1 Department of Histology and Cell Biology, Kagawa Medical University, Miki,
Kagawa 761-0793, Japan
2 Department of Biochemistry, Kagawa Medical University, Miki, Kagawa 761-0793,
Japan
3 Department of Microbiology and Immunology, University of Michigan Medical
School, Ann Arbor, MI 48109-0620, USA
* Author for correspondence (e-mail: naraki{at}kms.ac.jp)
Accepted 23 October 2002
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Summary |
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Key words: Myosin, Myosin light chain kinase, Actin, Phosphoinositide 3-kinase, Phagocytosis, Macropinocytosis
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Introduction |
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In Fc receptor (FcR)-mediated phagocytosis, phagocytic cup extension
along the surfaces of immunoglobulin G (IgG)-opsonized particles is mediated
by the sequential interactions between IgG Fc regions and FcR, in a process
referred to as the zipper closure model
(Greenberg and Silverstein,
1993
). Recent studies have indicated that the zipper closure model
is insufficient to explain phagocytosis. Phosphoinositide 3-kinase (PI3K)
inhibitors such as wortmannin and LY294002 inhibit the closure of phagosomes,
but not phagocytic cup formation along IgG-opsonized particles
(Araki et al., 1996
),
indicating that closure of phagocytic cups into phagosomes requires additional
mechanisms that employ PI3K. Cox et al.
(Cox et al., 1999
) pointed out
the importance of PI3K-dependent vesicle transport for maximal phagocytic cup
extension during phagocytosis of large particles. Furthermore, several events
in phagocytosis seem to lie downstream of PI3K
(Gold et al., 1999
;
Didichenko et al., 2000
;
Vieira et al., 2001
;
Cox et al., 2002
).
The myosin family of actin-based mechanochemical motors is an important
component of the phagocytic apparatus. Myosins I, II, V, IX and X have been
localized to macrophage phagosomes at various stages of their formation
(Stendahl et al., 1980;
Allen and Aderem, 1995
;
Swanson et al., 1999
;
Al-Haddad et al., 2001
;
Cox et al., 2002
;
Diakonova et al., 2002
;
Olazabal et al., 2002
),
suggesting that they play distinct roles in phagocytosis. Morphological
studies identified a PI3K-dependent contractile activity, capable of
constricting deformable erythrocytes caught between two macrophages
(Swanson et al., 1999
). Myosin
IC localized to the distal margin of phagocytic cups, suggesting that it
mediated a PI3K-dependent, purse-string-like contractile activity that closed
the cup aperture into an intracytoplasmic phagosome
(Swanson et al., 1999
).
Consistent with this model, Cox et al.
(Cox et al., 2002
) identified
a role for myosin X in phagocytosis, which is notable because myosin X
contains a pleckstrin-homology domain that recognizes
phosphatidylinositol(3,4,5)triphosphate. Although myosin II (known as
conventional myosin) has been localized around forming phagosomes
(Stendahl et al., 1980
;
Swanson et al., 1999
;
Diakonova et al., 2002
), its
precise role and regulation in phagocytosis remain to be elucidated. One aim
of this study is to elucidate the role of myosin II in the process of
phagocytosis and to clarify the relationship of myosin function of PI3K
signaling.
Macropinocytosis is a form of fluid-phase endocytosis that provides an
efficient route for non-selective uptake of extracellular solute
macromolecules (Swanson and Watts,
1995). Recent attention has been directed toward the mechanism of
macropinocytosis, because that route is used for MHC class I and class II
antigen presentation in dendritic cells and macrophages
(Norbury et al., 1995
;
Sallusto et al., 1995
;
Nobes and Marsh, 2000
). Unlike
micropinocytosis mediated by clathrin-coated vesicles and small uncoated
vesicles (
100 nm diameter), macropinocytosis is associated with active
cell-surface ruffling. Macropinosomes (0.5-5 µm diameter) arise from the
deformation of ruffles, which are actin-rich cell-surface protrusions.
Circular ruffles, formed by inward curving of peripheral ruffles, are
considered to be precursors of macropinosomes, in that they often close into
macropinosomes.
There are many similarities in the signaling for macropinocytosis and
FcR-mediated phagocytosis. Like FcR-mediated phagocytosis, macropinocytosis
involves Rac1, Cdc42 and Arf6
(Radhakrishna et al., 1996;
Zhang et al., 1999
;
Nobes and Marsh, 2000
;
West et al., 2000
), and the
closure of cup-shaped circular ruffles into macropinosomes requires PI3K
activity (Araki et al., 1996
).
However, some differences in their signaling, regulation and mechanism are
notable (Swanson and Baer,
1995
; Swanson and Watts,
1995
; Niewohner et al.,
1997
). For example, unlike phagocytosis, the cell-surface
protrusions forming circular ruffles can form without the guidance of any
particle surface. Therefore, one might expect that shaping ruffles into
macropinosomes would require spatial and temporal controls distinct from those
for phagocytic cup formation. Although myosin II has been shown to generate
force for cell movements, its role in macropinocytosis is not clear. It is of
interest to determine whether myosin II contributes similarly to
macropinocytosis and phagocytosis.
ML-7 is an inhibitor of MLCK (Saitoh et
al., 1987), and inhibition of MLCK results in selective
perturbation of myosin II function (Somlyo
and Somlyo, 1994
; Ruchhoeft
and Harris, 1997
). Although higher concentrations of ML-7 might
inhibit other protein kinases, the concentration of ML-7 we used does not much
affect other protein kinases (Mansfield et
al., 2000
). In the present study, we have compared the effects of
the MLCK inhibitor and inhibitors of PI3K on macropinocytosis and
phagocytosis. We describe a novel actomyosin-driven motion that squeezes
phagocytic cups and promotes circular-ruffle formation for macropinocytosis.
This now distinguishes a third activity of the actin cytoskeleton in
phagocytosis.
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Materials and Methods |
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Bone-marrow-derived macrophages were obtained from femurs of C3H HeJ mice
as previously described (Araki et al.,
1996). After 6 or 7 days of culture, macrophages were harvested
from dishes and plated onto coverslips or 24-well dishes, then incubated
overnight in DME-10F (DME with 10% heat-inactivated FBS). 30 minutes before
experiments, DME-10F was replaced with Ringer's buffer (RB) consisting of 155
mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM Na2HPO4,
10 mM glucose, 10 mM Hepes pH 7.2 and 0.5 mg ml-1 BSA. These
bone-marrow-derived macrophages were used for all but the transfection
experiments.
