(Received for publication, May 11, 1995; and in revised form, June 7, 1995)
From the
A major protein kinase C substrate, MacMARCKS (F52, MPR), was
examined for its role in phagocytosis. In macrophage-phagocytosing
zymosan particles, MacMARCKS was concentrated around nascent phagosomes
as detected by immunofluorescent microscopy. The effector domain of
MacMARCKS contains the phosphorylation sites, a calmodulin binding
site, as well as a putative actin binding site. Stable J774 macrophage
cell lines constitutively expressing effector domain deletion mutants
of MacMARCKS were generated. When given zymosan particles, these
transfectants showed approximately a 90% reduction in their phagocytic
capacity. The receptor-mediated endocytosis of acetylated low density
lipoproteins, however, was not affected by the mutant. These results
strongly suggest the involvement of MacMARCKS in macrophage
phagocytosis. Macrophage phagocytosis requires efficient signal transduction
from the phagocytic receptors to the actin-based cytoskeleton. Such
signals induce a massive membrane-cytoskeleton
rearrangement(1) , providing the motile force to drive the
internalization of particles. This process is accompanied by the
formation of pseudopodia (3, 4) or membrane
ruffles(5) , and F-actin is essential for the internalization
of ligand-coated particles(4, 6, 7) .
However, the signals that induce actin-cytoskeleton rearrangements and
the mechanism of such a rearrangement are not yet identified. A number
of cytoskeleton-associated proteins, such as talin (8) and
paxillin(9) , have been implicated in this process. Tyrosine
phosphorylation is part of the signal transduction
pathway(s)(10) . Another major signal transduction pathway
involved in macrophage phagocytosis requires protein kinase C
(PKC). Among the
PKC substrates, the MARCKS (myristoylated alanine-rich C kinase
substrate) family has received much attention. MARCKS is a
membrane-associated, actin-binding protein with ubiquitous distribution
(reviewed in (14) and (15) ). Its membrane association
and actin binding activities are regulated both by
Ca Besides MARCKS, macrophages also express another member of the
MARCKS family of PKC substrate,
MacMARCKS/F52/MPR(20, 21, 22) . The synthesis
and phosphorylation of MacMARCKS in macrophages are dramatically
increased when macrophages encounter bacteria(20) , a finding
that suggests the potential role of this protein in macrophage defense
against infection. A comparison of primary structure shows similar
domain organization between MacMARCKS and MARCKS. They both contain a
myristoylated membrane targeting domain that is important for the
membrane association of these
proteins(20, 23, 24) . These two proteins
also share an almost identical basic domain that contains PKC
phosphorylation sites and calmodulin and actin binding
sites(20, 21, 25) . This basic domain is
therefore called the effector domain. Invitro experiments have shown that MacMARCKS binds calmodulin in a
phosphorylation-dependent manner(20, 21) . Such
regulated calmodulin binding activity has been demonstrated with a
similar protein, neuromodulin(26) . However, whether MacMARCKS
interacts with actin is yet to be determined. The in vivo function of MacMARCKS is the focus of the current study. We report
that MacMARCKS protein is highly enriched on phagosomes containing
zymosan particles. We further generated an effector domain deletion
mutant of MacMARCKS (ED). Expression of this mutant showed a dominant
negative effect and dramatically reduced the phagocytic activity of
J774 macrophage cells toward zymosan particles.
As a control, cDNA encoding full-length MacMARCKS was
also inserted into vector p463 and transfected into J774 cells using
the above method. Again, for simplification, FM is used to refer to the
full length MacMARCKS.
To assay the
attachment index of macrophages, the cells were incubated with
25-µg zymosan particles at 12 °C for 45 min. After three washes
in PBS, the cells were fixed, and attached zymosan particles were
counted. To assay the phagocytic activity of macrophages, the cells
were given zymosan particles for 1 h at 37 °C to achieve maximum
uptake (32) and to verify that ED mutant cells did not
phagocytose more slowly than the FM control cells and parental J774
cells. After fixation, the internalized zymosan particles were counted
in samples from three parallel experiments. At least 300 cells were
counted in each sample. The internalized zymosan particles can be
distinguished easily by their refractile appearance in phagocytic
vacuoles(33) .
