* Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, Ospedale S. Luigi, 10043 Orbassano, Italy; Department
of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; § Max-Planck-Institut für
Biochemie, 82152 Martinsried, Germany; and
Leukosite, Inc., Cambridge, Massachusetts 02142
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Abstract |
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Chemotaxis and phagocytosis are basically
similar in cells of the immune system and in Dictyostelium amebae. Deletion of the unique G protein subunit in D. discoideum impaired phagocytosis but had
little effect on fluid-phase endocytosis, cytokinesis, or
random motility. Constitutive expression of wild-type
subunit restored phagocytosis and normal development. Chemoattractants released by cells or bacteria
trigger typical transient actin polymerization responses
in wild-type cells. In
subunit-null cells, and in a series
of
subunit point mutants, these responses were impaired to a degree that correlated with the defect in
phagocytosis. Image analysis of green fluorescent
protein-actin transfected cells showed that
subunit-
null cells were defective in reshaping the actin network
into a phagocytic cup, and eventually a phagosome, in
response to particle attachment. Our results indicate
that signaling through heterotrimeric G proteins is required for regulating the actin cytoskeleton during
phagocytic uptake, as previously shown for chemotaxis.
Inhibitors of phospholipase C and intracellular Ca2+
mobilization inhibited phagocytosis, suggesting the possible involvement of these effectors in the process.
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Introduction |
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IN chemotaxis, ameboid cells, like leukocytes and Dictyostelium cells, respond directionally to chemical gradients; in phagocytosis, they bind and engulf foreign organisms or apoptotic cells (Devreotes and Zigmond, 1988;
Rabinovitch, 1995
). Chemotaxis and phagocytosis seem to
be closely related, suggesting that the underlying signal transduction events and cytoskeletal responses have evolved in parallel (Metchnikoff, 1968
). In the simple eukaryote Dictyostelium discoideum, and in ameboid cells of the immune
systems of animals, chemotactic and phagocytic stimuli
elicit a remarkably similar spectrum of behavioral events
and biochemical reactions (Devreotes and Zigmond, 1988
;
Greenberg, 1995
). Foremost among these is the polymerization of actin into filaments that support the extension of
pseudopods and the formation of phagocytic cups (McRobbie and Newell, 1983
; Greenberg, 1995
; Zigmond, 1996
).
Chemotaxis and phagocytosis involve both G protein-
coupled and tyrosine kinase-linked signal transduction
pathways. Many chemoattractants interact with serpentine
receptors, such as cAMP receptors in Dictyostelium and
chemokine receptors in leukocytes (Parent and Devreotes,
1996; Murphy, 1996
). Agonists for receptor tyrosine kinases trigger actin polymerization and act as chemoattractants (Kundra et al., 1994
). With regard to phagocytosis,
bound particles activate protein tyrosine kinases, such as
syk, leading to actin polymerization and rearrangement,
possibly through involvement of the small G protein Rho
(Greenberg et al., 1994
, 1996
; Indik et al., 1995
; Hackam
et al., 1997
). Heterotrimeric G proteins have been involved in chemotactic activation of macrophages, which leads to phagocytosis (Thelen and Wirthmueller, 1994
),
and in phagosome-endosome fusion (Desjardins et al., 1994
;
Béron et al., 1995; Allen and Aderem, 1996
), whereas no
compelling evidence has been reported so far for a role of
G proteins in phagocytic uptake.
D. discoideum amebae contain a single G protein subunit; its deletion creates cells that lack functional G proteins (Lilly et al., 1993
; Wu et al., 1995
). These mutants are
severely defective in chemotaxis, aggregation, and development. When plated on bacterial lawns, they form smooth
plaques consisting of monolayers of undifferentiated cells.
These plaques are much smaller than those of wild type (Wu et al., 1995
). We report here that this slow growth reflects a severe defect in phagocytosis, which is primarily
due to a failure in organizing the actin meshwork into a
phagocytic cup. We have also used several inhibitors of G
protein-linked effectors, such as protein kinase A (PKA),1
protein kinase C (PKC), and phospholipase C (PLC) tyrosine kinases, phosphoinositide 3-kinase (PI3) and phosphoinositide 4-kinase (PI4), as well as signal transduction
mutants, to dissect the downstream components involved
in phagocytosis. We suggest that a signaling pathway mediated by heterotrimeric G proteins, possibly involving
PLC activation and mobilization of Ca2+ ions, is necessary
to regulate the actin cytoskeleton during phagocytosis.
