to lymphocytes, and the ability to contribute to tissue
repair by removing dead and damaged tissue (1, 2). Resting macrophages
are fusiform or stellate in shape, whereas activated macrophages have
extensive pseudopodia as well as short microvilli and lamellipodia (1).
The protrusions are apparently important for phagocytosis, as they
extend around the pathogens prior to their engulfment.
MARCO1 is a macrophage
receptor (3) constitutively expressed in a subpopulation of macrophages
in the marginal zone of the spleen and in the medullary cord of lymph
nodes, i.e. regions where macrophages are actively engaged
in the removal of pathogens and other foreign substances from the blood
and lymph fluid. This has implied a direct role for MARCO in the
removal of pathogens. This assumption is supported by studies showing
that COS, CHO, and fibroblast cells, which normally do not bind
bacteria, acquire binding activity for both Gram-positive and
Gram-negative bacteria following transfection and expression of MARCO
(3). Furthermore, it has been shown that during systemic bacterial
infections, expression of MARCO is induced in macrophages located in
other tissues, such as liver and lung (4). Together, these data
strongly indicate a role for MARCO in the host defense against microorganisms.
MARCO is structurally related to the macrophage class A scavenger
receptors (MSR-A) (5, 6). However, despite structural similarities, the
pattern of expression is different as MSR-A receptors are
constitutively expressed by macrophages in all tissues. All these
receptors are homotrimeric proteins with a short cytoplasmic domain, a
single transmembrane domain, and a large extracellular part. The
extracellular portion of MSR-A has both a triple helical coiled coil
and collagenous structures, whereas MARCO contains only a long
collagenous triple helix. Additionally, both MARCO and MSR-AI have a
C-terminal cysteine-rich domain composed of about 100 amino acid
residues, whereas MSR-AII lacks this domain completely (3, 5-7). The
bacteria-binding region of MARCO has been localized to this C-terminal
region (7). MSR-A has been shown to bind a wide variety of ligands,
such as modified low density lipoprotein and bacterial surface
components (8-11). Studies with MSR-A knock-out mice have shown that
this receptor contributes to the formation of atherosclerotic lesions
and plays a role in host defense against pathogens (12, 13), suggesting that the binding activities of MSR-A shown in vitro have
physiological significance. In addition, MSR-A has been implicated in
macrophage adhesion (14).
In our studies on MARCO function we observed that expression of this
protein in many different cell lines resulted in dramatic cell shape
changes. Typically, these changes include formation of large
lamellipodia-like structures, as well as of extensive plasma membrane
processes. By expressing various truncated forms of MARCO, we show that
a segment in the C-terminal cysteine-rich domain is crucial for this
activity. The MARCO-induced morphological changes are accompanied by
rearrangement of the actin cytoskeleton. Furthermore, they are
inhibited, but not completely abolished, when CHO cells are plated on
fibronectin-coated surface or when MARCO is co-expressed with a
dominant-negative form of the small GTPase Rac1. The morphoregulatory
activity of MARCO may contribute to efficient removal of pathogens from
the blood and lymph fluids.
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EXPERIMENTAL PROCEDURES |
Materials--
Cytochalasin D, butanedione monoxime,
streptavidin-agarose, p-phenylenediamine, vitronectin, and
fibronectin were obtained from Sigma. Laminin-1 was a kind gift from J. Engel (Biozentrum, Basel, Switzerland). Rat anti-mouse MARCO monoclonal
antibodies were kindly provided by L. van der Laan and G. Kraal (Free
University, Amsterdam, The Netherlands). Mouse anti-paxillin monoclonal
antibody (clone 349) was purchased from Transduction Laboratories, and mouse anti-Myc antibody (clone 9E10) was from Santa Cruz Biotechnology. Secondary antibodies were obtained from Dako. Rhodamine-conjugated streptavidin was obtained from Jackson ImmunoResearch. Sulfo-NHS-biotin was from Pierce, and rhodamine-conjugated phalloidin was from Molecular
Probes. Plasmid pEA1 encoding a full-length cDNA for the human
asialoglycoprotein receptor H1 subunit and rabbit antiserum against a
purified human asialoglycoprotein receptor were kind gifts from M. Spiess (Biozentrum, Basel, Switzerland). The expression plasmids
pEXV-Myc Cdc42N17 and pEXV-Myc Rac1V12N17 were
generously provided by A. Hall (University College, London, UK).