To visualize MacMARCKS protein, polyclonal
rabbit anti-MacMARCKS antibodies were affinity-purified as
described(31) , and their specificity was demonstrated by three
criteria. First, the antibodies immunoprecipitated a single smear band
from
Figure 1:
A, J774 cells were labeled with
J774 cells on
coverslips were allowed to phagocytose unopsonized zymosan for 15 min.
Using affinity-purified antibodies, MacMARCKS was seen concentrated
beneath the phagosomes at the periphery of the cells (Fig. 1B, MacMARCKS, arrowheads).
Using fluorescein-conjugated phalloidin, F-actin was also seen
concentrated beneath these same phagosomes (Fig. 1B, Actin, arrowhead). The phagosomal association of
MacMARCKS was selective. A number of perinuclear-distributed mature
phagosomes were seen in these cells (Fig. 1B, arrows). However, MacMARCKS and actin were not concentrated
around those phagosomes. F-actin has been shown to associate only with
nascent phagosomes(8) , and our results thus suggest that
MacMARCKS-decorated phagosomes are also nascent phagosomes. This
phagosomal association of MacMARCKS prompted us to further examine
whether it is involved in macrophage phagocytosis.
A
cDNA-encoding effector domain deletion mutant of MacMARCKS (ED) (Fig. 2A) was generated and introduced into J774 cells
as described under ``Experimental Procedures.'' J774 cells
expressing the ED mutant were selected by subjecting the cells to
selection with 800 µg/ml G418. Three stable ED cell lines were
randomly picked and expended as ED1, ED2, and ED3. Two-dimensional
IEF-SDS-PAGE was used to separate ED mutant (pI 3.84) from endogenous
MacMARCKS (pI 4.37), and the expression level of these proteins was
determined using immunoblot with anti-MacMARCKS antibodies. The ratios
of endogenous MacMARCKS to ED mutant were 1:1.06 for ED1, 1:0.81 for
ED2, and 1:0.96 for ED3 (Fig. 2C).
Figure 2:
A, a diagram illustrating the full-length
MacMARCKS (FM) and effector domain deletion mutant of
MacMARCKS (ED). B, lysates from FM control cells and
parental J774 cells were resolved by SDS-PAGE and transferred to PVDF
membrane. Expression level of MacMARCKS protein was determined by
immunoblotting with rabbit anti-MacMARCKS antiserum. C, lysate
from ED mutant cells was subjected to two-dimensional IEF-SDS-PAGE and
transferred to PVDF membrane. Endogenous MacMARCKS (Endo) and
ED mutant proteins were detected by immunoblotting with anti-MacMARCKS
antiserum.
As a control,
cDNA-encoding full-length MacMARCKS (FM) was also transfected into J774
cells. Because FM is identical with the endogenous MacMARCKS in J774
cells, we could not determine their expression separately. However, the
total amount of MacMARCKS in FM control cells was about twice as much
as in parental J774 cells (Fig. 2B), suggesting that FM
was expressed to the same level as endogenous MacMARCKS.
Figure 3:
Cells on coverslips were incubated with
unopsonized zymosan at 12 °C for 45 min followed by 1 h at 37
°C. The samples were then fixed and photographed as shown (lower panel). The upper panel shows the cells before
they were given zymosan. The binding activities were measured by
incubating cells with zymosan at 12 °C for 45 min without
proceeding to 37 °C (images not shown). The attached and ingested
particles were counted in samples from three parallel experiments (n = 3). At least 300 cells were counted in each
sample. The phagocytic/attachment indexes (number of ingested/attached
zymosan particles per 100 cells) are shown under corresponding
images.
