From the Beirne B. Carter Center for Immunology Research and the Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908
Received for publication, December 13, 2000
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ABSTRACT |
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Apoptosis or programmed cell death occurs in
multicellular organisms throughout life. The removal of apoptotic
cells by phagocytes prevents secondary necrosis and inflammation and
also plays a key role in tissue remodeling and regulating immune
responses. The molecular mechanisms that regulate the engulfment
of apoptotic cells are just beginning to be elucidated. Recent
genetic studies in the nematode Caenorhabditis elegans have
implicated at least six genes in the removal of apoptotic cell corpses.
The gene products of ced-2, ced-5, and ced-10
are thought to be part of a pathway that regulates the reorganization
of the cytoskeleton during engulfment. The adapter proteins CrkII and
Dock180 and the small GTPase Rac represent the mammalian orthologues of
the ced-2, ced-5 and ced-10 gene products,
respectively. It is not known whether CrkII, Dock180, or Rac proteins
have any role during engulfment in mammalian cells. Here we show, using
stable cell lines and transient transfections, that overexpression of
wild-type CrkII or an activated form of Rac1 enhances engulfment.
Mutants of CrkII failed to mediate this increased engulfment. The
higher CrkII-mediated uptake was inhibited by coexpression of a
dominant negative form of Rac1 but not by a dominant a negative Rho
protein; this suggested that Rac functions downstream of CrkII
in this process, which is consistent with genetic studies in the worm
that place ced-10 (rac) downstream of
ced-2 (crk) in cell corpse removal. Taken
together, these data suggest that CED-2/CrkII and CED-10/Rac are part
of an evolutionarily conserved pathway in engulfment of apoptotic cells.
Programmed cell death is a process that occurs in various tissues
of the body throughout ontogeny (1). The apoptosis of specific cells is
closely linked to the removal of apoptotic bodies/cell corpses (2-5).
The removal of the dying cells before the integrity of the cell
membrane is lost prevents the release of potentially harmful contents,
thus protecting the neighboring cells. Engulfment also plays a key role
in tissue remodeling and in regulating immune responses (2-5). In
mammals, macrophages and immature dendritic cells are thought to carry
out the majority of the apoptotic cell removal, although clearly
other cell types can perform this function in different tissues (1, 4,
6).
The dying cells expose several "eat-me" signals or apoptotic
markers that can be recognized by the phagocytic cells through one or
more cell surface receptors (2). The subsequent intracellular signaling
events in the phagocyte lead to cytoskeletal rearrangements that
facilitate the engulfment of the dying cells. The signaling during
engulfment also regulates the cytokine secretion by the phagocyte (1,
2). One of the eat-me signals is the exposure on the cell
surface of phosphatidylserine
(PS),1 a lipid normally found
only on the inner leaflet of the plasma membrane and that can be
detected by annexin V staining (7). PS recognition by the phagocytes
can occur via CD36 (8, 9) and CD14 (10) as well as the recently cloned
unique PS receptor (11). PS exposure on the phagocyte has also been
seen under certain conditions (12). Modifications to glycosylation
patterns on cell surface proteins and a change in the surface charge of the dying cells can also serve as apoptotic markers, although the
mechanisms for their recognition have been less well defined (13, 14).
Although progress has been made in identifying some of the phagocytic
receptors, relatively little is known about coordination of the
specific intracellular signaling events downstream of these receptors
and how they regulate engulfment of apoptotic cells.