Expression Constructs and Transient Transfections--
All MARCO
expression constructs were cloned into the mammalian expression vector
pcDNA3 (Invitrogen). DNA manipulations were carried out using
established molecular biological methods. Truncated forms M-419, M-436,
and M-441 of murine MARCO were created from the full-length cDNA
(3) by replacing the region encoding the C-terminal part of MARCO by
fragments encoding a stop codon after the codon for serine 419, valine
436, or glutamate 441, respectively. Version M-419 is a truncated form
completely lacking the C-terminal cysteine-rich domain (domain V),
whereas versions M-436 and M-441 also contain short segments of this
domain. Constructs "Bst" and "Eco" encode proteins with large
internal deletions. The version Bst lacks the last 40 Gly-X-Y triplets of the 270-residue long collagenous domain,
whereas Eco encodes a form, whose extracellular domain is composed of
the first four residues of the spacer domain, the last 22 triplets of
the collagenous domain, and an intact domain V. The construct "Del
422-433" encodes a version of MARCO that lacks the first 12 residues
of domain V (residues 422-433). The various constructs are summarized
in a schematic form in Fig. 4. The cDNA encoding the full-length
human asialoglycoprotein receptor was removed from the pEA1 vector and
inserted in the pcDNA3 vector.
Cells were transfected using the calcium-phosphate method. 20 µg of
DNA was used per 100-mm dish. In co-transfections, 10 µg + 10 µg of
the expression plasmids was used. Precipitates were incubated on cells
overnight, and cells were seeded on glass coverslips 24 h after
transfection. In some experiments, transfected cells were taken into
suspension 24 h after transfection and were incubated in
suspension for 12-16 h before plating on glass coverslips. Suspension
culture plates were prepared by coating 100-mm culture dishes with a
layer of 1% agarose sterilized by boiling for 30 min. The agarose
layer was allowed to harden, and the dishes were equilibrated by two
overnight incubations with 10 ml of Dulbecco's minimum essential
medium, 10% FCS.
When examining cell morphology 5 min after plating, cells grown in
suspension were plated directly from the suspension culture onto
coverslips. Alternatively, cells were collected by centrifugation, washed, and resuspended in Dulbecco's minimum essential medium, 1%
bovine serum albumin, 20 mM Hepes, pH 7.4, before plating
on coverslips. Cells were plated either on uncoated coverslips or on
coverslips coated with FCS, fibronectin, laminin-1, or vitronectin. When coating with FCS, a 10% solution was incubated on coverslips for
30 min at room temperature. Under these conditions, vitronectin is the
primary extracellular matrix (ECM) component adsorbed to the glass
(15). When coating with purified ECM components, a 40 µg/ml solution
was incubated on coverslips overnight at 4 °C. Before plating cells,
coverslips were further incubated in 2% bovine serum albumin in PBS
for 2 h at 37 °C.
Cell Surface Labeling and Streptavidin Precipitation--
Two
sparse 100-mm dishes of CHO cells were transfected per each construct.
Cells were pooled to one dish 24 h after transfection, and they
reached about 50% confluency 40 h post-transfection. At this time
point, monolayers were rinsed twice with ice-cold PBS, once with
ice-cold Hepes-buffer, pH 8.0, containing 150 mM NaCl, 0.2 mM CaCl2, and 0.2 mM
MgCl2 and then cooled in this buffer for 10 min in an
ice-water bath. Cell-surface proteins were labeled by incubating cells
with sulfo-NHS-biotin (0.5 mg/ml in Hepes buffer, 3 ml/plate) for 30 min in an ice-water bath with gentle intermittent agitation. Monolayers
were then washed twice with ice-cold Hepes buffer containing 100 mM glycine and further incubated in the same solution for
15 min in an ice-water bath. Cells were lysed by adding 1 ml of
ice-cold RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5%
sodium deoxycholate, 0.1% SDS, 10 µg/ml aprotinin, 1 mM
phenylmethylsulfonyl fluoride, 1 mM
N-ethylmaleimide), and proteins were solubilized by
incubating the plates on ice for 30 min with constant agitation. Lysed
cells were then scraped with a cell scraper, transferred to
microcentrifuge tubes, and further incubated on ice for 30 min with
mixing. Insoluble material was removed by centrifugation (15,000 rpm
for 15 min at 4 °C). Cleared lysates were incubated with
streptavidin-agarose (40 µl of beads) for 5-6 h on ice with constant
mixing. The streptavidin-agarose was pelleted and washed four times
with the RIPA buffer. Precipitated proteins were eluted by boiling the
beads in 50 µl of 2× SDS sample buffer for 10 min with intermittent
agitation. A second round of streptavidin precipitation was carried out
in some experiments. However, subsequent Western blotting with an
anti-MARCO antibody demonstrated that practically no MARCO protein
precipitated at this time, indicating that the first precipitation was
quantitative. Therefore, the second precipitation was not carried out
in every experiment.