The phagocytic index (number of ingested particles
per 100 cells) was obtained by counting the ingested
particles(33) . The ingested zymosan particles appear
refractile, while those outside the cells are dark and can be counted
easily. We observed that parental J774 cells phagocytosed unopsonized
zymosan with a phagocytic index of 325 ± 58 (n =
3) (Fig. 3). A similar phagocytic index of unopsonized zymosan
by J774 cells had been reported earlier(33) . However, the ED
mutant cells showed a 10 To minimize the possible
miscounting due to human error, a modified flow cytometry method
described by Liao et al.(34) was used to measure the
phagocytosis of zymosan particles. In brief, fluorescein-conjugated
zymosan particles were given to ED mutant cells, FM control cells, and
parental J774 cells. After 1 h, cell surface-bound zymosan particles
were removed by extensive trypsin digestion, and cell-associated
fluorescence was measured using flow cytometry. Microscopy showed that
the cells appeared as a dark shadow around bright fluorescent zymosan
particles (Fig. 4, upper panel). Most FM control cells
and parental J774 cells contained more than one fluorescent zymosan
particle, whereas most of the ED mutant cells remained dark (no zymosan
inside) or contained only one particle. Analyzed with flow cytometer,
parental J774 cells and FM cells showed fluorescence with relative
average intensities of 29.9 and 37.1, respectively (Fig. 4, lower panel). With ED mutant cells, only a small percentage of
cells showed fluorescence with relative average intensities of only
1.58 for ED1, 1.15 for ED2, and 1.32 for ED3.
Figure 4:
Cells were allowed to ingest
fluorescein-conjugated unopsonized zymosan for 1 h. The bound zymosan
particles were released from the cell surface by extensive trypsin
digestion. A fraction of the cells was examined under a microscope (upper panel). With both visible and fluorescent light sources
on, the cells appeared as a dark shadow and zymosan as a bright spot (as the circle and arrow indicate). The lower panel shows the results of the flow
cytometry analysis of these samples.
Both ED mutant cells
and FM control cells showed similar binding capacity toward zymosan
particles (Fig. 3) as their parental J774 cells. In addition,
the surface expression of Fc
Figure 5:
Cells were allowed to endocytose AcLDL for
30 min as described under ``Experimental Procedures.'' The
cells were then fixed in 10% formalin and subjected to flow cytometry
analysis.
The results of our study suggest that MacMARCKS plays an
important role in macrophage phagocytosis. It has been demonstrated
previously that the activation of PKC strongly enhances
phagocytosis(11, 13) . As a major PKC substrate,
MacMARCKS is a logical candidate to be examined for its potential
involvement in this process. The phagosomal association of MacMARCKS
initially suggested that MacMARCKS might be involved in phagocytosis.
This enrichment of MacMARCKS appears only on nascent phagosomes,
whereas perinuclear-distributed mature phagolysosomes are devoid of
MacMARCKS (Fig. 1B). Such a distribution of MacMARCKS
very much resembles the selective enrichment of actin on nascent
phagosomes(8) . Our data indeed showed that MacMARCKS and actin
are colocalized on the same phagosomes (Fig. 1B).
F-actin was demonstrated to be essential for macrophage phagocytosis
(reviewed in (1) ). A number of other cytoskeleton-associated
proteins, such as talin (8) and paxillin (9) , showed
similar phagosomal association as well. Since MacMARCKS protein
contains a putative actin binding domain, its phagosomal association
and colocalization with actin encouraged a more detailed investigation
into its potential role in phagocytosis. A more direct suggestion of
the role of MacMARCKS in phagocytosis comes from the expression of an
effector domain deleted mutant of MacMARCKS (ED). The effector domain
is a basic domain that contains PKC phosphorylation sites(20) ,
a calmodulin binding site(25) , and a putative actin binding
site(17) . Deletion of this domain abolished PKC-mediated
phosphorylation and calmodulin binding. MARCKS protein has been
shown to bind and cross-link actin filaments (17) and possibly
regulates membrane-cytoskeleton interaction(14) . MacMARCKS
contains an almost identical actin binding domain (within the effector
domain) as MARCKS, and deletion of this domain might therefore affect
actin-based cytoskeleton and thereby causes a defect in phagocytosis.