For transfection with an enhanced GFP (EGFP)-fused actin expression plasmid (pEGFP-actin, CLONTECH, Palo Alto, CA), macrophage-like RAW264.1 cells, obtained from Riken Cell Bank (Tsukuba, Japan), were plated onto 25 mm circular coverslips in 35 mm dishes filled with DME-10F. The pEGFP-actin was transfected into the cells using a DNA transfection reagent, TransFast (Promega, Madison, WI). Fluorescence was observed by video microscopy 48 hours after transfection, as described below.
Phagocytosis and microscopy
For phagocytosis, sheep erythrocytes were opsonized with rabbit anti-sheep
erythrocyte IgG (1:200 dilution, Organon Teknika-Cappel) and resuspended in
PBS at 109 erythrocytes ml-1. 10 µl of a suspension
of IgG-opsonized sheep erythrocytes (IgG-Es) was added to each 24-well dish
containing macrophages on 12 mm circular coverslips. Macrophages were then
incubated for various times at 37°C, to allow phagocytosis. For
quantitative assay of phagocytosis, after a 30-minute incubation with IgG-Es,
cells on coverslips were dipped into distilled water for 30 seconds to disrupt
extracellularly exposed IgG-Es by low osmolarity, then fixed with 4%
paraformaldehyde in a buffer. For the binding assay, cells were incubated with
IgG-Es for 30 minutes at 4°C, briefly washed in cold PBS to remove unbound
IgG-Es and fixed. The number of IgG-Es in 100 macrophages was counted under a
phase-contrast microscope and the number of bound or internalized IgG-Es per
macrophage was expressed as the binding or phagocytic index, respectively.
For fluorescence microscopy of phagocytosis combined with filamentous actin (F-actin) staining, cells were fixed for 30 minutes at room temperature with 4% paraformaldehyde and 0.1% glutaraldehyde in 100 mM phosphate buffer, pH 7.4, containing 6.8% sucrose (PB/sucrose). To distinguish extracellularly exposed IgG-Es from intraphagosomal IgG-Es, macrophages were incubated with FITC-labeled anti-rabbit IgG (1:50 dilution in PBS), which was not accessible to intraphagosomal IgG-Es, for 15 minutes at room temperature. Then, F-actin was stained with rhodamine phalloidin (Molecular Probe, 5 unit ml-1 PBS containing 0.25% Triton X-100).
For immunocytochemistry, cells on 12-mm coverslips were fixed with 4% paraformaldehyde in PB/sucrose for 30 minutes, rinsed in PBS, and permeabilized with 0.25% Triton X-100 in PBS followed by treatment with a blocking buffer (0.5% BSA, 0.2% gelatin, 0.25% Triton X-100 in PBS). Then, cells were incubated with rabbit polyclonal anti-myosin-II antibody (1:100 dilution; Biomedical Technologies, Stoughton, MA) or goat polyclonal anti-phosphomyosin-light-chain (P-MLC) antibody (1:100 dilution; Santa Cruz Biotechnology) for 90 minutes at room temperature. After rinsing in PBS, cells were labeled with FITC-anti-rabbit IgG and/or Texas-red anti-goat IgG (Vector, Burlingame, CA).
Coverslips were mounted on glass slides using a FluoroGuard (BioRad) and observed with an epifluorescence microscope (Nikon TE 300) operated by MetaMorph Imaging System (Universal Imaging, West Chester, PA).
For scanning electron microscopy (SEM) of phagocytosis, macrophages on
small coverslips were fixed with 2% glutaraldehyde in PB/sucrose for 1 hour at
room temperature. Coverslips were then rinsed in a buffer, post-fixed with 1%
osmium tetroxide, treated with 1% tannic acid, conventionally processed for
SEM and observed by a Hitachi S-900 SEM as previously described
(Araki et al., 1996).
Spectrofluorometric assay of macropinocytosis
Macropinocytosis was stimulated by the addition of human recombinant M-CSF
(2000 unit ml-1) to the macrophage culture
(Racoosin and Swanson, 1992;
Araki et al., 1996
).
After 30-minute incubation with 1.0 mg ml-1 FDx150 to allow fluid-phase pinocytosis, dishes were drained and rinsed twice in 1 1 PBS plus 1 mg ml-1 BSA and then once in 1 1 PBS, each at 4°C for 5 minutes. Cells were lysed in a lysis buffer consisting of 0.1% Triton X-100 and 50 mM Tris pH 8.5. The amount of fluorescence in cell lysates was measured by a spectrofluorometer (Hitachi 650-40) and normalized by the total cell protein.
Video microscopy and digital image analysis
Macrophages were plated onto 25 mm circular coverslips in 35 mm culture
dishes. The culture medium was replaced with RB 1 hour before experiments. The
coverslip was assembled into a stainless steel cell chamber (Atto Instruments,
Rockville, MD) and placed in a temperature-controlled stage at 37°C on an
inverted microscope (Nikon TE300). Living cells were observed using a 100 lens
with phase-contrast optics. Before and after addition of drugs, time-lapse
video images were collected using a cooled CCD camera (IFG-300, Dage-MTI,
Michigan City, IN) and MetaMorph Imaging System. These images were revealed as
8-bit digital images consisting of pixels with 256 shades of gray (0-255 gray
value). Time-lapse video microscopic images taken at a 10-second interval
using MetaMorph were assembled to QuickTime movies. Videos accompany
Fig. 4A,B,
Fig. 7A-D and are available in
the online version at
http://jcs.biologists.org/supplemental.
|
|
Ruffling activity was quantified using a subtraction-based image analysis
developed with MetaMorph software (Araki et
al., 1996). In phase-contrast images of living cells, ruffles were
seen as phase-dense bands that grew in length and migrated centripetally along
the upper surface of the cells. Using 20-second interval time-lapse series,
one frame of an image was subtracted, pixel by pixel, from the frame taken 20
seconds later. A gray value of 120 was then added to all pixels of the
subtracted image. Change of phase density in a 20-second interval produced
contrast in the subtracted image. The average of standard deviations of gray
values in the cell region of 10 subtracted images was calculated as the
ruffling activity index.