Western Blotting--
Western blotting was carried out using
established procedures. The rat monoclonal antibody 1.18, which
recognizes an epitope within the cytoplasmic domain of MARCO (16), was
used to probe the different forms of MARCO. The filters were stripped
with 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM
2-mercaptoethanol, before probing with an anti-paxillin monoclonal antibody.
Immunofluorescence--
For immunofluorescence analysis, cells
grown on glass coverslips were fixed in 4% paraformaldehyde solution
for 20 min at room temperature and then permeabilized in 0.1% Triton
X-100/PBS for 5 min. After rinsing twice in PBS, free aldehyde groups
were quenched by incubating cells in PBS containing 50 mM
NH4Cl for 10 min. Thereafter, cells were incubated in PBS
containing 2% bovine serum albumin (blocking solution). All antibodies
were diluted in the blocking solution. When staining for MARCO, cells were incubated either with rat monoclonal antibody 1.18 or 7.21 (10 µg/ml) for 1-2 h. The epitope of the anti-MARCO antibody 7.21 is
located in domain V (16). After rinsing several times in PBS, cells
were incubated with FITC-labeled rabbit anti-rat IgG. Cells were rinsed
again several times in PBS, and coverslips were mounted by inverting
them into PBS containing 50% glycerol and 1 mg/ml
p-phenylenediamine. For filamentous actin staining,
rhodamine-conjugated phalloidin was added to the secondary antibody solution.
For double labeling for MARCO and paxillin, cells were fixed and
permeabilized as above and then incubated for 2 h with mouse monoclonal antibody against paxillin (1 µg/ml). After several washes
in PBS, cells were incubated with biotinylated rabbit anti-mouse IgG (5 µg/ml) for 30 min, rinsed again in PBS, and incubated with rhodamine-conjugated streptavidin for 20 min (5 µg/ml). After this,
cells were fixed again in the paraformaldehyde solution and stained for
MARCO as described above.
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RESULTS |
Expression of MARCO Induces Formation of Cellular Processes and
Rearrangement of the Actin Cytoskeleton--
An interesting change in
cell shape was noticed when cells not normally expressing MARCO were
transfected with a plasmid encoding the full-length protein. When cells
were grown overnight on glass coverslips in complete medium and
examined by immunofluorescence microscopy about 40 h after
transfection, MARCO-expressing cells often had processes with lengths
several times the size of the cell body. Cell lines studied included
CHO, HeLa, NIH3T3, and 293 cells. There was some variation in the
morphological effects of MARCO between the cell lines so that the
processes were longest in 293 cells and that the other cell lines
mentioned above often also had large lamellipodia-type structures (from
which the long processes extended). Examples of MARCO-expressing CHO
and HeLa cells are shown in Fig. 1
(A, C, and D) and also in Figs. 6 and 7. In
contrast, COS7 cells responded quite differently to MARCO transfection.
In these cells, MARCO expression led to formation of short pine
needle-like protrusions (not shown).

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Fig. 1.
Expression of MARCO induces formation of long
plasma membrane processes. CHO (A-C) and HeLa cells
(D) were transiently transfected with an expression plasmid
encoding the full-length MARCO and examined 40 h after
transfection. Cells were double-stained for MARCO (FITC) and
filamentous actin (rhodamine). Double-staining is shown in A,
C, and D. B represents the same field as
A but shows only the actin filament staining.
MARCO-expressing cells are not stained as strongly as untransfected
cells by rhodamine-conjugated phalloidin. This exposure does not reveal
the weak phalloidin staining of the long processes.