That the expression of ED mutant did not affect receptor-mediated
endocytosis of AcLDL seems to favor such a hypothesis, because the main
difference between phagocytosis and endocytosis is their dependence on
actin-based cytoskeleton. Phagocytosis depends on the actin-based
cytoskeleton, whereas endocytosis does not. In addition, upon adding
zymosan, ED mutant cells failed to spread out as normal J774 cells do,
thus appearing smaller. Such spreading is important for macrophage
phagocytosis, and the actin-based cytoskeleton is primarily responsible
for such a spreading process(5) . The difference in spreading
was more dramatic when Salmonella was added to these
cells.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)Activation of PKC with phorbol esters
profoundly enhances phagocytosis mediated both by C3 receptors (11) and Fc receptors(12, 13) . However,
little is known about the events after PKC activation, nor is it known
how the PKC signal is transduced to the cytoskeleton to initiate the
internalization of particles. One approach to these questions is to
investigate the role of PKC substrates in these processes.
/calmodulin and by PKC-mediated
phosphorylation(16, 17) . As an actin-binding protein,
it is involved in a number of actin-cytoskeleton-related cellular
activities, such as motility (18) and secretion(19) .
Materials
The J774.a1 macrophage cell line was
obtained from ATCC and cultured in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum. Zymosan A
(Catalogue No. Z4250) and fluorescein-conjugated phalloidin (Catalogue
No. P5282) were purchased from Sigma. Fluorescein-conjugated zymosan
particles (Catalogue No. Z2841), Dil-conjugated low density lipoprotein
(LDL) (Catalogue No. L3484), and Texas Red-conjugated goat
anti-rabbit IgG (Catalogue No. T2767) were purchased from Molecular
Probes. Rabbit anti-MacMARCKS antibody was originally generated in Dr.
A. Aderem's laboratory (Rockefeller University). Anti-Fc
(2.4G2) and anti-CD11b (M1/70) were kindly provided by Dr. R. Steinman
(Rockefeller University).
Expression of Mutant MacMARCKS in J774 Macrophage Cell
Line
To obtain a cDNA construct encoding the effector
domain-deleted MacMARCKS, two fragments flanking this domain were
generated using polymerase chain reaction. One fragment encoded amino
acids 1-85, and another encoded amino acids 109-200 of
MacMARCKS. The two fragments were then ligated through the MluI sites. The mutant DNA was then inserted into a eukaryotic
expression vector (p463) (27) that was kindly provided by Dr.
M. Nussenzweig (Rockefeller University). The final DNA construct was
then electroporated into J774 cells as described(28) . The
transfected cells were subjected to G418 (800 µg/ml) selection 48 h
later. After selection, positive clones were randomly picked, and the
expression was examined by two-dimensional isoelectrofocusing and
SDS-polyacrylamide gel electrophoresis
(IEF-SDS-PAGE)(29, 30) . Then the proteins were
transferred to PVDF membrane (Millipore Catalogue No. IPVH304F0)
followed by immunoblotting with anti-MacMARCKS antibodies. The
anti-MacMARCKS antiserum recognizes both mutant protein and the wild
type protein with the same efficiency (data not shown). For
simplification, ED is used to refer the effector domain deletion mutant
of MacMARCKS.Uptake of Zymosan Particles and
Immunofluorescence
Parental J774 cells, FM control cells, and ED
mutant cells were cultured on coverslips overnight. Zymosan particles
were added to the culture at a final concentration of 25 µg/ml. The
cells were allowed to bind zymosan particles for 45 min at 12 °C,
and ingestion was allowed to proceed for 15 min at 37 °C. After
fixation with 10% formalin in phosphate-buffered saline (PBS) for 15
min at 4 °C, the cells were permeabilized with acetone at -20
°C for 5 min. MacMARCKS was visualized with affinity-purified
rabbit anti-MacMARCKS antibody followed by Texas
Red-conjugated goat anti-rabbit IgG as
described(31) . To visualize F-actin at the same time,
fluorescein-conjugated phalloidin was added to cells at a concentration
of 10 ng/µl together with secondary antibody.