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Results |
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To analyze the effects of these inhibitors on the mechanism of phagocytosis, we used fluorescence microscopy to examine the morphologies of IgG-Es and F-actin during phagocytosis. After 30 minutes with IgG-Es in the presence or absence of inhibitors, macrophages were fixed and incubated with FITC-conjugated anti-rabbit IgG (FITCanti-IgG) to label extracellularly exposed IgG-Es, then with rhodamine-phalloidin to label F-actin. In control macrophages, IgG-Es were internalized into intracellular compartments and most F-actin was dissociated from the particle-adherent membranes. FITCanti-IgG could not access the compartments containing IgG-Es, indicating that these compartments were closed phagosomes or phagolysosomes (Fig. 2A-C). In ML-7- or wortmannin-treated cells, however, IgG-Es remained associated with F-actin-rich structures, presumed phagocytic cups. Most of the IgG-Es associated with these cells were labeled with FITC, indicating that these IgG-Es surrounded by F-actin-rich protrusions were partially exposed to extracellular fluid (Fig. 2D-I). Differences between PI3K-inhibitor- and MLCK-inhibitor-treated cells were not evident under light microscopy. These data indicated that both PI3K inhibitors and MLCK inhibitor perturbed phagocytic-cup closure but not F-actin assembly for forming phagocytic cups. Unlike in PI3K-inhibitor- or MLCK-inhibitor-treated cells, F-actin assembly was not induced by the binding of IgG-Es in cytochalasin-D-treated cells. Consequently, neither phagocytic-cup formation nor internalized IgG-Es were observed in these cells (not shown).
|
Role for myosin in the phagocytic-cup squeezing
Consistent with light-microscopic observations, SEM revealed that, after 30
minutes, no IgG-Es were evident on the dorsal surface of control macrophages,
indicating that IgG-Es were all intracellular (not shown). However, cells
fixed 7 minutes after adding IgG-Es showed phagocytic cups at various stages
of formation. These cups engulfing IgG-Es frequently appeared cylindrical
rather than spherical (Fig.
3A). In PI3K-inhibitor-treated cells, many IgG-Es were observed in
phagocytic cups on the dorsal cell surface, because cup closure was arrested
(Araki et al., 1996). The
phagocytic cups formed in PI3K-inhibitor-treated cells were tightly fitted to
the surface of IgG-Es and their distal margins remained open even at 30
minutes after beginning phagocytosis (Fig.
3B). The configurations of the phagocytic cups in PI3K-inhibited
cells were similar to those observed in control cells fixed 7 minutes after
addition of IgG-Es (Fig.
3A,B).
|
In ML-7-treated cells, phagocytic cups appeared to be somewhat shallower than those in PI3K-inhibitor-treated cells. Even though phagocytic cups formed around IgG-Es, the inner side face of phagocytic cups was not closely adherent to the surface of the IgG-Es, so phagocytic cups looked like flowers in bloom (Fig. 3C). This unique configuration of phagocytic cup indicated that cup side wall could extend without guidance from sequential IgG-FcR binding, and that myosin might be unnecessary for phagocytic cup formation but necessary for the close apposition of the cup side wall against opsonized particles. We scored the morphology of phagocytic cups in ML-7- and wortmannin-treated cells (100 of each). In wortmannin-treated cells, 82% of phagocytic cups apparently showed tight apposition to IgG-Es and others were loosely open or undefined. By contrast, in ML-7-treated cells, 45% of phagocytic cups were loosely open, 48% were short or irregular and only 7% were closely fitted against the IgG-Es. When F-actin polymerization was inhibited by cytochalasin-D treatment, IgG-Es bound to the dorsal cell surface but no phagocytic cup formation was evident (Fig. 3D). These SEM data also indicated that phagocytic cup side wall extension requires F-actin polymerization but not myosin II.
To explore further the actomyosin-driven constriction of phagocytic cups, we observed actin dynamics during phagocytosis in living RAW264.1 cells transfected with pEGFP-actin. Video microscopy of control cells showed that binding of IgG-Es induced GFP-actin accumulation underneath the plasma membrane near the particle. F-actin-enriched protrusions then extended along the surfaces of IgG-Es. In a side view of the phagocytic cup, a dense band of F-actin moved from the bottom toward the top of the phagocytic cup during extension. In a movie [Video 1A (http://www.jcs.biologists.org/supplemental)], it was notable that the IgG-Es in the phagocytic cup deformed along the axis of the phagocytic-cup extension, making it look like a deformable particle passing through a tight ring of F-actin. When the phagocytic cup was fully extended to enclose the particle, the F-actin band closed the top aperture of phagocytic cup to form an intracellular phagosome. Most F-actin then appeared to be immediately dissociated from the phagosomal membrane. The process of phagocytosis generally took 3-5 minutes from F-actin assembly to F-actin dissociation [Fig. 4A, Video 1A (http://www.jcs.biologists.org/supplemental)].
Phagocytic-cup extension also occurred in ML-7-treated cells but seemed to be somewhat slower than that in control cells. The phagocytic cups did not constrict or squeeze IgG-Es. Rather, they repeatedly elongated and shortened their side walls without closing into phagosomes. IgG-Es remained in unclosed phagocytic cups even at 30 minutes or more after IgG-E binding [Fig. 4B, Video 1B (http://www.jcs.biologists.org/supplemental)]. The effect of ML-7 on phagocytosis was reversible, because phagocytosis proceeded normally after washout of the drug (not shown). The cups formed in PI3K-inhibitor-treated cells did not close into phagosomes but they could deform IgG-Es during cup formation (not shown).
Next, we examined the localization of myosin II and phosphorylated myosin light chain (P-MLC) in macrophages. In macrophages stimulated with M-CSF, myosin II abundantly localized in active ruffles on the cell dorsal surface (Fig. 5A). Using an antibody specific to P-MLC, it was shown that myosin II light chain was phosphorylated in active ruffles (Fig. 5B). In control macrophages during phagocytosis, P-MLC was detected in some early stages of phagocytic cups (Fig. 5C,D). In ML-7-treated cells, P-MLC was scarcely seen in phagocytic cups, suggesting that ML-7 inhibited phosphorylation of MLC (Fig. 5E,F).
|
Role of myosin in macropinocytosis and ruffling
To analyze the myosin contribution to macropinocytosis, we first measured
the effects of inhibitors for PI3K, MLCK and F-actin on pinocytosis of FDx150,
a fluid-phase probe that is preferentially taken up by macropinocytosis
(Araki et al., 1996;
Araki and Swanson, 1998
).
