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Cell Motility and the Cytoskeleton 37:253–262 (1997)
Actin in the Parasite Toxoplasma gondii
is Encoded by a Single Copy Gene,
ACT1 and Exists Primarily in a Globular Form
Janice M. Dobrowolski, Ingrid R. Niesman, and L. David Sibley*
Department of Molecular Microbiology,
Washington University School of Medicine, St. Louis, Missouri
Actin is a highly conserved microfilament protein that plays an important role in
the invasion of host cells by the protozoan parasite Toxoplasma gondii. We have
characterized the ACT1 gene and localized the conventional isoform of actin that it
encodes within T. gondii. The predicted amino acid sequence of ACT1 was most
similar to two other parasite actins, Plasmodium falciparum Pfact-1 (93.1%
identical) and Cryptosporidium parvum actin (88.1%): among vertebrate actins,
ACT1 was most closely related to the mammalian b and g (83%) actin isoforms.
Actin-specific antibodies and fluorescently labeled DNAse I were used to localize
actin in T. gondii tachyzoites by immunofluorescence and immunoelectron
microscopy. Actin was detected beneath the parasite cell membrane and in clusters
scattered within the cytosol of T. gondii tachyzoites. Actin filaments were not
detected in detergent-solubilized parasites separated by high speed centrifugation,
indicating that actin exists primarily in a globular form in T. gondii. Cell Motil.
Cytoskeleton 37:253–262, 1997. r 1997 Wiley-Liss, Inc.
Key words: microfilament; motility; invasion; cytoskeleton; parasite
INTRODUCTION
Toxoplasma gondii is an obligate intracellular parasite that can invade all types of nucleated cells from a
wide variety of mammalian hosts. Several lines of
evidence indicate that T. gondii enters host cells by an
active process. First, T. gondii tachyzoites have the ability
to invade cells that are not normally phagocytic including
fibroblasts, endothelial, and epithelial cells [Werk, 1985].
Additionally, light fixation of macrophages with glutaraldehyde, which prevents phagocytosis, does not block
invasion of T. gondii tachyzoites [Nichols and O’Connor,
1981]. Finally, T. gondii invasion occurs much more
rapidly than phagocytosis and proceeds without host cell
membrane ruffling or actin filament rearrangement
[Morisaki et al., 1995].
T. gondii tachyzoites are capable of movement
although they lack flagella, cilia, or other organelles
normally associated with locomotion. Tachyzoite movement is substrate-dependent and is characterized by a
helical rotation of the organism as it glides forward along
the surface [Russell and Sinden, 1981]. Cytochalasins,
r 1997 Wiley-Liss, Inc.
which interfere with microfilament assembly by destabilizing actin polymerization [Cooper, 1987], have profound effects on parasite motility and invasion. Cytochalasin D blocks invasion of T. gondii into a variety of
mammalian cell types including mouse peritoneal macrophages, bladder tumor 4934 cells [Ryning and Remington, 1978], human fibroblasts [Schwartzman and Pfefferkorn, 1983], and LA-9 cells [Silva et al., 1982]. The
related compound cytochalasin B blocks invasion of red
blood cells by P. knowlesi merozoites [Miller et al., 1979]
and blocks the gliding motility and invasion by Eimeria
sporozoites [Russell and Sinden, 1981; Russell, 1983].
The ability of cytochalasins to inhibit invasion of these
Contract grant sponsor: National Institutes of Health; Contract grant
number: AI 34036; Contract grant number: AI 07172; Contract grant
sponsor: Burroughs Wellcome.
*Correspondence to: Dr. L. David Sibley, Dept. of Molecular Microbiology, Box 8230, Washington University School of Medicine, St.
Louis, MO 63110.
Received 6 January 1997; accepted 6 March 1997.
254
Dobrowolski et al.
related protozoan parasites implies they share a common
invasion mechanism that depends on actin polymerization as has recently been shown for T. gondii [Dobrowolski and Sibley, 1996].
Previous studies have reported the localization of
actin in T. gondii using antisera to heterologous actins
from Entamoeba histolytica [Cintra and De Souza, 1985],
Ascaris body wall [Yasuda et al., 1988], or chicken
smooth muscle [Endo et al., 1988]. By indirect immunofluorescence, actin was reported to be primarily concentrated in the anterior one-third of the tachyzoite and in a
diffuse staining pattern throughout the cytoplasm [Cintra
and De Souza, 1985; Endo et al., 1988]. Immunoelectron
microscopy (EM) staining with heterologous antisera
localized actin at the apical end of the parasite in the
conoid and associated with subpellicular microtubules
[Yasuda et al., 1988]. Myosin has also been localized to
the apical end of tachyzoites by immunofluorescence
using antisera to cricket muscle myosin [Schwartzman
and Pfefferkorn, 1983]. While these studies provide
preliminary evidence for the presence of microfilament
proteins in T. gondii, they are based exclusively on
heterologous reagents from distantly related organisms.
To provide improved reagents for examining the
distribution of actin in T. gondii, we have isolated and
characterized the ACT1 gene. Furthermore, we have
localized actin in T. gondii by immunofluorescence,
immuno-EM, and cell fractionation using highly specific,
affinity-purified antibodies.