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Materials and Methods |
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Cell Cultures
The following D. discoideum strains were used throughout: wild-type
strains AX2 and AX3; subunit-null strains LW6 and LW14, or
subunit
point mutants S1, S2, S3, I1, I2; "rescue" mutants LW18 and LW20. Transformation, selection, and developmental phenotype of the mutants have
been described by Lilly et al. (1993)
and Wu et al. (1995)
.
For axenic growth, cells were cultured in AX2 medium (Watts and
Ashwort, 1972) under shaking at 150 rpm and 23°C. G418 at a concentration of 20 µg/ml was added to cultures of LW14, point mutants, LW18,
and LW20. For starvation, cells were washed twice in 0.017 M Na-K Soerensen phosphate buffer, pH 6.0, and then shaken in the same buffer at a
concentration of 107 per ml.
Phagocytosis Microassays
Phagocytosis was measured by a modification of a previously described
assay (Bozzaro et al., 1987), using cells starved for 30 min. Cells were vortexed and mixed with cold FITC-labeled Escherichia coli B/r, or in some
cases Salmonella minnesota R595, at a final concentration of 4 × 106 cells
and 5 × 109 bacteria per ml, respectively, in a final volume of 0.1 ml in 5-ml polystyrene tubes. The tubes were placed on a rack mounted on a
vortex equipped with speed control (model Reax 2000; Heidolph, Kelheim, Germany), and shaken at 200 vibrations per minute. At various
time points, cells were washed free of unbound bacteria by dilution with 5 ml
of cold 0.050 M Na-phosphate buffer, pH 9.2, and centrifugation at 110 g
for 3 min. After additional washing, the cell pellet was resuspended in 1 ml
of Na-phosphate buffer containing 0.2% Triton X-100 and then lysed for
30 min. Fluorescence in cell lysates was measured in a spectrofluorimeter
(model SFM25; Kontron, Schliereu, Switzerland) at an excitation wavelength of 470 nm and emission of 520 nm. To determine the number of
bacteria, a calibration curve was made by serial dilutions of FITC-labeled bacteria lysed with 1% SDS for 2 min at 90°C (Vogel, 1987
).
For measuring phagocytosis of latex beads, cells at a final concentration of 4 × 106 per ml were mixed with a suspension of 1-µm-diam standard Dow latex beads (Serva, Heidelberg, Germany) at a final OD of 1.5, in a total volume of 0.1 ml Soerensen phosphate buffer. At the end of incubation, cells were washed free of beads by dilution and washing as above. The cell pellet was resuspended in 1-ml Soerensen phosphate buffer containing Triton X-100, lysed, and then the OD of the suspension was measured. The percentage of engulfed beads was determined by reference to a serial dilution curve of latex beads.
Phagocytosis of yeasts was studied under similar conditions, by mixing
2 × 106 cells per ml with a fivefold excess of TRITC-labeled, heat-killed yeast particles. At the end of the incubation, 0.9 ml of cold Soerensen
phosphate buffer was added to each sample, followed by the addition of
0.1 ml of trypan blue (2 mg/ml in 0.02 M citrate buffer containing 0.15 M
NaCl) for quenching the fluorescence of noningested particles (Hed, 1986;
Maniak et al., 1995
). After 15 min of incubation under shaking, excess trypan blue was removed by centrifugation and washing, and then fluorescence determined at 544/577 nm in the spectrofluorimeter.
FITC bacteria were freshly prepared by incubating the bacteria at a final concentration of 1010/ml with 0.1 mg/ml FITC (Sigma Chemical Co.,
St. Louis, MO) as described (Vogel, 1987), except that the incubation time
and temperature were 90 min and 23°C. TRITC yeasts were prepared by
labeling 2 × 1010 heat-killed yeast particles in 20 ml of 0.05 M Na2HPO4,
pH 9.2, containing 2 mg/ml TRITC (Sigma Chemical Co.) and incubating
at 37°C for 30 min under shaking. After extensive washing, the TRITC yeasts were aliquoted in 0.017 M Na-K Soerensen phosphate buffer, pH
6.0, and stored at
20°C until use.