Elegant genetic studies in the nematode Caenorhabditis
elegans have implicated at least six genes segregated into two
partially redundant complementation groups as important in the
engulfment of apoptotic cells (with ced-1, ced-6,
and ced-7 in one group and ced-2,
ced-5, and ced-10 in another) (15-19). Recent
identification of the mammalian orthologues of five of the six genes
(19-23) has provided an exciting opportunity to better understand the
molecular events involved in mammalian engulfment. In the first
complementation group, ced-6 encodes an adapter protein,
hCED-6, containing a PTB (phosphotyrosine binding) domain,
ced-7 encodes the homologue of the mammalian ATP-binding
cassette transporter protein, ABC1, whereas the identity of
ced-1 has not yet been reported (17, 18, 22). Recent studies
indicate that both hCED-6 and ABC1 can play a role in mammalian
engulfment (20, 21, 24). In the second complementation group,
ced-2 encodes the homologue of the mammalian adapter protein
CrkII, ced-5 the homologue of the large adapter protein
Dock180, and ced-10 the small GTPase Rac (19). A role for
Crk has been recognized in the migration of fibroblasts (25). CrkII,
via its SH3 domain, interacts with Dock180, and Dock180 itself has been
shown to bind to nucleotide-free Rac (26, 27). Moreover, overexpression
of CrkII and Dock180 in 293T cells can enhance the formation of
GTP-bound Rac (27, 28). The activated GTP-bound form of Rac has been
shown to regulate the cytoskeleton in many systems including
macrophages (29, 30). Thus, based on the genetic studies in the worm,
it has been proposed that the
ced-2/ced-5/ced-10 gene products may
be part of pathway that regulates reorganization of the cytoskeleton during engulfment (19). However, it is not known whether CrkII, Dock180, or Rac proteins play a role in engulfment in mammalian cells.
In this report, we examined the role of the adapter protein CrkII and
the GTPase Rac1 in mammalian engulfment. We observe that overexpression
of CrkII or Rac in the LR73 cell line enhances phagocytosis and that
the CrkII-mediated increased uptake is dependent on downstream
signaling through Rac. These data suggest that CED-2/CrkII and
CED-10/Rac are part of an evolutionarily conserved pathway in
engulfment of apoptotic cells.
Cells--
The phagocytic LR73 Chinese hamster ovary cell line
was cultured in Alpha's modified Eagle's medium supplemented with
10% fetal calf serum and
penicillin/streptomycin/L-glutamine as described previously
(31).
Plasmids and Antibodies--
The plasmids encoding Myc-tagged wt
or mutant CrkII were kindly provided by Michiyuki Matsuda
(Tokyo, Japan). The plasmids encoding Rac1L61, Rac1N17, and
RhoN19 were provided by Dr. J. T. Parsons (University of Virginia),
and the PEZ-EGFP3 plasmid was provided by Dr. Ian Macara (University of
Virginia). pEBG-Shc, from our laboratory, has been described
previously (32). The anti-Myc and anti-CrkII antibodies were from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA).
Transfections--
For stable transfections, the LR73 cells were
transfected using the calcium phosphate method with 20 µg of
linearized plasmids encoding Myc-tagged rat CrkII wt or mutants (28)
along with 0.5 µg of a puromycin plasmid for selection. Stable single
colonies were picked and analyzed for CrkII expression by
immunoblotting using anti-c-Myc or anti-CrkII. Immunoprecipitation and
immunoblotting were performed as described previously (31). For
transient transfections, LipofectAMINE-2000 reagent (Life Technologies,
Inc.) was used as recommended by the manufacturer. At least two wells
were independently transfected for each condition/plasmid combination,
the duplicate samples were processed separately in the rest of the
experiment, and the mean and standard deviation were calculated. All
transient transfection experiments were performed multiple times with
the data presented being representative of at least three independent experiments. Briefly, 150,000 LR73 cells/well were plated in 24-well plates. The cells were transfected the next day by incubation with
plain medium containing 2 µl LipofectAMINE-2000 and 0.8 µg of each
plasmid for 6 h, washed, and incubated with fresh medium (10%
serum and no antibiotic) for 24 h before the phagocytosis assays
were performed as described below.
Engulfment Assays--
20,000 LR73 cells/well were plated in
24-well plates and used 2-3 days later. Apoptotic thymocytes used as
targets were labeled in one of two ways. Thymocytes from 6-week-old
mice were harvested and labeled with the lipophilic red fluorescent dye
PKH-26 (6 µM for 5 min). The excess dye was quenched and
the viable cells were purified using Lympholite M gradient (Cedarlane).