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Process formation was not dependent on the transfection method, since a
similar effect was observed when MARCO-expressing DNA was introduced
either by calcium-phosphate method or by retroviral infection (not
shown). To test if the process formation was due to overexpression of a
membrane protein, we expressed the H1 subunit of the human
asialoglycoprotein receptor which is, similar to MARCO, a type II
membrane protein. Expression of this protein did not induce any
morphological changes (not shown), demonstrating that cell shape
changes are not simply a consequence of expression of a cell membrane
protein. In further studies, it turned out that MARCO-expressing cells
could be identified in cell populations simply by staining for actin
filaments (Fig. 1B). Compared with untransfected cells,
MARCO-expressing cells stained only weakly by the filamentous
actin-staining rhodamine-conjugated phalloidin. Furthermore,
filamentous actin appeared to be organized differently in these two
populations of cells. Examination of cells by a higher magnification
revealed that untransfected cells had an organized actin cytoskeleton
with numerous long stress fibers (Fig.
2). In contrast, the MARCO-expressing
cells almost completely lacked stress fibers. Instead, there were fine
actin filaments extending from the cell periphery into the processes,
although the phalloidin staining was very weak in these processes (not
shown). The processes were frequently heavily branched, and examination
of the branching pattern revealed that these processes were
"protrusive filopodia" instead of trailing tails, being left behind
by migratory cells.

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Fig. 2.
Effect of MARCO expression on distribution of
filamentous actin. Untransfected CHO cells have an organized actin
cytoskeleton with numerous stress fibers. A MARCO-expressing cell
(lower left corner) appears to lack stress fibers. Instead,
there are fine actin filaments protruding from cell periphery.
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In order to study the early phases of the process formation, we focused
on transiently transfected CHO cells, since these cells did not
aggregate when held overnight in suspension. Cells were taken into
suspension 24 h after transfection and were then plated 12-16 h
later on FCS-coated coverslips. When cells were fixed 5 min after
plating, all of them were round, and the MARCO-expressing ones were
morphologically indistinguishable from untransfected cells (not shown).
However, when cells were fixed 30 min after plating, untransfected
cells were still fairly round, but most of the MARCO-expressing cells
were already well spread (Fig.
3A). In addition, there were
lamellipodia-like protrusions extending from the periphery of some of
the MARCO-expressing cells (Fig. 3B). Examination of cells
1 h after plating revealed that most of the MARCO-expressing cells
had protrusions (Fig. 3C). Furthermore, many of the cells
sent out thin dendritic processes. After a few hours the processes
could be several times longer than the cell body (3 h after plating in
Fig. 3D). It is of interest to note that the cell spreading
is fairly symmetrical and that forming lamellipodia appear to extend
randomly in different directions. However, soon thereafter
MARCO-expressing cells acquired polarity so that the long processes
extended only from one pole of the cell.

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Fig. 3.
Time course of MARCO-promoted cell shape
changes. Transiently transfected CHO cells were taken into
suspension 24 h after transfection and plated 16 h later on
FCS-coated glass coverslips. Cells were fixed after 30 min
(A and B), 1 h (C), or 3 h
(D) and stained for MARCO (FITC) and actin filaments
(rhodamine). When examined 30 min after plating, MARCO-expressing cells
can already be distinguished from untransfected cells by their flatter
appearance (A). Sometimes MARCO-expressing cells have
extensive lamellipodia-type protrusions already at this time point
(B). Lamellipodia-type protrusions extend randomly in
different directions (B and C), whereas the long
processes extend only from one of the protrusions (D). The
arrow in B indicates lamellipodia.
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The MARCO-induced morphological effects were efficiently inhibited when
cells were plated on FCS-coated coverslips for 45 min in the presence
of 2 µM cytochalasin D (not shown), demonstrating the
crucial role of actin polymerization in this process (at a 2-h time
point cytochalasin D was found to have a toxic effect). On the other
hand, 10 mM butanedione monoxime, a known myosin inhibitor,
did not have significant effects during the initial cell spreading,
whereas it markedly inhibited the process formation (not shown). In
this experiment, cells were assayed 45 min and 2 h after plating
on FCS-coated coverslips.
Segment of Domain V Is Important for the Morphoregulatory Activity
of MARCO--
In order to identify specific regions of the MARCO
polypeptide required for the morphological effects, different truncated forms of MARCO were expressed in CHO cells (Fig.