Growth Assays on Bacteria or Axenic Medium
For measuring growth on shaken bacteria, cells were inoculated at 5 × 104 per ml in a suspension of 1010 E. coli B/r per ml, and cell number was counted every 3 h for a total of 36 h. Colony growth on bacterial lawns was assessed by inoculating cells with a toothpick on a lawn of E.coli B/2 cultivated on nutrient agar and then measuring the diameter of the plaque every 12 h for a total of 48 h.
For growth in axenic medium, growing cells were diluted to 105 per ml in fresh axenic medium and counted every 8 h for a total of 56 h.
Fluid-Phase Endocytosis Assay
Fluid-phase endocytosis was measured as described by Aubry et al. (1993)
using FITC-labeled dextran (70,000; Sigma Chemical Co.) as a marker, except that the final volume was 0.1 ml per time point.
Actin Polymerization Assay
Chemoattractant-induced F-actin formation was measured as described
(Hall et al., 1988). Briefly, cells were shaken at 2 × 107/ml in Soerensen
phosphate buffer. At various time points after addition of 10
6 M cAMP
or supernatant from bacterial growth medium, 0.1-ml cell suspension was
transferred to 0.9-ml stop solution containing 3.7% formaldehyde, 0.1%
Triton X-100, 0.25 µM TRITC-phalloidin in 20 mM K-PO4, 10 mM
Pipes, 5 mM EGTA, 2 mM MgCl2, pH 6.8. After staining for 1 h, samples
were centrifuged for 5 min in a microfuge (model Biofuge A; Heraeus,
Hanau, Germany), pellets were extracted with 1 ml methanol for 20 h, and
then fluorescence (540/575 nm) was read in a spectrofluorimeter. Bacteria-conditioned medium was prepared by clarification of a stationary
phase culture of Klebsiella aerogenes grown at 22°C. Medium was used as
a stimulus at a dilution of 1:100.
Transfection with Green Fluorescent Protein-Actin
Fusion of the coding region of the D. discoideum actin with the red-shifted
green fluorescent protein (GFP) S65T mutant has been described (Westphal et al., 1997). Two vectors for expression of the GFP-actin fusion
were constructed, one in which the fusion product was inserted in the
pDEX H vector containing the resistance to G418 (Faix et al., 1992
), and a
second based on the pBsr2 vector containing the resistance to blasticidin
(Sutoh, 1993
). The first vector was used for transforming AX2, AX3, and
LW6, and the second vector for LW20, which is already resistant to G418.
Transformation was done by electroporation and transformants were selected on plates in nutrient medium containing 20 µg/ml G418 or 10 µg/ml blasticidin. Individual clones were used in the experiments.
Light and Fluorescence Microscopy of Living and Fixed Cells
Phagocytosis of yeast particles by single cells incubated on Petriperm dishes (no. 26136906; Heraeus) was followed for 2 h by time-lapse videomicroscopy using an Axiovert microscope (model 35; Carl Zeiss, Inc., Oberkochen, Germany) equipped with a 100× Neofluar objective and a Zeiss charge-coupled device videocamera (model ZVS-47DE) connected to a Panasonic videorecorder (model 6050; Osaka, Japan). Images were recorded at an interval of 0.5 s.
Cells incubated with TRITC-labeled yeast particles on glass coverslips
were fixed with picric acid and formaldehyde and postfixed with 70% ethanol as described by Humbel and Biegelmann (1992). The fixed cells were
labeled with 0.5 µg/ml FITC-phalloidin (Sigma Chemical Co.) for 30 min
at room temperature, and confocal sections taken on an inverted Zeiss microscope (model LSM 410) with a 100× Neofluar 1.3 oil-immersion objective. For excitation, the 488-nm band of an argon-ion laser line was used
and its emission collected with a 510-525-nm bandpass filter for FITC and
a longpass filter of 570 nm for TRITC.