Alternatively, thymocytes were washed and resuspended in Hanks'
balanced salt solution (Life Technologies, Inc.) at 5 million cells in
100 µl, and mixed v/v with Hanks' balanced salt solution
containing 240 µM of the cytoplasmic cell tracker dye
CM-orange (5-(and-6)-4-chloromethyl-benzoylamino tetramethylrhodamine)
(Molecular Probes, Eugene, OR). After 2 min of incubation at room
temperature, the cells were incubated at 37 °C for 25 min followed
by a further incubation with fetal calf serum (v/v) for 25 min at
37 °C. Thymocytes labeled with either dye were induced to undergo
apoptosis by incubation with 2 µM dexamethasone for
4 h. Under these conditions, about 40-50% of the thymocytes were
Annexin V positive and less than 10% of Annexin V positive cells were
propidium iodide positive (i.e. necrotic). The thymocytes
were washed once and overlayed (at 500,000 cells/well in 300 µl of
growth medium) on LR73 cells. Routinely, the assays were carried out in
duplicates or triplicates for each condition with a 50-min incubation
for thymocytes and a 2-h incubation for beads. The wells were then
aspirated and washed twice with cold phosphate-buffered saline. The
cells on the plate were trypsinized, resuspended in cold medium (with
0.2% sodium azide), and analyzed by flow cytometry. Unengulfed
thymocytes were gated out by their forward and side scatter. Routinely,
10,000-20,000 events were collected and the data analyzed using
CellQuest software. The controls included the use of live thymocytes
(not treated with dexamethasone) and apoptotic thymocytes incubated
with LR73 cells at 4 °C. Although we trypsinized the cells prior to
analysis, the FACS assay cannot distinguish between fluorescence
derived from bound versus engulfed thymocytes. However, as
determined by confocal microscopy, the majority of the fluorescent
phagocytes scored in the FACS assay represents cells that have engulfed
the thymocytes or are in the process of engulfment (data not shown). For engulfment assays with 2 or 0.1 µm carboxylate-modified beads (Sigma), indicative of phagocytosis and pinocytosis, respectively, the
beads were incubated with cells for 2 h, washed extensively, and
analyzed as described above. Forward scatter was used to gate out the
unbound beads. To test the involvement of Rho family GTPases, Clostridium difficile toxin B (provided by Dr. Chang Hahn,
University of Virginia) was preincubated with the phagocytes at 10 ng/ml for 15 min and was present throughout the engulfment assay.
Overexpression of CrkII Enhances Phagocytosis--
Genetic studies
in C. elegans have indicated a role for the CED-2 orthologue
CrkII in the removal of apoptotic bodies (19). To test the role of Crk
proteins in engulfment in mammalian cells, we overexpressed Myc-tagged
CrkII in the phagocytic LR73 cell line. Stable LR73 clones expressing
various levels of CrkII proteins (Crk-LR73) were established. Compared
with parental LR73 cells, Crk-LR73 cells had an increased uptake of
apoptotic cells when the ratio of apoptotic versus live
cell uptake was assessed (an increase of 1.5-2-fold in four
independent experiments, p
To determine whether the enhanced uptake due to CrkII expression
reflects increased binding or internalization, we measured the
engulfment in the presence of sodium azide (Fig. 2b).
Because phagocytosis is an energy-dependent process, we
expected that incubation with azide would still allow binding without
the subsequent internalization. For both parental LR73 and Crk-LR73
cells, compared with incubation at 4 °C, we could clearly detect
"binding" in the presence of azide at 37 °C. However, the
difference in binding between these two cell populations was
minimal, whereas there was a much greater uptake by the Crk-LR73 cells
than by parental cells at 37 °C without azide. This suggested that
the increased uptake by CrkII overexpressing cells is primarily because
of enhanced internalization. The primary flow cytometry data used
to obtain the bar graphs are presented in the bottom panel of Fig.
2b.
CrkII is composed of one Src homology 2 (SH2) and two Src homology 3 (SH3) domains (Fig. 3). We compared the
engulfment by LR73 cells stably expressing either wt CrkII or CrkII
carrying mutations in either the SH2 domain (R38V) or the first SH3
domain (W169L), the latter being implicated in Dock180 binding (Fig. 3). Although LR73 clones expressing the lowest levels of Myc-tagged wt
CrkII still enhanced engulfment, this was not seen with LR73 expressing
the highest levels of the mutants. There was no difference between LR73
cells expressing wt and mutant CrkII when assessed for uptake of 0.1 µm beads (data not shown). It is possible that both the SH2 and the
first SH3 domain of CrkII are critical for Crk recruitment/function in
this system and that mutation of either domain may render the protein
functionally inactive and may explain the lack of enhancement or
inhibition with either mutant.