4). First, we tested truncations M-436
and M-441, which almost completely lacked the C-terminal cysteine-rich
domain V and contained, respectively, only the first 16 and 21 residues
of this domain. Both forms effectively promoted the morphological
change, demonstrating that most of the domain V is not needed for this
activity. We next produced a version of MARCO that lacks the C-terminal
half of the collagenous domain (version Bst, lacking residues
299-419), and the morphological change of cells was observed upon
expression of this protein, too. Since this indicated that the
C-terminal part of the collagenous domain is not needed for the
morphological effects, we next tested a form of MARCO, whose
extracellular part is composed of the first 4 residues of the spacer
domain, the last 65 residues of the collagenous domain, and an intact
domain V (version Eco). Somewhat surprisingly, this variant also
promoted process formation, albeit not as efficiently as the other
truncated forms. Taken together, the only region of the extracellular
domain that is common for all these effective truncated versions is a
segment of domain V before its first cysteine residue (residues
420-436). Therefore, we predicted that expression of the form M-419,
which does not contain any segments of domain V, would not result in
the change of the cell shape. Indeed, this was the case, indicating
that the segment of domain V encompassing its first 15-20 residues is
important for this activity. We tried to confirm this result by
expressing a version of MARCO in which only this region is deleted
(version Del 422-433). However, this protein did not appear on the
cell surface (see below) but was apparently retained in the secretory
pathway.

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Fig. 4.
Schematic illustrations of the MARCO variants
used in transfection experiments. M-419, M-436, and M-441 are
C-terminal truncations. Bst, Eco, and Del422-433 contain internal
deletions. The deleted regions are delineated by dashed
lines and are additionally highlighted by hatch marks.
cyto, cytoplasmic domain; TM, transmembrane
domain; SRCR, scavenger receptor cysteine-rich domain
(domain V); C, C-terminal end; N, N-terminal end;
w.t., wild-type MARCO.
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Since the truncation M-419 did not promote any morphological changes,
we wanted to ensure that it was still expressed on the cell surface. In
order to examine this, cells were biotinylated with a
membrane-impermeable reagent sulfo-NHS-biotin, after which biotinylated
proteins were precipitated by streptavidin-agarose and analyzed by
Western blotting using an antibody recognizing the cytoplasmic domain
of the MARCO protein. This experiment demonstrated that the form M-419
and the full-length MARCO were expressed at equal levels on the cell
surface (not shown) and that some of the morphologically active
truncations, such as M-436, are not as abundantly expressed on the cell
surface (Fig. 5A). Moreover, along with the results obtained in the transfection studies with a
plasmid encoding the H1 subunit of the human asialoglycoprotein receptor (see above), these results indicate that the morphological change is not a nonspecific effect, due only to overexpression of a
cell-surface protein. In accordance with this conclusion, we have
observed that cells change their morphology also when expression of
MARCO is driven from a weak promoter, the minimal cytomegalovirus
promoter (not shown). To confirm that only cell-surface proteins were
labeled by sulfo-NHS-biotin, we wanted to verify that paxillin, a
component of focal adhesions at the inner site of the plasma membrane,
was not precipitated by streptavidin-agarose. This was the case, even
though an anti-paxillin antibody gave a strong signal with the total
cell lysate (Fig. 5B). Similarly, one of the MARCO
truncations, Del 422-433, was not precipitated by
streptavidin-agarose, although it was abundant in the total cell
lysate. Thus, the absence of these two proteins, the MARCO form Del
422-433 and paxillin, in the streptavidin precipitates indicates that
sulfo-NHS-biotin did not leak into cells during the course of the
experiment.

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Fig. 5.
Analyses of proteins in transiently
transfected CHO cells. A, amounts of the different
MARCO forms on the cell surface. Cell-surface proteins were
biotinylated on ice followed by the precipitation with
streptavidin-agarose. The precipitated proteins were analyzed by
Western blotting using an anti-MARCO antibody recognizing the
cytoplasmic domain (monoclonal antibody 1.18). CHO cells were
transfected with expression vectors encoding forms Del422-433
(lane 1), M-419 (lane 2), M-436 (lane
3), and M-441 (lane 4). B, two of the
samples analyzed in A, M-419 and M-436, were also analyzed
for the presence of paxillin in the streptavidin precipitate.
Lane 1, total cell lysate from cells expressing the form
M-419 (1/100 portion of the total lysate); lane 2, total
cell lysate from cells expressing the form M-436 (1/100 portion).
Lanes 3 and 4 represent streptavidin precipitates
and show that paxillin was not precipitated by streptavidin-agarose,
indicating that only cell-surface proteins were biotinylated during the
course of the experiment. One-quarter of the precipitated proteins were
loaded in the gel.
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Taken together, these results demonstrate that the MARCO-induced
morphological change is not a nonspecific effect due to overexpression of a cell-surface protein and that the region of domain V before its
first cysteine residue is important for this activity of MARCO.