Macropinocytosis was stimulated by the addition of M-CSF to the medium
(Racoosin and Swanson, 1989
).
Cells were then incubated with 1 mg ml-1 FDx150 for 30 minutes in
the absence or presence of inhibitors. Quantitative fluorometric analysis
indicated that ML-7 significantly reduced intracellular accumulation of
FDx150, although their inhibitory extent was somewhat less than that of PI3K
inhibitors. Cytochalasin D perturbed fluid-phase pinocytosis of FDx150 most
severely (Fig. 6). Even though
the accumulation of FDx150 might be slightly influenced by F-actin-independent
micropinocytosis, the comparative quantitation indicated that myosin II
inhibition attenuated macropinocytosis. Similarly, fluorescence microscopy
revealed that macropinosomes labeled with fixable FDx10 for 5 minutes were
largely diminished by all of these inhibitors (not shown).
|
We next used time-lapse video microscopy to identify myosin-dependent
activities associated with ruffling and macropinocytosis in living cells.
Consistent with earlier observations (Araki
et al., 1996), wortmannin-treated macrophages displayed dorsal
surface ruffling and circular ruffle formation much like control cells.
However, circular ruffles receded without closing into macropinosomes
[Fig. 7A,B, Video 2A,B
(http://www.jcs.biologists.org/supplemental)].
Unlike the PI3K inhibitors, however, MLCK inhibitor reduced most ruffle
movement. In the ML-7-treated cells, the ruffles infrequently raised from the
cell edge and showed a slow centripetal movement on the dorsal surface.
Circular ruffle formation was not seen in MLCK-inhibited cells, indicating
that such simple ruffling was insufficient for macropinocytosis
[Fig. 7C, Video 2C
(http://www.jcs.biologists.org/supplemental)].
Ruffling activity was quantified by digital image analysis of time-lapse
phase-contrast images, which indicated that wortmannin or LY294002 reduced
ruffling movements much less than ML-7-treatment did
(Fig. 8,
Table 1). These observations
indicated that myosin II acts earlier than PI3K in the coordination of
ruffling for macropinocytosis.
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Discussion |
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In FcR-mediated phagocytosis, phagocytic-cup extension along the surface of
IgG-opsonized particles has been explained by the `zipper model', which
implies sequential binding between cell surface FcR and Fc region of IgG on
the particle surface (Greenberg and
Silverstein, 1993). The interaction between IgG and FcR triggers
the phosphorylation of specific tyrosine residues in the receptor within
motifs termed ITAMs (immunoreceptor tyrosine-based activation motif) by the
Src-family tyrosine kinases (Ghazizadeh et
al., 1994
; Greenberg et al.,
1994
; Cox et al.,
1996
; Cox et al.,
1997
; Crowley et al.,
1997
; Isakov,
1997
). These early signals from ligated receptors lead to further
signaling pathways involving lipid kinases, phospholipases and GTPases.
(reviewed by Aderem and Underhill,
1999
; Kwiatkowska and Sobota,
1999
; Lennartz,
1999
). Downstream, Rho-family members of monomeric GTPases such as
Rac, Rho and Cdc42 induce F-actin polymerization and reorganization
(Cox et al., 1997
;
Massol et al., 1998
;
Castellano et al., 2000
;
Chimini and Chavrier, 2000
),
changes which are also regulated by phosphoinositides such as
PI(4,5)P2 and PI(3,4,5)P3
(Hackam et al., 1997
;
Botelho et al., 2000
;
Marshall et al., 2001
). Many
actin-binding proteins (e.g. actin-nucleating factors and F-actin bundling and
severing proteins) and myosins are involved in particle engulfment to form
nascent phagosomes (Greenberg et al.,
1990
; Allen and Aderem,
1996b
; Swanson et al.,
1999
; Titus, 1999
;
Araki et al., 2000
;
May et al., 2000
). This
complexity indicates that the zipper closure mechanism must be integrated by
supplemental mechanisms. PI3K inhibition perturbs phagocytic-cup closure but
not F-actin assembly or phagocytic-cup formation
(Araki et al., 1996
;
Cox et al., 1999
;
Diakonova et al., 2002
).
Dynamin 2 recruitment to phagocytic cups is controlled by PI3K
(Gold et al., 1999
), although
the function of dynamin in phagosome closure has not been determined.
Furthermore, focal exocytosis of VAMP3-positive recycling vesicles to the site
of particle uptake might be required for phagocytic-cup extension sufficient
to enclose large particles (Banjo et al.,
2000
). Such full phagocytic-cup extension also depends on PI3K
signaling (Cox et al.,
1999
).
To complete phagosome formation, some forces would be required for
sequential binding and extension of phagocytic cups along particle surfaces.
In neutrophils, regulation of phagocytosis by MLCK indicates that myosin II is
involved in FcR-mediated phagocytosis of IgG-Es
(Mansfield et al., 2000).
Myosin II and MLCK contribute to many cell motilities such as lamellipodial
movement and cytokinesis, and our quantitative measurements indicated that
myosin II was required for FcR-mediated phagocytosis. Furthermore, our
immunofluorescence localized P-MLC in phagocytic cups, and showed that
phosphorylation of MLC in phagocytic cups was inhibited by ML-7. These
immunolocalizations strongly supported the notion that myosin II plays an
important role in the formation of phagosomes during FcR-mediated phagocytosis
in macrophages. On the other hand, de Lanerolle et al.