Confocal serial images of living cells expressing GFP-actin and incubated with TRITC-labeled yeast particles on glass coverslips were obtained by scanning at intervals of 34 s, using a 100× Plan-Neofluar objective. To avoid damaging the cells upon intense light exposure, the laser intensity was attenuated to 1/100 of its maximal power. Since the yeast particles were strongly labeled, the small fraction of their emission obtained by excitation as 448 nm was sufficient to record their fluorescence together with that of GFP. The two contributions to the emission signal could be separated by using a bandpass filter of 510-525 nm for GFP and a longpass filter of 570 nm for TRITC. Phase contrast was recorded simultaneously. The images from the green channel in Fig. 7 were pseudocolored with a color code ranging from cyan to yellow, and then superimposed to the red channel using Applied Visualization Systems (Waltham, PA) software.
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Nuclei of cells growing in axenic medium were labeled with 4',6-diamidino-2-phenylindole (Sigma Chemical Co.) after cell fixation and postfixation as above. The number of nuclei per cell was determined on a Leitz fluorescence microscope (model Laborlox D; Wetzlar, Germany) using a LH102Z filter.
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Results |
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Phagocytosis and Growth on Bacteria Are Impaired in Subunit-null cells and Restored in Rescue Mutants
Phagocytosis was assessed in wild-type and in subunit-
null (g
) cells by measuring the uptake rate of fluorescently-labeled E. coli B/r or latex beads. Shaken wild-type
cells engulfed ~300 bacteria per hour under our conditions, a value in the range of previously published data
(Gerisch, 1959
; Vogel et al., 1980
; Bozzaro et al., 1987
). In
cells lacking the G protein
subunit, the initial uptake rate
of bacteria or latex beads was decreased to 20 or 40-60%
of the wild-type rate, respectively (Fig. 1, A and B). Constitutive expression of wild-type
subunit cDNA in g
cells
restored nearly normal rates of phagocytosis (Fig. 1, A and
B). Replacing E. coli B/r with Salmonella minnesota R595, which is known to adhere stronger to the surface of Dictyostelium cells (Malchow et al., 1967
; Bozzaro and Gerisch,
1978
; Niewöhner et al., 1997
), did not significantly alter the
uptake rates of
subunit-null mutants, rescue mutants, or
wild-type cells (data not shown). It is open to what extent
uptake of different types of bacteria, or of inert particles,
such as latex beads, is mediated by different receptors in
Dictyostelium, though some genetic evidence favors the hypothesis that both specific, lectin-type receptors and nonspecific receptors contribute to phagocytosis (Vogel et al.,
1980
; Bozzaro and Ponte, 1995
; Chia, 1996
). The defect in phagocytosis of g
cells seems, however, to be independent of particle binding to a particular receptor, since the
uptake of E.coli, S. minnesota R595 as well as latex beads
was inhibited in the mutant and restored in rescue cells.
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We measured growth of gand rescue cells both in
shaken suspension or on a lawn of E. coli. Consistent with
the phagocytosis defect, the doubling time on shaken suspensions was increased from 3 to 10 h in g
cells and the
rate of plaque expansion on bacterial lawns was decreased
from 0.6 to 0.1 mm/h. Normal rates of growth under both conditions were found for the rescue cells (Table I).
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We also examined individual clones expressing different
randomly mutagenized subunit cDNAs. None of the mutant proteins rescued the developmental defects of the
g
cells; the individual clones all formed smooth plaques.
Rates of phagocytosis and doubling times in suspensions
of E. coli closely correlated with their plaque sizes (Table
I), presumably depending on the severity of the mutation.
The diameters of these plaques were small like those of
the g
cells, intermediate, or large like those of rescued
mutants (Table I).
Pinocytosis, Cytokinesis, and Motility Are Normal in Subunit-null Cells
Although the g cells were severely defective in chemotaxis and phagocytosis, they were competent in cytokinesis, pinocytosis, and random motility. Pinocytosis was
directly monitored by measuring uptake of FITC-conjugated dextran. The initial rates of uptake were essentially
identical in mutant, wild-type, and rescued cells (Fig. 1 C).
Doubling times and cytokinesis of g
and wild-type cells
in liquid media were also similar (Table I). We also monitored the random motility of undifferentiated cells on hydrophilic Petriperm surfaces and found that it was 3.9 (±1.6 SD) and 3.6 (±1.8 SD) µm/min for wild-type and
g
cells, respectively. Finally, no differences in cell adhesion to polystyrene surface were detected between undifferentiated g
and control cells (data not shown). Taken
together, these results strongly indicate that the small
plaque size of g
cells on bacterial lawns is solely due to a
defect in phagocytosis and not to additional defects in cytokinesis or motility.