Effects of MARCO Expression on the Formation of Focal
Adhesions--
The observation that actin filaments were rearranged in
the MARCO-expressing cells (Fig. 2) raised the question whether these cells still have intact focal adhesions. To examine this, transiently transfected CHO and HeLa cells were plated on glass coverslips 24 h post-transfection and were fixed and double-labeled for MARCO and
endogenous paxillin 16 h later. The staining revealed that MARCO-expressing CHO cells with extensive processes do not have focal
adhesions (not shown). MARCO-expressing CHO cells with short processes
have some kind of focal adhesions, but they are not well formed
compared with focal adhesions in untransfected cells (Fig.
6, A and B). There
are no focal adhesions in the cellular processes. Furthermore, it
appears that focal adhesions do not follow the margins of the cell
body, indicating that the MARCO-induced lamellipodia-like structures
are devoid of focal adhesions. The same also holds true for
MARCO-expressing HeLa cells, as shown in Fig. 6, C and
D.

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Fig. 6.
Effects of MARCO expression on focal adhesion
assembly. CHO (A and B) and HeLa cells
(C and D) transiently expressing MARCO were
double-stained for MARCO (A and C) and endogenous
paxillin (B and D). A and B
and C and D, respectively, represent the same
field. Cells were examined 40 h post-transfection, after growing
them for 16 h on glass coverslips. Focal adhesions do not follow
cell margins in the MARCO-expressing cells. Note that CHO cells
(B) not expressing MARCO have distinct paxillin-positive
focal adhesions (arrows). It is not shown here, but often
there are no focal adhesions in those MARCO-expressing cells that have
very long extensions, or, as shown in B, if there are focal
adhesions, they are not necessary well formed.
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Effects of Different ECM Substrata on the Formation of the Long
Plasma Membrane Processes--
To examine the effects of different
substrata on the MARCO-induced cell shape changes, cells were plated on
coverslips precoated with FCS or with purified ECM components
laminin-1, vitronectin, and fibronectin. As a comparison, cells were
plated on uncoated glass. Transiently transfected CHO cells kept in
suspension overnight were plated in serum-free medium, and cells were
fixed and stained for MARCO and actin filaments 3 h later. This
staining showed that extensions on the MARCO-expressing cells were
longest when cells were plated on uncoated glass and that plating on
different ECM components did not prevent formation of the extensions.
However, they were clearly much shorter on the fibronectin-coated
surface (not shown). On that surface, both untransfected and
MARCO-expressing cells spread extensively.
Dominant-Negative Rac1 Inhibits the Morphological Effects of MARCO
in CHO Cells--
Small GTPases of the Rho family have been shown to
control rearrangement of the actin cytoskeleton (17). Specifically,
activated Cdc42 promotes formation of filopodia, whereas the activated
form of Rac1 induces cell spreading and lamellipodia formation. Since the MARCO-induced cell shape changes somewhat resemble the phenotypic changes caused by activated Cdc42 and Rac1, we investigated whether these GTPases play any role in the MARCO-induced cell spreading/process formation. In order to study this, we co-expressed MARCO and
dominant-negative forms of Cdc42 and Rac1 in CHO cells (Fig.
7). Transiently transfected CHO cells
were taken into suspension 24 h after transfection and were plated
12 h later on FCS-coated coverslips. Cells were fixed after 30 min, since it was known that the MARCO-expressing cells are already
extensively spread at this time point (Fig. 3A). Cells co-expressing MARCO and dominant-negative Cdc42 did not differ morphologically from the MARCO-expressing cells. In contrast, dominant-negative Rac1 markedly inhibited the MARCO-induced cell spreading (not shown). We also examined cells that were plated on
coverslips for 16 h. Again, dominant-negative Cdc42 did not have
inhibitory activity (Fig. 7B), but Rac1V12N17
clearly inhibited the MARCO-induced morphological changes (Fig. 7,
C and D). However, Rac1V12N17 did not
completely abolish the MARCO-induced effects. There were a few large
MARCO-positive cells but no cells with long processes. The
dominant-negative forms of Cdc42 and Rac1 contain a Myc epitope tag at
their N termini, and analysis of total cell lysates by Western blotting
using an anti-Myc antibody demonstrated that these GTPase mutants were
produced at equal levels (Fig. 7F).

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Fig. 7.