(de Lanerolle et al., 1993
)
reported that myosin phosphorylation did not significantly increase in J774
macrophages during phagocytosis of opsonized yeast; and electroinjection of
anti-MLCK did not inhibited phagocytosis. However, they measured the total
amount of phosphorylated myosin but not the net amount involved in
FcR-mediated phagocytosis (de Lanerolle et
al., 1993
). Moreover, unlike IgG-Es, opsonized yeast would be
phagocytosed by a nonopsonic, lectin-sugar (ß-glucan and mannan)
recognition mechanism even if FcR-mediated phagocytosis were inhibited
(Le Cabec et al., 2000
). It
was also reported that Dictyostelium myosin-II-null mutant did not
appear to have any defects in phagocytosis of living Escherichia coli
(Maselli et al., 2002
). This
discrepancy might be due to differences in the cell types and/or the
phagocytic targets. Some types of phagocytosis of nonopsonized particles or
living bacteria might not require myosin II activity. Most recently, Olazabal
et al. (Olazabal et al., 2002
)
showed that ML-7 also inhibited complement-receptor-mediated phagocytosis as
well as actin recruitment to the site of phagocytosis. They further indicated
that myosin II was required for FcR-mediated phagocytosis but not actin
recruitment (Olazabal et al.,
2002
). Their findings on FcR-mediated phagocytosis were totally
consistent with our data presented here. Therefore, myosin II and its
phosphorylation seem to be required for FcR-mediated phagocytosis and
complement-receptor-mediated phagocytosis. However, the role of myosin II
might differ between the two types of phagocytosis, because they use different
cytoskeletal components and signaling pathways during the engulfment of
particles (Allen and Aderem,
1996b
). Importantly, our morphological data now suggest a role for
myosin II in phagocytic-cup squeezing during FcR-mediated phagocytosis.
Our SEM of control cells revealed that the side wall of phagocytic cups
appeared tightly apposed to the IgG-Es and deformed IgG-Es in the phagocytic
cups. This deformation of erythrocytes might be explained by a myosin-based
contractile activity that squeezes particles during phagocytic cup formation.
In ML-7-treated cells fed with IgG-Es, phagocytic cups were formed but
remained unclosed, as in the PI3K inhibitor-treated cells. It was notable
that, unlike PI3K-inhibited or normal phagocytic cups, their side walls were
not closely apposed to the surface of particles. This unique configuration of
bloomed flower-like phagocytic cups in MLCK-inhibited cells indicated, first,
that phagocytic cup formation occurred without myosin activated by MLCK, and,
second, that the sequential binding of FcR to IgG Fc was not required for
phagocytic-cup extension. This free side wall extension of phagocytic cups
might occur by directional actin polymerization pushing against plasma
membrane (Theriot, 2000).
Video microscopy of living cells transfected with pEGFP-actin indicated that
an actin ring squeezed the phagocytic cup, resulting in deformation of the
IgG-Es. ML-7 perturbed such squeezing activity without affecting actin
polymerization at the site of IgG-E binding. This suggests that myosin II
contributes to the squeezing, although other classes of myosins might also
participate. In addition, upon myosin inhibition, phagocytic-cup extension was
slowed and shortened, resulting in failure of phagocytic-cup closure. These
findings indicate that myosin-II activity and sequential ligand-FcR binding
co-operate to facilitate maximal phagocytic-cup extension for the engulfment
of large particles. Such cooperative contractile activity might reinforce
binding between the particle surface and the plasma membrane, particularly
when the opsonization is weak. Moreover, because such squeezing would push
extra-particle fluid out of phagosomes, this mechanism might decrease
phagosomal volume and consequently increase lumenal concentrations of
superoxide and protons needed for bacterial killing.
The phagocytic cups formed in myosin-inhibited cells resemble spacious
phagosomes induced by bacteria such as Salmonella typhimurium
(Alpuche-Aranda et al., 1994).
In contrast to opsonized pathogens, which enter macrophages in tightly fitted
phagosomes and are killed rapidly, these pathogens enter and survive inside
phagosomes containing extracellular fluid. S. typhimurium might
create spacious phagosomes by inhibiting the myosin-driven squeezing of the
phagocytic cups. Alternatively, S. typhimurium might enter cells
simply by inducing macropinocytosis locally. Other types of phagocytosis,
besides FcR-mediated phagocytosis, show distinct characteristics in
morphology, machineries and signaling
(Rittig et al., 1999
;
Greenberg, 2001
), and it would
be of interest to elucidate the contributions of myosins to these
processes.
In contrast to FcR-mediated phagocytosis, macropinocytosis efficiently
engulfs extracellular fluid. Myosin II localized in active ruffles might be
required to coordinate ruffling to form macropinosomes. Our video microscopic
analysis indicated that M-CSF-stimulated ruffling was much reduced by ML-7,
but was not much affected by PI3K inhibitors. Upon inhibition of MLCK, ruffle
movements became much slower than those observed in control cells, and
circular ruffles did not form. These findings suggest that myosin II is
required for circular ruffle formation, which can be regarded as the initial
process of macropinocytosis. However, myosin-based motility in
macropinocytosis is likely to be highly complex, involving different isoforms
and classes of myosins that perform distinct functions. Other classes of
myosins appear to participate in later stages of macropinocytosis than
circular ruffle formation. For instance, Dictyostelium
myosin-I-knockout strains show increased circular ruffles, probably because of
defects in later stages of macropinocytosis
(Novak et al., 1995;
Jung et al., 1996
). Perhaps
myosin I contributes to macropinosome closure in macropinocytosis, similar to
the phagocytic-cup closure in phagocytosis.
Because PI3K inhibitors attenuated both phagocytosis and macropinocytosis
at later stages than the MLCK inhibitor did, we infer that the relevant
myosin-II activity is independent of PI3K. Other signaling pathways than the
PI3K cascade might modulate myosin-II function. Rac1 and Cdc42 are implicated
in the actin cytoskeleton remodeling during phagocytosis and macropinocytosis
(Ridley et al., 1992;
Cox et al., 1997
;
Massol et al., 1998
;
Dumontier et al., 2000
;
Nobes and Marsh, 2000
), and
p21-activated kinase 1 (PAK1), which is a direct target for Rac1 and Cdc42, is
located in phagocytic cups and circular ruffles
(Dharmawardhane et al., 1999
).