The Phagocytosis Defect Is Correlated with Impaired F-actin Formation in Response to Chemoattractants
Chemoattractants induce rapid, transient increases in cellular levels of filamentous actin (McRobbie and Newell,
1983; Hall et al., 1988
; Tardif et al., 1995
). As previously
reported, g
cells are not attracted to folic acid, cAMP, or
bacteria-conditioned medium containing a broad range of
chemoattractants. Overexpression of the surface receptor
cAR1 does not restore sensitivity to cAMP (Lilly et al.,
1993
). Consistent with these results, the actin polymerization responses triggered by these compounds are undetectable in g
cells (Fig. 2). We also examined the series
of
subunit point mutants for actin polymerization elicited by bacteria-conditioned medium or cAMP. The magnitudes of these responses correlated with the rates of phagocytosis as well as growth rates on shaken suspensions or
lawns of bacteria (Table I; Fig. 3 and data not shown).
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The Subunit-null Cells Are Defective in Reshaping
the Actin Cytoskeleton into a Phagocytic Cup
The correlation between actin polymerization and bacteria uptake prompted us to examine actin localization during phagocytosis in g cells. Labeling with phalloidin or
anti-actin mAbs failed to reveal major differences in actin
distribution between g
, rescue, or wild-type cells incubated with bacteria (data not shown). Consistent with the
reduced uptake rates of particles, g
cells contained
fewer bacteria in their cytoplasm, some of which were surrounded by actin, probably representing freshly ingested phagosomes.
To recognize details, we replaced bacteria by the larger
heat-killed yeasts. Like uptake of bacteria, engulfment of
suspended yeast was strongly inhibited in the mutant (Fig.
4). Yeast uptake by cells incubated on a hydrophilic Petriperm surface was recorded. In parallel, cells were fixed
at different times and labeled with FITC-phalloidin. A serial image sequence of individual wild-type or g cells incubated with yeast particles is shown in Fig. 5. Both cell
types extended leading edges, and bound yeast particles, which they came in contact with, without detectable differences. Binding either led to formation of a phagocytic cup
around a particle or was followed by detachment. Similarly, when a phagocytic cup was formed, the cup could
progress up to engulfment of the particle or a new leading
front was formed, with subsequent cell detachment from
the yeast particle. This behavior is consistent with a zipper model of phagocytosis (Swanson and Baer, 1995
), which
has been shown to apply to Dictyostelium (Maniak et al.,
1995
). All these events occurred in wild-type and g
cells,
but with significantly different probabilities. As shown in
Table II, the number of yeast-cell attachment events leading to successful phagocytic cup formation and engulfment
was strongly reduced in g
cells. The mutant was defective
in both steps, phagocytic cup, and phagosome formation,
with an inhibition of 35 and 80%, respectively, compared
with wild-type cells. However, no qualitative differences in
phalloidin labeling between wild-type and mutant cells
could be detected in fixed parallel samples; the cell cortex
of both control and g
cells was decorated with actin, as
were phagocytic cups and freshly ingested phagosomes
(Fig. 6).
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To follow the dynamics of actin assembly during phagocytosis in individual cells, actin was tagged with GFP
(Westphal et al., 1997). Serial image analysis of GFP-actin-producing cells showed that g
cells, similarly to the other
cell lines (AX2, AX3, and LW20), formed leading edges,
in which actin was enriched, and often switched from one
front to another, to which actin was newly recruited (Fig.