Dominant-negative Rac1 inhibits the
MARCO-induced process formation in CHO cells. CHO cells were
transfected with a plasmid encoding MARCO together with either an empty
expression vector, an expression vector encoding a Myc-tagged
dominant-negative form of Cdc42, Cdc42N17, or with an
expression vector encoding a Myc-tagged dominant-negative form of Rac1,
Rac1V12N17. A-D, cells were seeded on glass
coverslips 24 h post-transfection and were fixed and stained for
MARCO and filamentous actin 36 h after transfection. The photos
represent the following transfections: MARCO + empty vector
(A), MARCO + Cdc42N17 (B), and MARCO + Rac1V12N17 (C and D). The
dominant-negative Rac1 significantly inhibits the MARCO-induced process
formation. E and F, cells were grown on tissue
culture plates and were analyzed 40 h post-transfection.
E, cell-surface level of MARCO was examined as described
under "Experimental Procedures" and in Fig. 5. The lanes represent
the following transfections: MARCO + Rac1V12N17 (lane
1), MARCO + Cdc42N17 (lane 2), and MARCO + empty vector (lane 3). F, total cell lysates were
analyzed by Western blotting for the presence of the GTPase mutants
using an anti-Myc antibody. Results from two different transfection
experiments are shown. The lanes represent the following transfections:
MARCO + Rac1V12N17 (lanes 1 and 4),
MARCO + Cdc42N17 (lanes 2 and 5), and
MARCO + empty vector (lanes 3 and 6). The results
shown in E and F demonstrate that different
cotransfectants express similar levels of MARCO on the cell surface
(E) and that both of the GTPase mutants are expressed at
high levels (F).
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One possible explanation for the different phenotypic effects of the
dominant-negative Rac1 and Cdc42 was that MARCO/Rac1V12N17
transfectants expressed MARCO at lower levels than
MARCO/Cdc42N17 transfectants. In order to study if this was
the case, we analyzed the cell-surface levels of MARCO using the
biotinylation/streptavidin-agarose precipitation procedure followed by
Western blotting using an anti-MARCO antibody. In three different
experiments the MARCO/Rac1V12N17 transfectants were found
to have at least as much MARCO on the cell surface as the
MARCO/Cdc42N17 transfectants (Fig. 7E). These
results demonstrate that the phenotypic effects of dominant-negative
Rac1 are not simply due to an inhibitory effect on the cell-surface
expression of MARCO. As in previous studies, we confirmed that the
biotinylation reagent labeled only cell-surface proteins by using an
anti-paxillin antibody (not shown).
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DISCUSSION |
In this study, we have shown that expression of the
macrophage MARCO receptor results in extensive changes in cell
morphology through induction of dendritic plasma membrane processes. Of
the five cell lines tested, COS7 cells were the only exception, since in these cells MARCO expression had minor morphological effect by
leading to the formation of short pine needle-like protrusions. The
reason for this different response is unclear, but it could be related
to the strength of adhesion between cells and serum-coated surface.
Supporting this notion, we found that processes were longest in 293 cells, which are weakly adhering cells, whereas COS7 cells spread well
and adhere tightly to serum-coated glass surface.
Several lines of evidence indicate that the morphological effects are
specific effects of the MARCO receptor itself. By using various
truncated forms of MARCO, it was shown that the part of domain V before
its first cysteine residue is needed for this activity. This conclusion
is based, for example, on the following two observations. First, a
MARCO form completely lacking domain V did not have any effect on cell
morphology, although it was expressed at high levels on the cell
surface. Second, a form that extends only 16 residues to domain V was
found to be about as active as the full-length MARCO. In a previous
study, we showed that when cells are transfected with expression
vectors encoding these two forms, only cells expressing the longer form
bind heat-killed bacteria (7). This result is another indication that
the region of domain V proximal to the collagenous domain is
functionally important in MARCO. Our previous work also suggested that
this region is exposed on the MARCO protein since it was very sensitive for proteolysis when expressed as a fusion protein (7).
It remains to be investigated whether the cytoplasmic domain and/or the
transmembrane domain are also needed for the morphological activity of
MARCO. Thus far, we have tested a truncated form lacking the N-terminal
half of the cytoplasmic domain, and the morphological effects are not
abolished. The use of chimeric proteins is needed to determine the role
of these domains, since MARCO is a type II membrane protein, and the
membrane-proximal region, which contains several positively charged
residues, is very likely important for a correct topological
orientation of MARCO.