Rac Functions Downstream of CrkII in Mammalian
Engulfment--
Genetic studies using Drosophila Myoblast
city (a homologue of CED-5/Dock180) and overexpression studies
in 293T cells have indicated that CrkII binds to Dock180 via its SH3
domain and that overexpression of Crk and Dock180 can enhance the
formation of GTP-bound Rac, leading to cytoskeletal reorganization
(26-28). In wt Crk-LR73 cells, as expected, the immunoprecipitation of Myc-tagged CrkII co-precipitated Dock180 (data not shown). When we
attempted to generate Dock180-overexpressing cell lines to test the
role of Dock180 in engulfment, we were unable to obtain clones
expressing good levels of Dock180 protein. To determine whether the
enhanced phagocytosis due to CrkII overexpression is a result of
Crk signaling via Rac1, we examined the effect of C. difficile toxin B, which has been shown to inactivate Rho family
GTPases including Rac1 (35). Toxin B treatment abolished the engulfment
by parental as well as Crk-LR73 cells (Fig.
4a). To directly test the role
of Rac, we transiently transfected LR73 cells with plasmids coding for
CrkII, the constitutively active Rac1L61, or the dominant negative
Rac1N17. In all cases, we cotransfected a plasmid coding for the green
fluorescent protein (GFP) as a marker for transfected cells. We then
examined the engulfment of "red"-labeled 2 µm carboxylate beads
by the GFP positive "green" cells and scored the percentage of
"double positive" cells within the transfected population by flow
cytometry. Compared with GFP only transfected cells, transient
expression of CrkII enhanced the engulfment of carboxylate-modified
beads by about 4-fold, whereas Rac L61 increased the uptake by
3-4-fold (Fig. 4b). Under the same conditions, the
expression of Rac1N17 inhibited the uptake to about 50% of the GFP
control transfection (representative of three independent experiments).
Similarly, when LR73 cells transiently transfected with Rac1N17 and GFP
marker were tested for their ability to take up CM-orange-labeled
apoptotic thymocytes, there was a significant decrease in the uptake of
dying cells compared with control plasmid transfected cells (Fig.
4c). Under the same conditions, as seen in stable
transfectants (Fig. 1a), Crk overexpression had an increased
uptake of dead cells (170% compared with Shc; data not shown). These
data suggested that functional endogenous Rac proteins are important
for basal engulfment of apoptotic cells.
We then tested whether the increased uptake due to Crk overexpression
would also be sensitive to inhibition by Rac1N17 coexpression. The
N17Rac1 inhibited the Crk-mediated enhancement, suggesting that Rac1
functions downstream of CrkII in this process (Fig. 4d). In
contrast, expression of the dominant negative form of Rho (RhoN19)
along with CrkII did not inhibit Crk-mediated enhanced uptake.
Moreover, under these conditions, expression of another adapter
protein, Shc, did not increase the uptake or affect the RacL61- or
CrkII-mediated enhanced engulfment (Fig. 4d).