7). Thus,
subunit-null cells are not defective in rapid, spontaneous accumulation of actin at leading edges or
other sites of the membrane. We monitored several events
of cell-yeast particle adhesion to determine whether adhesion resulted in local actin recruitment and whether the local actin meshwork was converted into a phagocytic cup
with the same probabilities in wild-type and g
cells. As
shown in Table III, actin was found to accumulate at sites
of cell-yeast particle adhesion in ~50% of the cases in both wild-type and g
cells. These data are consistent with
the hypothesis that particle binding does not automatically
trigger actin recruitment, though the low number of cases
observed might have obscured differences between wild-type and mutant cells. However, independently of whether
or not binding stimulates actin recruitment, the ratio of
particle engulfment versus events in which actin was enriched at sites of cell-yeast adhesion was found to be significantly reduced in the mutant (Table III). In most cases
no phagocytic cup, and in a few cases only a half cup was
formed, followed by actin dissociation from the adhesion
site after 30-90 s (Fig. 7, D and E). It is worth mentioning
that this was usually a sufficient time interval for successful phagosome formation, both in the wild-type and in the
mutant. Fig. 7 E further shows that particles can attach to
the surface of mutant cells for a much longer time period.
Thus, g
cells seem to be impaired in reshaping the actin
cytoskeleton at adhesion sites into a phagocytic cup and
eventually a phagosome. We never observed, neither in
wild-type nor in the mutant, successful uptake in the absence of locally enriched actin.
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Downstream Transduction Pathways Regulating Phagocytosis
In a preliminary attempt to dissect the cascade of signal
transduction that leads from the G protein to phagocytic
uptake, we tested the effects of several inhibitors on phagocytosis. Most of the drugs interfere with main effectors
linked to G protein, such as PKA, PLC, and PKC, or with
influx or intracellular mobilization of Ca2+ ions. Table IV
summarizes the results obtained with these drugs as well
as phospholipase A2 (PLA2), tyrosine kinase, and PI3/PI4 kinase inhibitors. Phagocytosis was strongly affected by
PLC inhibitors, such as U73122 and manoalide, and by the
intracellular Ca2+ chelator BAPTA-AM. Interestingly,
U73122 interferes with G protein-dependent activation of
PLC (Smith et al., 1990) and inhibits IP3-induced mobilization of Ca2+ (Willems et al., 1994
; Schaloske et al., 1995
).
Manoalide inhibits primarily PLA2, but several other PLA2
inhibitors, active in Dictyostelium (Schaloske and Malchow, 1997
), were ineffective, suggesting that PLC is the
target of the observed inhibitory effect. Mobilization of intracellular Ca2+, but not Ca2+ influx, seems to also be required for efficient phagocytosis, not however via activation of PKC. Neither genistein nor wortmannin or LY294002
affected phagocytosis, which question the involvement of
protein tyrosine or PI3 kinases in the process. Consistent with results obtained with the PKA inhibitors, mutants defective in activation of adenylyl cyclase or PKA, or mutants expressing constitutively PKA, showed normal rates
of phagocytosis (not shown). It is worth mentioning that
none of the drugs at the maximal concentration tested interfered with random cell motility or with stream formation during aggregation (data not shown). In contrast, inhibitors of phosphotyrosine phosphatases, such as phenylarsine oxide or benzylphosphonic acid-(AM)2, inhibited
phagocytosis at concentrations that correlated with rounding up of the cells (data not shown).
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Discussion |
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G Protein Controls an Early Step in Phagocytic Uptake
Deletion of the unique G protein subunit in Dictyostelium generates a cell that lacks functional heterotrimeric G
proteins. This leads to defects in multiple chemoattractant-induced responses, and, as reported here, in phagocytosis. The
subunit mutation affects an early step of the
phagocytic uptake, as evidenced by the strongly reduced
efficiency of individual cells in forming phagocytic cups
and phagosomes around yeast particles, and by the finding
that the initial rate of phagocytosis in shaken suspension is
reduced. These results indicate an involvement of the heterotrimeric G protein in particle uptake, which is distinct
from its potential role in intracellular phagosome-endosome fusion suggested for macrophages (Desjardins et al.,
1994
; Béron et al., 1995). The phenotype of the g
mutant
differs also in many respects from Dictyostelium phagocytosis mutants previously described by us and others. Mutants
defective in particle adhesion (Vogel et al., 1980
; Ceccarelli and Bozzaro, 1992
; Cohen et al., 1994
) or talin-null
mutants (Niewöhner et al., 1997
) fail to phagocytose in
shaken cultures but do so on bacterial lawns. In contrast,
g
cells are impaired both under shaking conditions and
on a solid substratum. Unlike phagocytosis mutants with
defects in F-actin cross-linking proteins (Rivero et al., 1996
;
Cox et al., 1996
), the g
mutant does not display their
pleiotropic defects in cell-substratum adhesion, locomotion, or cytokinesis. This also distinguishes the
subunit
deficiency from elimination of coronin, which has been
suggested to lay downstream of heterotrimeric G protein
and to act as an integrator of incoming signals to the cytoskeleton (Gerisch et al., 1995
; Maniak et al., 1995
).