MARCO was shown to induce process formation on different surfaces, such
as uncoated glass, laminin-1, and vitronectin. In fact, the processes
were longest when the cells were plated in serum-free media on uncoated
glass. Of the purified ECM components tested, fibronectin was the only
protein that clearly had an inhibitory effect on the MARCO-induced
process formation. Thus, cell adhesion on fibronectin and MARCO
expression seem to have opposite effects on the organization of the
actin cytoskeleton. Indeed, cell adhesion on fibronectin has been
reported to promote rapid actin stress fiber and focal adhesion
formation (18), whereas the present study shows that expression of
MARCO promotes dissolution of stress fibers and focal adhesions.
It is not yet known whether MARCO binds glass or different ECM
proteins. We are currently producing a soluble MARCO for the binding
studies. In this context, it is noteworthy that a protein related to
MARCO, macrophage scavenger receptor A, has been shown to function as
an adhesion receptor, whose ligand is present in fetal calf serum (14).
Interestingly, thioglycollate-elicited peritoneal macrophages from
MSR-A knock-out mice have also been found to spread on glass slower
than wild-type macrophages (13). It is, however, possible that MARCO
exerts its effects by interacting with another cell-surface protein
expressed on the same cell, as was found to be the case for integrin
6A
1 in embryonic stem cells. In these
cells expression of
6A
1 integrin induces
changes in the morphology (filopodia formation) and migration without having to engage with its ECM ligand (19). Instead, the
6A
1-induced motility appears to depend on
the association of
6A
1 with CD81, a
member of the tetraspanin superfamily of cell-surface molecules (19).
Dominant-negative Rac1 partially inhibited the MARCO-induced cell shape
changes in CHO cells. We believe that this is not due to nonspecific
toxic effects since these cells were still capable of functioning
normally, at least if judged by the amount of MARCO they produced and
transported to the cell surface. The partial inhibition suggests that
exogenous dominant-negative Rac1 could not completely inhibit the
activity of the endogenous Rac1 or Rac1-related protein. On the other
hand, it is known that activated Rac1 causes a phenotype that differs
from that induced by MARCO. It promotes cell spreading and formation of
lamellipodia but does not induce formation of long plasma membrane
processes. Thus, the Rac1 pathway is not the only pathway activated by
MARCO. Surprisingly, dominant-negative Rac1 did not have any effect on
MARCO-induced cell shape changes in HeLa cells. We do not know the
reason for this, but it is conceivable that MARCO activates some
Rac1-related protein in these cells and that dominant-negative Rac1 is
not able to interfere with the activity of this protein.
Several protein kinases, such as p21 (Cdc42/Rac)-activated kinase aPAK,
have been shown to play a role either in the formation or dissolution
of stress fibers and focal adhesions (18, 20). Also, lipid kinases have
been shown to be involved in the actin remodeling. For example,
expression of type I phosphatidylinositol-4-phosphate 5-kinase induces
formation of pine needle-like protrusions in COS7 cells (21).
Interestingly, these protrusions resemble those induced by MARCO
expression in COS7 cells. The effects of
phosphatidylinositol-4-phosphate 5-kinase on cell morphology are most
likely due to elevated levels of phosphatidyl 4,5-biphosphate which is
able to uncap actin filament barbed ends, thereby promoting actin
polymerization (22). Recent evidence also suggest that the Rho family
GTPases are able to regulate phosphatidyl 4,5-biphosphate levels. For
example, Rac was shown to induce rapid synthesis of phosphatidyl
4,5-biphosphate in permeabilized platelets (22).
Our efforts to elucidate the signaling pathways activated by MARCO have
been partly hampered by the lack of a population of cells uniformly
expressing MARCO. CHO transfectants appear to lose most of their
MARCO-expressing cells during their propagation. It is possible that
the morphological effects of MARCO hinder cell division, and we wish to
overcome this problem by the use of an inducible expression system.
MARCO is expressed constitutively only in macrophages in lymph nodes
and marginal zone of the spleen (3). The marginal zone macrophages are
thought to be important in the trapping and clearance of microorganisms
from the bloodstream (23). Our results indicate that MARCO has several
functions that contribute to this activity of the marginal zone
macrophages. First of all, as shown previously (3, 7), MARCO is able to
bind bacteria. Second, the morphoregulatory activity resulting in the
formation of large lamellipodia-like structures and long plasma
membrane extensions provides an enlarged cell-surface area. This, in
turn, facilitates efficient trapping of pathogens by cell-surface receptors.