Moreover, a constitutively active PAK1 mutant induced the formation of
circular ruffles (Dharmawardhane et al.,
1997
). Interestingly, PAK1 affects the phosphorylation state of
MLC and eventually regulates cell movements by myosin II
(Sells et al., 1999
). These
observations, together with the fact that actinmyosin-II interactions
are regulated by the phosphorylation of MLC
(Somlyo and Somlyo, 1994
),
indicate that myosin II participates in Rac1 and Cdc42-induced cellular
movements for FcR-mediated phagocytosis and macropinocytosis. It is likely
that Ca2+ is also required for FcR-mediated phagocytosis, because
MLCK activity depends on Ca2+/calmodulin
(Saitoh et al., 1987
).
Although it is known that FcR ligation is accompanied by rises in cytosolic
free Ca2+ concentration, the requirement for Ca2+ in
FcR-mediated phagocytosis is still controversial
(Edberg et al., 1995
;
Hackam et al., 1997
;
Greenberg, 2001
). Further
studies will be needed to clarify the role of Ca2+ in phagocytosis
and macropinocytosis.
The PI3K-independent activity described here is distinct from the
PI3K-dependent contractile activities described previously
(Araki et al., 1996;
Swanson et al., 1999
). Myosins
I or X might close circular ruffles and phagocytic cups in a PI3K-dependent
manner. It is conceivable that the binding of myosin I to plasma membrane is
regulated by phosphoinositides such as PI(3,4)P2 and
PI(3,4,5)P3 (Adams and Pollard,
1989
; Zhou et al.,
1998
). Moreover, myosin X contains a pleckstrin-homology domain
that recognizes PI(3,4,5)P3
(Berg et al., 2000
). Cox et
al. (Cox et al., 2002
) have
shown that myosin X localizes phagosomes in a wortmannin-sensitive manner.
These results are consistent with myosins IC or X serving as the
PI3K-dependent contractile proteins that close phagosomes. In conclusion, we
now distinguish three component activities of the actin cytoskeleton during
phagocytosis and macropinocytosis. (1) Stimulated actin polymerization for
phagocytic cup extension. (2) PI3K-independent, myosin-II-dependent
contractile activities that squeeze phagocytic cups and curve ruffles. (3)
PI3K-dependent contractile activities that constrict the distal margins of
cup-shaped protrusions and complete phagosome/macropinosome closure.
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Acknowledgments |
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Footnotes |
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References |
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---|
Adams, R. J. and Pollard, T. D. (1989). Binding of myosin I to membrane lipids. Nature 340,565 -568.[CrossRef][Medline]
Aderem, A. and Underhill, D. M. (1999). Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 17,593 -623.[CrossRef][Medline]
Al-Haddad, A., Shonn, M. A., Redlich, B., Blocker, A.,
Burkhardt, J. K., Yu, H., Hammer, J. A., 3rd, Weiss, D. G., Steffen, W.,
Griffiths, G. et al. (2001). Myosin Va bound to phagosomes
binds to F-actin and delays microtubule-dependent motility. Mol.
Biol. Cell 12,2742
-2755.
Allen, L. A. and Aderem, A. (1996a). Mechanisms of phagocytosis. Curr. Opin. Immunol. 8, 36-40.[CrossRef][Medline]
Allen, L. A. and Aderem, A. (1996b). Molecular definition of distinct cytoskeletal structures involved in complement- and Fc receptor-mediated phagocytosis in macrophages. J. Exp. Med. 184,627 -637.[Abstract]
Allen, L. H. and Aderem, A. (1995). A role for MARCKS, the alpha isozyme of protein kinase C and myosin I in zymosan phagocytosis by macrophages. J. Exp. Med. 182,829 -840.[Abstract]
Alpuche-Aranda, C. M., Racoosin, E. L., Swanson, J. A. and Miller, S. I. (1994). Salmonella stimulate macrophage macropinocytosis and persist within spacious phagosomes. J. Exp. Med. 179,601 -608.[Abstract]
Araki, N., Hatae, T., Yamada, T. and Hirohashi, S.
(2000). Actinin-4 is preferentially involved in circular ruffling
and macropinocytosis in mouse macrophages: analysis by fluorescence ratio
imaging. J. Cell Sci.
113,3329
-3340.
Araki, N., Johnson, M. T. and Swanson, J. A. (1996). A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages. J. Cell Biol. 135,1249 -1260.[Abstract]
Araki, N. and Swanson, J. A. (1998). Labeling of endocytic vesicles using fluorescent probes for fluid-phase endocytosis. In Cell Biology: A Laboratory Handbook, Vol.2 (ed. J. E. Celis), pp.495 -500. San Diego: Academic Press.
Banjo, L., Peng, X.-R., Schreiber, A. D., Moore, H.-P., Trimble,
W. S. and Grinstein, S. (2000). Focal exocytosis of
VAMP3-containing vesicles at sites of phagosome formation. J. Cell
Biol. 149,697
-705.
Berg, J. S., Derfler, B. H., Pennisi, C. M., Corey, D. P. and
Cheney, R. E. (2000). Myosin-X, a novel myosin with
pleckstrin homology domains, associates with regions of dynamic actin.
J. Cell Sci. 113,3439
-3451.
Botelho, R. J., Teruel, M., Dierckman, R., Anderson, R., Wells,
A., York, J. D., Meyer, T. and Grinstein, S. (2000).
Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites
of phagocytosis. J. Cell Biol.
151,1353
-1368.
Castellano, F., Montcourrier, P. and Chavrier, P.
(2000). Membrane recruitment of Rac1 triggers phagocytosis.
J. Cell Sci. 113,2955
-2961.
Chimini, G. and Chavrier, P. (2000). Function of Rho family proteins in actin dynamics during phagocytosis and engulfment. Nat. Cell Biol. 2,E191 -E196.[CrossRef][Medline]
Cox, D., Chang, P., Kurosaki, T. and Greenberg, S.
(1996). Syk tyrosine kinase is required for immunoreceptor
tyrosine activation motif-dependent actin assembly. J. Biol.
Chem. 271,16597
-16602.
Cox, D., Chang, P., Zhang, Q., Reddy, P. G., Bokoch, G. M. and
Greenberg, S. (1997). Requirements for both Rac1 and Cdc42 in
membrane ruffling and phagocytosis in leukocytes. J. Exp.
Med. 186,1487
-1494.
Cox, D., Tseng, C. C., Bjekic, G. and Greenberg, S.
(1999). A requirement for phosphatidylinositol 3-kinase in
pseudopod extension. J. Biol. Chem.