The data presented here indicate that the adapter protein CrkII,
through a pathway that involves the downstream signaling via Rac,
regulates engulfment of apoptotic cells. Together with the genetic
studies in the worm, the data presented here suggest that CrkII and Rac
proteins play an evolutionarily conserved role in the clearance of
apoptotic cells. The enhancement of engulfment due to wt CrkII
overexpression suggests that this signaling pathway may be one of the
rate-limiting steps. It was surprising to us that the CrkII proteins
with a mutation in either the SH2 domain or the first SH3 domain failed
to function as a dominant negative in engulfment. One possibility is
that both the SH2 and the first SH3 domain of CrkII may be critical for
Crk recruitment/function and that mutation of either domain may render
the protein "null." This likelihood may explain the lack of
enhancement or failure of inhibition of the basal engulfment due to
expression of either mutant. At present, the receptor(s) that function
upstream of CrkII during phagocytosis is not precisely known. Genetic
studies in C. elegans have not identified a receptor in the
second complementation group of genes (which encode the Crk, Dock180,
and Rac orthologues). Because some members of the integrin family of
surface receptors have been shown to signal via Crk (25, 36), the
potential role of an integrin receptor functioning upstream of CrkII
during engulfment seems plausible. During the review of this
manuscript, Albert et al. (37) reported that
Our observations that Rac overexpression alone can enhance engulfment
and that CrkII-mediated enhanced uptake is inhibited by dominant
negative RacN17 expression are consistent with the genetic studies in
the worm, where the deficiency of cell corpse removal in
ced-2 (crk)-deficient animals was rescued by
ced-10 (rac) overexpression. Interestingly, we
also observe that CrkII, Dock180, and Rac proteins can localize to the
glycosphingolipid-enriched membrane microdomains referred to as lipid
rafts (data not shown). Moreover, the disruption of lipid rafts
inhibited the engulfment of apoptotic
cells.2
Because actin polymerization has been linked to the lipid rafts, and
occurs downstream of the CrkII/Dock180/Rac proteins, our working hypothesis is that engagement of the engulfment receptor(s)
prelocalized or recruited to these microdomains may lead to the
initiation of signaling events via the Crk/Dock180/Rac signaling
pathway. In turn, through already known or yet to be defined Rac
effectors, this could lead to a reorganization of the actin
cytoskeleton and facilitate engulfment. Testing this model and defining
the specific localization of molecules during engulfment of apoptotic cells might lead to a better understanding of the molecular details of
this fundamentally important biological process.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
0.01) (Fig.
1a). CrkII-overexpressing
cells also showed an enhanced uptake of 2 µm carboxylate-modified
latex beads, which are thought to mimic the negative charge on dying
cells and have been used previously as a surrogate for apoptotic cells
(33, 34). The increased phagocytosis was specific, as there was no
detectable difference between parental and Crk-LR73 cells in the uptake
of Escherichia coli (Fig. 1b) or 0.1 µm beads
(indicative of pinocytosis) (data not shown). An analysis of multiple
clones expressing different levels of wt CrkII showed a roughly
dose-dependent increase in engulfment of 2 µm
carboxylate-modified beads (2-5-fold in 10 independent experiments)
(Fig. 2a). The primary flow
cytometry data from the engulfment assay that was used to generate the
bar graphs are shown also in Fig. 2a (bottom
panel). This enhancement in phagocytosis was specific for wt CrkII
overexpression, as stable clones expressing glutathione S-transferase
or another adapter protein, Shc, did not show enhanced phagocytosis
(data not shown; see also in Fig. 4).
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Fig. 1.
Overexpression of CrkII in LR73 cells
enhances phagocytosis. a, stable LR73 clone expressing
Myc-tagged CrkII (clone 32) or the control LR73 cells were
incubated for 50 min with fluorescently labeled apoptotic thymocytes
(see "Experimental Procedures"). The fraction of LR73 cells that
became fluorescent due to engulfment of thymocytes was analyzed by flow
cytometry. The backgrounds after incubation with live thymocytes have
been subtracted. The engulfment by control LR73 was set at 100%, and
the engulfment from four independent experiments (each performed on
duplicate samples) is shown (p < 0.01, n = 4). b, control LR73 cells and Crk-LR73
(clone 32) were analyzed for engulfment of 2 µm carboxylate beads or
E. coli bacteria (50 particles/cell) by flow
cytometry.
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Fig. 2.
CrkII overexpression causes a roughly
dose-dependent increase in uptake. a, six
different Crk-LR73 clones expressing different levels of the CrkII
protein were analyzed for engulfment of 2 µm beads as described in
the legend for Fig. 1b. The expression of Myc-tagged CrkII
was analyzed by immunoblotting (middle panel). We often see
two bands for c-Myc CrkII with the less intense upper band
corresponding to a phosphorylated form (data not shown). The primary
data obtained by flow cytometry to derive the data presented in the
bar graphs is also shown (bottom panel). In the
histograms, in the gate setting to determine the percentage of cells
containing engulfed particles, we excluded the sharp first peak as it
was seen even in the presence of azide, suggesting that this may
reflect binding rather than internalization (see data in Fig.