Flow Cytometry Analysis of Phagocytosis
This assay
was adopted from Liao et al.(34) . J774 cells cultured
in 60-mm dishes were allowed to ingest fluorescein-conjugated zymosan
particles for 1 h at 37 °C. Most of the excess zymosan particles
was removed by three washes in PBS. The cells were then treated with
trypsin/EDTA for 1.5 h at 37 °C to free any bound zymosan from the
cell surface and cells from the plate. After fixation with 10%
formalin, the cells were analyzed using flow cytometry at 488 nm. Free
zymosan particles were distinguished from cells containing zymosan by
their different light scattering behavior.Assay of the Endocytosis of AcLDL
This assay was
adopted from Suzuki et al.(35) . J774 cells in 60-mm
dishes were starved in serum-free Dulbecco's modified
Eagle's medium for 30 min before adding acetylated LDL (AcLDL).
Dil-conjugated AcLDL was then added to the culture to a final
concentration of 10 µg/ml. The cells were cultured for an
additional 30 min before being washed in PBS twice and fixed in 10%
formalin. The uptakes of Dil-AcLDL were determined by flow cytometry.
MacMARCKS Associates with Phagosomes in J774 Macrophage
Cells
We first examined the subcellular localization of
MacMARCKS during uptake of zymosan particles. The J774 cell line was
used in this study for two reasons. First, J774 cells express
endogenous MacMARCKS. Second, they have been used in previous studies
of the phagocytosis of zymosan(33) . Unopsonized zymosan
particles are phagocytosed by macrophages through multiple receptors
such as complement and mannose receptors, as well as through potential
unidentified receptors(33, 36) . Thus, these particles
are particularly suitable for simultaneously monitoring phagocytosis
via multiple receptors.P
-labeled total J774 cell lysate (Fig. 1A). Second, they recognized a single protein
(doublet) on a Western blot of total J774 cell lysate resolved on
SDS-PAGE (Fig. 1A). MacMARCKS has been shown to appear
as a smear band or doublet on SDS-PAGE(20, 37) .
Third, the affinity-purified antibodies did not stain free zymosan
particles outside the cells (Fig. 1B).
P
, and MacMARCKS was specifically
immunoprecipitated (IP) with affinity-purified rabbit
polyclonal anti-MacMARCKS antibodies as described. The
affinity-purified antibodies also specifically recognized MacMARCKS
(doublet) on immunoblot (Blot) of total cell lysate resolved
by SDS-PAGE. B, unopsonized zymosan was added to J774 cells on
a coverslip at 12 °C for 45 min. The cells were moved to 37 °C
to start phagocytosis. After 15 min, the cells were fixed and stained
for both MacMARCKS and actin as described under ``Experimental
Procedures.'' The arrowheads indicate the phagosomes that
were decorated with MacMARCKS and actin. The arrows indicate
mature phagosomes that lacked staining.
Expression of MacMARCKS Mutant Proteins in J774
Macrophage Cells
The effector domain of MacMARCKS is a basic
domain that contains the PKC phosphorylation sites(20) ,
calmodulin binding site (25) , and a putative actin binding
site(17) . Thus, deleting this domain is likely to generate a
nonfunctional MacMARCKS that might serve as a dominant negative mutant in vivo to compete with endogenous wild type MacMARCKS.