274,1240
-1247.
Cox, D., Berg, J. S., Cammer, M., Chinegwundoh, J. O., Dale, B. M., Cheney, R. E. and Greenberg, S. (2002). Myosin-X is a downstream effector of PI(3)K during phagocytosis. Nat. Cell Biol. 4,469 -477.[Medline]
Crowley, M. T., Costello, P. S., Fitzer-Attas, C. J., Turner,
M., Meng, F., Lowell, C., Tybulewicz, V. L. and DeFranco, A. L.
(1997). A critical role for Syk in signal transduction and
phagocytosis mediated by Fcgamma receptors on macrophages. J. Exp.
Med. 186,1027
-1039.
de Lanerolle, P., Gorgas, G., Li, X. and Schluns, K.
(1993). Myosin light chain phosphorylation does not increase
during yeast phagocytosis by macrophages. J. Biol.
Chem. 268,16883
-16886.
Dharmawardhane, S., Sanders, L. C., Martin, S. S., Daniels, R.
H. and Bokoch, G. M. (1997). Localization of p21-activated
kinase 1 (PAK1) to pinocytic vesicles and cortical actin structures in
stimulated cells. J. Cell Biol.
138,1265
-1278.
Dharmawardhane, S., Brownson, D., Lennartz, M. and Bokoch, G. M. (1999). Localization of p21-activated kinase 1 (PAK1) to pseudopodia, membrane ruffles, and phagocytic cups in activated human neutrophils. J. Leukocyte Biol. 66,521 -527.[Abstract]
Diakonova, M., Bokoch, G. and Swanson, J. A.
(2002). Dynamics of cytoskeletal proteins during Fc
receptor-mediated phagocytosis in macrophages. Mol. Biol.
Cell 13,402
-411.
Didichenko, S. A., Segal, A. W. and Thelen, M. (2000). Evidence for a pool of coronin in mammalian cells that is sensitive to PI 3-kinase. FEBS Lett. 485,147 -152.[CrossRef][Medline]
Dumontier, M., Hocht, P., Mintert, U. and Faix, J.
(2000). Rac1 GTPases control filopodia formation, cell motility,
endocytosis, cytokinesis and development in Dictyostelium. J. Cell
Sci. 113,2253
-2265.
Edberg, J. C., Lin, C. T., Lau, D., Unkeless, J. C. and
Kimberly, R. P. (1995). The Ca2+ dependence of
human Fc gamma receptor-initiated phagocytosis. J. Biol.
Chem. 270,22301
-22307.
Ghazizadeh, S., Bolen, J. B. and Fleit, H. B.
(1994). Physical and functional association of Src-related
protein tyrosine kinases with Fc gamma RII in monocytic THP-1 cells.
J. Biol. Chem. 269,8878
-8884.
Gold, E. S., Underhill, D. M., Morrissette, N. S., Guo, J.,
McNiven, M. A. and Aderem, A. (1999). Dynamin 2 is required
for phagocytosis in macrophages. J. Exp. Med.
190,1849
-1856.
Greenberg, S. (2001). Diversity in phagocytic
signaling. J. Cell Sci.
114,1039
-1040.
Greenberg, S., Burridge, K. and Silverstein, S. C. (1990). Colocalization of F-actin and talin during Fc receptor-mediated phagoctosis in mouse macrophages. J. Exp. Med. 172,1853 -1856.[Abstract]
Greenberg, S., Chang, P. and Silverstein, S. C.
(1994). Tyrosine phosphorylation of the gamma subunit of Fc gamma
receptors, p72syk, and paxillin during Fc receptor-mediated phagocytosis in
macrophages. J. Biol. Chem.
269,3897
-3902.
Greenberg, S. and Silverstein, S. C. (1993). Phagocytosis. In Fundamental Immunology, 3rd edn (ed. W. E. Paul), pp. 941-964. New York: Raven Press.
Hackam, D. J., Rotstein, O. D., Schreiber, A., Zhang, W. and
Grinstein, S. (1997). Rho is required for the initiation of
calcium signaling and phagocytosis by Fcgamma receptors in macrophages.
J. Exp. Med. 186,955
-966.
Isakov, N. (1997). Immunoreceptor tyrosine-based activation motif (ITAM), a unique module linking antigen and Fc receptors to their signaling cascades. J. Leukocyte Biol. 61,6 -16.[Abstract]
Jung, G., Wu, X. and Hammer, J. A., 3rd (1996). Dictyostelium mutants lacking multiple classic myosin I isoforms reveal combinations of shared and distinct functions. J. Cell Biol. 133,305 -323.[Abstract]
Kwiatkowska, K. and Sobota, A. (1999). Signaling pathways in phagocytosis. BioEssays 21,422 -431.[CrossRef][Medline]
Le Cabec, V., Cols, C. and Maridonneau-Parini, I.
(2000). Nonopsonic phagocytosis of zymosan and Mycobacterium
kansasii by CR3 (CD11b/CD18) involves distinct molecular determinants and
is or is not coupled with NADPH oxidase activation. Infect.
Immun. 68,4736
-4745.
Lennartz, M. R. (1999). Phospholipases and phagocytosis: the role of phospholipid-derived second messengers in phagocytosis. Int. J. Biochem. Cell Biol. 31,415 -430.[CrossRef][Medline]
Mansfield, P. J., Shayman, J. A. and Boxer, L. A.
(2000). Regulation of polymorphonuclear leukocyte phagocytosis by
myosin light chain kinase after activation of mitogen-activated protein
kinase. Blood 95,2407
-2412.
Marshall, J. G., Booth, J. W., Stambolic, V., Mak, T., Balla,
T., Schreiber, A. D., Meyer, T. and Grinstein, S. (2001).
Restricted accumulation of phosphatidylinositol 3-kinase products in a
plasmalemmal subdomain during Fc receptor-mediated phagocytosis.
J. Cell Biol. 153,1369
-1380.
Maselli, A., Laevsky, G. and Knecht, D. A.
(2002). Kinetics of binding, uptake and degradation of live
fluorescent (DsRed) bacteria by Dictyostelium discoideum.Microbiology 148,413
-420.
Massol, P., Montcourrier, P., Guillemot, J. C. and Chavrier,
P. (1998). Fc receptor-mediated phagocytosis requires CDC42
and Rac1. EMBO J. 17,6219
-6229.