G Protein Regulates the Actin Cytoskeleton during Phagocytosis
We have failed to detect differences between wild-type
and g cells in cell adhesion to a substrate surface, in motility or in spontaneous actin accumulation to leading
edges or other sites of the membrane. This indicates that
rapid reorganization of the actin cytoskeleton is not impaired in
subunit-null cells.
The g cells, and to a varying degree the
subunit
point mutants, fail to undergo rapid, transient actin assembly upon stimulation with cAMP or bacterial chemoattractants. This impaired actin response is correlated with the
phagocytosis defect, suggesting that the G protein is involved in regulating the actin cytoskeleton during phagocytosis. The results with GFP-actin are interesting, in this context, with respect to three questions: (a) they show that
spontaneous actin accumulation at leading edges or other
membrane extensions is not defective in the mutant, and
this is consistent with the absence of a general defect in adhesion to substrate, fluid-phase endocytosis, or cytokinesis; (b) they further show that cell contact with a yeast particle does not automatically trigger local actin assembly,
neither in wild-type nor in the mutant. Actin accumulation occurs only in ~50% of the adhesion events with no differences between wild-type and mutant; and (c) they finally
show that the g
cells, even when actin is enriched at cell-
yeast adhesion sites, are strongly inhibited in their ability
to reshape the actin meshwork into a phagocytic cup, and
eventually a phagosome.
We thus propose that a G protein-linked process regulates an appropriate actin assembly beneath the plasma
membrane leading to phagocytic cup and phagosome formation. Some evidence suggests that phagosome formation requires a different cytoskeletal organization as, for
example, cell spreading (Cannon and Swanson, 1992). G protein-linked signal transduction may be required for
this reorganization as occurs for actin assembly in chemotactically-induced pseudopods.
Cross-Talk between Chemotactic and Phagocytic Stimuli
There are multiple chemoattractant receptors and G subunits in Dictyostelium that are responsible for processing a
variety of chemotactic stimuli (Parent and Devreotes,
1996
). Double deletion of the chemoattractant receptors,
cAR1 and cAR3, or of the
subunit G
2 linked to these
receptors, blocked responses to the chemoattractant cAMP,
and deletion of the G
4 subunit blocked responses to folic acid, but none of these mutations eliminated the chemotactic responses to bacteria-conditioned medium nor did
they affect phagocytosis (data not shown). We have also
not observed smaller plaques that would suggest a defect
in phagocytosis in single null mutants of any of the
subunits (G
6 has not been examined), nor did we find reduced phagocytosis rates in G
2 and G
4 null mutants. It
is possible that the
subunits act redundantly in transducing these responses, which would make it difficult to recognize potential receptors required for phagocytosis.
Nevertheless, our results indicate that regulation of the
actin cytoskeleton is a common feature in processing
chemotactic and phagocytic stimuli, which are both transduced by the G protein subunit. This is consistent with
the finding that phagocytic and chemotactic stimuli compete in recruiting coronin and actin-binding proteins (Maniak et al.,1995). In contrast to chemotaxis, however, which is completely blocked in g
cells, phagocytosis is
only partially inhibited in these cells. This may suggest either the existence of a G protein-independent pathway
mediating phagocytosis or that the G protein acts at different levels in regulating the actin cytoskeleton in chemotaxis
or phagocytosis.
Chemotaxis and phagocytosis in Dictyostelium exhibit a
major distinct feature that also points to a different regulation of actin polymerization, or of cytoskeletal reorganization, by the subunit. Local chemotactic stimuli trigger
pseudopod formation quite automatically and lead to
chemotactic locomotion, as long as the stimulus persists.