J774 Cells Expressing the ED Mutant of MacMARCKS Are
Impaired in the Phagocytosis of Zymosan Particles
In the resting
state, ED mutant cells, FM control cells, and their parental J774 cells
appeared round and refractile, with no obvious difference in their
appearance (Fig. 3, upper panel). Once the zymosan
particles were added, FM control cells and parental J774 cells quickly
spread out and started to phagocytose. However, little phagocytosis
occurred in ED mutant cells, despite the apparent attachment of zymosan
particles to the surface of these cells (Fig. 3, lower
panel). During the 1-h incubation with zymosan, the ED mutant
cells remained round and refractile and therefore appeared smaller than
those control cells that spread out and phagocytosed zymosan particles (Fig. 3).
reduction in phagocytic capacity toward
unopsonized zymosan. The phagocytic indexes were 16 ± 8 (n = 3) for ED1, 23 ± 5 (n = 3) for
ED2, and 19 ± 9 (n = 3) for ED3 (Fig. 3),
whereas FM control cells showed approximately same phagocytic index of
349 ±48 (n = 3) as parental J774 cells (Fig. 3). This observation suggests that the reduction of
phagocytosis indeed resulted from the expression of ED mutant, not from
the integration of transfected foreign DNA.
and complement receptor (CD11b) were
similar to parental J774 cells as well (data not shown). Therefore, we
conclude that the ED mutant blocked phagocytosis at the internalization
stage.
Receptor-mediated Endocytosis of AcLDL Is Not Affected by
Mutant MacMARCKS
We examined whether ED mutant affected general
membrane trafficking as measured by receptor-mediated endocytosis. The
uptake of AcLDL is mediated both by the LDL receptor and macrophage
scavenger receptor (38) through receptor-mediated phagocytosis.
It was therefore used to measure the effect of MacMARCKS mutant on
endocytosis. After endocytosing Texas Red-conjugated AcLDL
for 30 min, the cells were washed, fixed, and subjected to flow
cytometry analysis as described(35) . All ED mutant cells
showed similar amounts of AcLDL uptake compared to FM control cells and
parental J774 cells (Fig. 5). This similarity in the uptake by
all the cell lines was confirmed by fluorescence microscopy (data not
shown). Thus, the defect in phagocytosis caused by MacMARCKS mutant did
not seem to affect receptor-mediated AcLDL uptake.
(
)Our
data showed that all three randomly selected ED mutant cell lines
expressed approximately the same amount of mutant protein with a ratio
to endogenous MacMARCKS of about 1:1. All three ED mutant cell lines
showed approximately a 90% reduction in phagocytosis of zymosan
particles compared with FM control cells. This result strongly suggests
that MacMARCKS plays a role in macrophage phagocytosis and that the ED
mutant acts as a ``dominant negative'' suppressor of
MacMARCKS functions. In general, phagocytosis consists of two steps:
binding and internalization of particles. Our data showed that there
were as many bound zymosan particles on the mutant cells as on the
control cells, implying a normal ligand binding of ED mutant cells (Fig. 3). Therefore, the ED mutant specifically blocks
phagocytosis at the internalization step.
Salmonella induced the formation of massive
membrane ruffles in macrophages as part of its entry mechanism, and
this process is actin-dependent(39) . We observed that Salmonella induced extension of lamellipodia in control cells
as soon as 30 s, whereas no change was observed in ED mutant
cells.
Therefore, we speculate that the MacMARCKS may be
involved in regulating membrane-cytoskeleton rearrangement during
macrophage phagocytosis.
We thank Dr. A. Aderem for providing us with MacMARCKS
cDNA and antibody against MacMARCKS protein. We also thank Dr. M.
Nussenzweig for providing expressing vector and Dr. R. Steinman for
providing antibodies against Fc and CD11b. We appreciate Dr. M.
Doctor and Dr. G. Majumdar for the flow cytometry measurement. We thank
Dr. D. Armbruster, an author's editor, for help in editing this
manuscript. Most of all, our thanks go to Dr. R. Steinman, Dr. S. D.
Wright, and Dr. Y. Liu for their fruitful discussion and advice, as
well as their comments on the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.