May, R. C., Caron, E., Hall, A. and Machesky, L. M.
(2000). Involvement of Arp2/3 complex in phagocytosis mediated by
FcR or CR3. Nat. Cell Biol.
2, 246-248.[CrossRef][Medline]
May, R. C. and Machesky, L. M. (2001).
Phagocytosis and the actin cytoskeleton. J. Cell Sci.
114,1061
-1077.
Niewohner, J., Weber, I., Maniak, M., Muller-Taubenberger, A.
and Gerisch, G. (1997). Talin-null cells of
Dictyostelium are strongly defective in adhesion to particle and
substrate surfaces and slightly impaired in cytokinesis. J. Cell
Biol. 138,349
-361.
Nobes, C. and Marsh, M. (2000). Dendritic cells: new roles for Cdc42 and Rac in antigen uptake? Curr. Biol. 10,R739 -R741.[CrossRef][Medline]
Norbury, C. C., Hewlett, L. J., Prescott, A. R., Shastri, N. and Watts, C. (1995). Class I MHC presentation of exogenous soluble antigen via macropinocytosis in bone marrow macrophages. Immunity 3,783 -791.[Medline]
Novak, K. D., Peterson, M. D., Reedy, M. C. and Titus, M. A. (1995). Dictyostelium myosin I double mutants exhibit conditional defects in pinocytosis. J. Cell Biol. 131,1205 -1221.[Abstract]
Olazabal, I. M., Caron, E., May, R. C., Schilling, K., Knecht,
D. A. and Machesky, L. M. (2002). Rho-kinase and myosin-II
control phagocytic cup formation during CR, but not FcR, phagocytosis.
Curr. Biol. 12,1413
-1418.[CrossRef][Medline]
Racoosin, E. L. and Swanson, J. A. (1989). Macrophage colony-stimulating factor (rM-CSF) stimulates pinocytosis in bone marrow-derived macrophages. J. Exp. Med. 170,1635 -1648.[Abstract]
Racoosin, E. L. and Swanson, J. A. (1992). M-CSF-induced macropinocytosis increases solute endocytosis but not receptor-mediated endocytosis in mouse macrophages. J. Cell Sci. 102,867 -880.[Abstract]
Radhakrishna, H., Klausner, R. D. and Donaldson, J. G. (1996). Aluminum fluoride stimulates surface protrusions in cells overexpressing the ARF6 GTPase. J. Cell Biol. 134,935 -947.[Abstract]
Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D. and Hall, A. (1992). The small GTP-binding protein Rac regulates growth factor-induced membrane ruffling. Cell 70,401 -410.[Medline]
Rittig, M. G., Wilske, B. and Krause, A. (1999). Phagocytosis of microorganisms by means of overshooting pseudopods: where do we stand? Microbes Infect. 1, 727-735.[CrossRef][Medline]
Ruchhoeft, M. L. and Harris, W. A. (1997). Myosin functions in Xenopus retinal ganglion cell growth cone motility in vivo. J. Neurobiol. 32,567 -578.[CrossRef][Medline]
Saitoh, M., Ishikawa, T., Matsushima, S., Naka, M. and Hidaka,
H. (1987). Selective inhibition of catalytic activity of
smooth muscle myosin light chain kinase. J. Biol.
Chem. 262,7796
-7801.
Sallusto, F., Cella, M., Danieli, C. and Lanzavecchia, A. (1995). Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J. Exp. Med. 182,389 -400.[Abstract]
Sells, M. A., Boyd, J. T. and Chernoff, J.
(1999). p21-activated kinase 1 (Pak1) regulates cell motility in
mammalian fibroblasts. J. Cell Biol.
145,837
-849.
Somlyo, A. P. and Somlyo, A. V. (1994). Signal transduction and regulation in smooth muscle. Nature 372,231 -236.[CrossRef][Medline]
Stendahl, O. I., Hartwig, J. H., Brotschi, E. A. and Stossel, T. P. (1980). Distribution of actin-binding protein and myosin in macrophages during spreading and phagocytosis. J. Cell Biol. 84,215 -224.[Abstract]
Swanson, J. A. and Baer, S. C. (1995). Phagocytosis by zippers and triggers. Trends Cell Biol. 5,89 -93.[CrossRef]
Swanson, J. A. and Watts, C. (1995). Macropinocytosis. Trends Cell Biol. 5, 424-428.[CrossRef]
Swanson, J. A., Johnson, M. T., Beningo, K., Post, P., Mooseker,
M. and Araki, N. (1999). A contractile activity that closes
phagosomes in macrophages. J. Cell Sci.
112,307
-316.
Theriot, J. A. (2000). The polymerization motor. Traffic 1,19 -28.[CrossRef][Medline]
Titus, M. A. (1999). A class VII unconventional myosin is required for phagocytosis. Curr. Biol. 9,1297 -1303.[CrossRef][Medline]
Vieira, O. V., Botelho, R. J., Rameh, L., Brachmann, S. M.,
Matsuo, T., Davidson, H. W., Schreiber, A., Backer, J. M., Cantley, L. C. and
Grinstein, S. (2001). Distinct roles of class I and class III
phosphatidylinositol 3-kinase in phagosome formation and maturation.
J. Cell Biol. 155,19
-25.
West, M. A., Prescott, A. R., Eskelinen, E. L., Ridley, A. J. and Watts, C. (2000). Rac is required for constitutive macropinocytosis by dendritic cells but does not control its downregulation. Curr. Biol. 10,839 -848.[CrossRef][Medline]
Zhang, Q., Calafat, J., Janssen, H. and Greenberg, S.
(1999). ARF6 is required for growth factor- and Rac-mediated
membrane ruffling in macrophages at a stage distal to Rac membrane targeting.
Mol. Cell. Biol. 19,8158
-8168.
Zhou, K., Pandol, S., Bokoch, G. and Traynor-Kaplan, A. E.
(1998). Disruption of Dictyostelium PI3K genes reduces
[32P]phosphatidylinositol 3,4 bisphosphate and
[32P]phosphatidylinositol trisphosphate levels, alters F-actin
distribution and impairs pinocytosis. J. Cell Sci.
111,283
-294.