In contrast, cell contact with a yeast particle does not always induce local membrane extensions to form a phagocytic cup. In addition, despite prolonged attachment of a
yeast particle to the cell surface, or even formation of a
phagocytic cup, the phagocytic process remains reversible,
as shown by Maniak et al. (1995)
, who have provided evidence in support of a zipper mechanism (Swanson and
Baer, 1995
) for phagocytosis in Dictyostelium. The present
results further confirm these observations. It is, however,
possible that yeast particle uptake might differ in some important aspects from bacterial uptake, thus some caution is required in extending these conclusions to phagocytosis of
bacteria.
The preliminary finding that both the intracellular Ca2+
chelator BAPTA-AM and U73122, a specific inhibitor of
receptor-stimulated PLC in neutrophils (Smith et al.,
1990) blocked phagocytosis suggests a role for IP3 and
Ca2+ in this process, and raises the possibility that the defect in the
subunit-null cells could be linked to the inability to activate PLC. To confirm this hypothesis it will
be necessary to determine which step in phagocytosis is
blocked upon cell treatment with these drugs. We cannot
exclude that these inhibitors affect the phagocytosis rate
indirectly, by interfering with intracellular processes, such
as phagosome-endosome fusion or receptor recycling, whereas we have shown that the
subunit-null cells are
blocked in actin reorganization in the phagocytic cup. Interestingly, the same inhibitors did not significantly influence chemotactic motility during aggregation (data not
shown), suggesting that downstream pathways leading to
phagocytosis and chemotaxis might be partially different.
Activation Mechanisms of the G Subunit
A G protein-dependent step in particle engulfment raises
a question: what are the signals for G protein-linked actin
reorganization during phagocytic cup formation? The correlation found between chemoattractant-stimulated F-actin
formation and phagocytosis points to a common role for
the G protein in regulating the actin cytoskeleton in chemotaxis and phagocytosis, but is no evidence that chemotactic
stimuli are the triggering signals for phagocytosis. With regard to latex beads, whose uptake is decreased in g cells
and rescued by expression of the
subunit, the possibility of chemoattractants that may be released from bacteria is
excluded. The impaired uptake of heat-killed yeast particles by g
cells also makes this hypothesis unlikely.
There are in principle three possibilities: (a) autocrine
chemokines may exist in Dictyostelium, which are secreted
upon particle binding and stimulate G protein-mediated
actin reorganization; (b) the G protein, or its subunit,
could be activated in response to particle binding to yet
unknown receptors; and (c) the
subunit might have an
integrator function in intracellular propagation of signals
arising from the site of initial particle attachment, and this
function might be independent of any interactions of G
proteins with specific cell-surface receptors. Activation
could occur through a clustering effect resulting, for example, from the local geometry of adhesion between cell and
particle, as proposed for syk tyrosine kinase involvement
in phagocytosis (Greenberg et al., 1996
). The correlation
found between phagocytosis and chemoattractant-stimulated actin assembly, as well as the mutual competition between phagocytic cups and leading edges described by Maniak et al. (1995)
, support the notion that the
subunit of
the heterotrimeric G protein converts signals originating
from different processes into activities of the cytoskeleton.
![]() |
Footnotes |
---|
Received for publication 26 March 1998 and in revised form 27 May 1998.
This paper was supported by grants of the European Union and Ministero Universita E Ricerca Scientifica to S. Bozzaro (ERBCHRXCT930250), the National Institutes of Health to P.N. Devreotes (GM 28007), and Deutsche Forschungsgeheinschaft to G. Gerisch (SFB266/C6).We thank A. Noegel (Institute of Biochemistry, University of Cologne, Cologne, Germany) for anti-actin mAb, R. Albrecht and J. Murphy (both from Max Planck Institute for Biochemistry, Martinsried, Germany) for help with image processing, and S. Zigmond (University of Pennsylvania, Philadelphia, PA), S. Van Es, and N. Zhang (both from Johns Hopkins School of Medicine, Baltimore, MD) for stimulating discussions.
![]() |
Abbreviations used in this paper |
---|
g, G
-null mutants;
GFP-actin, green fluorescent protein-actin fusion protein;
PLA2, phospholipase
A2;
PLC, phospholipase C;
PKA, protein kinase A;
PKC, protein kinase
C;
PI3 kinase, phosphoinositide 3-kinase;
PI4 kinase, phosphoinositide
4-kinase.
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