2b below). b, to distinguish whether the enhanced
engulfment seen was due to greater binding or internalization, parental
LR73 or Crk-LR73 cells were incubated with 2 µm carboxylate beads in
the presence or absence of 1% sodium azide at 4 °C or 37 °C.
Compared with cells incubated at 4 °C, the greater percentage of
fluorescent cells at 37 °C in the presence of azide is considered to
reflect cells with bound fluorescent beads, whereas incubation at
37 °C without azide reflects both binding and internalization. The
primary flow cytometric data is presented below the bar
graphs.
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Fig. 3.
CrkII-mediated enhanced engulfment requires
both the SH2 and SH3 domains of Crk. Different stable LR73 clones
expressing either the wt, W169L (mutant in the SH3 domain), or R38V
(mutant in the SH2 domain) were analyzed for engulfment with 2 µm
carboxylate beads. The wt clones are the same ones shown in Fig.
2a above. The relative expression levels of the transfected
proteins are shown in the middle panel. The bottom
panel depicts the schematic diagram of wt CrkII and the
mutants.
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Fig. 4.
CrkII-mediated enhancement of phagocytosis
depends on functional Rac proteins. a, parental or
Crk-LR73 cells (clones #32 and #1 shown in Fig. 2a,
high and low expressing clones, respectively) were incubated with
C. difficile toxin B (10 ng/ml final) for 15 min before
incubation with 2 µm carboxylate beads. b, LR73 cells were
transiently transfected, in duplicates, with an EGFP plasmid along with
CrkII, Rac1L61, and Rac1N17. 24 h post-transfection the cells were
analyzed for engulfment as described under "Experimental
Procedures" using 2 µm carboxylate-modified beads. The percentage
of GFP positive cells ranged from 20-30% in the individual
transfections. Within these GFP positive cells, the fraction that
became "double positive" because of uptake of the red beads is
expressed as a percentage after normalizing for transfection
efficiency. c, LR73 cells transiently transfected (in
duplicates) with plasmids encoding either Shc (control) or Rac1N17
along with an EGFP marker plasmid were tested for the engulfment of
CM-orange-labeled apoptotic thymocytes by flow cytometry as described
under "Experimental Procedures." The percent uptake between samples
was normalized for transfection efficiency (as determined by the % of
GFP positive cells), and the uptake by the control Shc-transfected
cells was set at 100%. The Rac1N17 data presented here represent the
mean ± S.D. for two independent experiments (the S.D. is not
visible, as it was too small). d, LR73 cells were
transiently transfected in duplicates with an EGFP plasmid in different
combinations with CrkII, Rac1L61, Rac1N17, RhoN19, and Shc plasmids and
analyzed as described for panel b.
v
5 integrin may play a role in signaling
upstream of Crk during engulfment of apoptotic cells. Moreover, using a
model system of HEK 293T cells as phagocytes, these authors
observed that overexpression of CrkII caused a decreased uptake of
apoptotic cells. Whether the discrepancy between our results and those
of Albert et al. reflects the different cell types being
used as phagocytes, or perhaps the much greater overexpression of
transfected plasmids achievable in T antigen-expressing 293T cells
compared with LR73 cells, remains to be determined. Nevertheless, both
sets of data implicate a function for CrkII-mediated signaling during engulfment.
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ACKNOWLEDGEMENTS |
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We thank Michiyuki Matsuda for the CrkII reagents and Chang Hahn for providing the C. difficile toxin B.
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FOOTNOTES |
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* This work was supported by grants from the National Institutes of Health (to K. S. R.) and a fellowship from the Association pour la Recherche contre le Cancer (to A.-C. T.-T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Beirne Carter Ctr. for
Immunology Research, University of Virginia, Bldg. MR4, Rm. 4012F, HSC,
Charlottesville, VA 22908-1386. Tel.: 804-243-6093; Fax: 804-924-1221;
E-mail: Ravi@virginia.edu.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M011238200
2 A.-C. Tosello-Trampont and K. S. Ravichandran, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: PS, phosphatidylserine; SH2/SH3, Src homology 2/3; wt, wild type; FACS, fluorescence-activated cell sorter; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein.
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