MATERIALS AND METHODS
Growth of Parasites and Isolation of DNA
Tachyzoites of T. gondii strain RH88 [Pfefferkorn
and Pfefferkorn, 1976] were grown in human foreskin
fibroblasts (HFF) maintained in Dulbecco’s modified
Eagle’s medium supplemented with 10% fetal calf serum,
2 mM glutamine and gentamycin (25 µg/ml). Tachyzoites
were isolated by syringe passage, filtration through 3.0
micron filters and centrifugation in HBSS supplemented
with 10 mM HEPES and 10 U/ml heparin [Sibley and
Boothroyd, 1992]. Genomic DNA was isolated from
intact HFF cells or purified parasites by sodium dodecyl
sulfate (SDS)/proteinase K lysis followed by phenolchloroform extraction and ethanol precipitation [Sibley
and Boothroyd, 1992].
Genomic Southern Analysis
Genomic DNAs from HFF cells or T. gondii were
digested with restriction endonucleases, electrophoresed
in a 1% agarose gel, and transferred to a nylon membrane
by alkaline capillary transfer [Maniatis et al., 1982]. Blots
were probed overnight with 32P-labeled pTact-1 (a partial
cDNA clone containing two-thirds of the ACT1 gene) at
high stringency (42°C in 6 3 SSPE, 0.5% SDS, 50%
formamide) and washed at 55°C in 0.1 3 SSPE, 0.5%
SDS. Filters were exposed to Kodak XAR film at 270°C
for autoradiography.
Contour-Clamped Homogeneous Electric Field
(CHEF) Gel Electrophoresis
Chromosome plugs from T. gondii strain RH88 and
Plasmodium falciparum strain HB3 were separated on a
0.7% chromosome-grade agarose gel in 0.25 3 TBE (13
is 90 mM Tris-borate, 2 mM EDTA pH 8.0) using the
CHEF-MAPPER apparatus (BioRad Laboratories, Hercules, CA). Gels were run at 14°C for 72 hours at 1.8
V/cm, with a fixed angle of 1100, and a 15 minute pulse
time. The chromosomal DNA was depurinated 2 3 30
minutes in 0.25 M HCl and transferred to nylon membranes by alkaline capillary transfer [Sibley and
Boothroyd, 1992]. Blots were probed with 32P-labeled
pTact-1 at 42°C in 6 3 SSPE, 0.5% SDS, 50% formamide
and washed at 50°C in 0.1 3 SSPE, 0.5% SDS.
Northern Blot Analysis
RNA was isolated from T. gondii tachyzoites by
disruption in 6 M guanidine thiocyanate, 0.1 M Tris, and
1% 2-mercaptoethanol [Maniatis et al., 1982]. Approximately 5 µg of total RNA per lane was separated on a 1%
agarose gel containing 6.6% formalydehyde in buffer
consisting of 20 mM 3-[N-morpholino] propanesulfonic
acid, 8 mM sodium acetate, and 1 mM EDTA. Gels were
capillary-transferred to nitrocellulose in 20 3 SSC,
hybridized overnight at 42°C in 6 3 SSPE, 0.5% SDS,
50% formamide with 32P-labeled pTact-1 as a probe, and
washed at room temperature in 0.2 3 SSPE, 0.5% SDS.
Production of Antisera and Western Blotting
The complete cDNA coding sequence of the T.
gondii actin gene was cloned into the pGEX-3X vector
system [Smith and Johnson, 1988]. The resulting ACT1GST fusion protein produced in E. coli was purified on
glutathione-agarose beads (Sigma Chemical. Inc., St.
Louis, MO) and used to immunize a rabbit. A second
rabbit was immunized with GST alone. IgGs were
purified using Protein A-sepharose (Bio-Rad Laboratories). Affinity-purified antibodies, referred to as antiACT1, were purified on recombinant GST-sepharose
(flow-through fraction) followed by ACT1-GST-sepharose (eluted fraction). The specificity of antisera was
tested by western blotting [Maniatis et al., 1982] of
proteins separated by SDS-PAGE [Laemmli, 1970]. Following transfer to nitrocellulose, membranes were blocked
with 1% nonfat dried milk (NFDM) in phosphatebuffered saline (PBS). Primary antibodies were diluted in
1% NFDM in PBS and incubated for 1 hr at the following
dilutions: C4 1:200; rabbit anti-GST 1:500, rabbit anti-
Characterization of Toxoplasma Actin
ACT1 1:500. Membranes were washed and incubated
with 125I-goat anti-mouse IgG or 125I-goat anti rabbit IgG
(ICN, Pharmaceuticals Inc, Costa Mesa, CA) followed by
washing and autoradiography.
Indirect Immunofluorescence (IF) and Direct
Fluorescence Microscopy
Parasites were fixed with 2.5% formalin, 0.5%
glutaraldehyde, 0.02% saponin in actin stabilization buffer
(60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM
MgCl2 and 0.125 M KCl; P. Bridgeman, Washington
University, personal communication). Following fixation,
slides were extracted by submersion in cold acetone
(220°C) for 2 min followed by rinsing in PBS. For IF,
slides were blocked in PBS containing 10% FBS, incubated with primary antibodies diluted in PBS-1% fetal
bovine serum (FBS), washed, and incubated in FITClabeled goat anti-rabbit IgG or goat anti-mouse IgG
antibodies (Sigma Chemical, Inc.) diluted in PBS-1%
FBS. For direct fluorescence staining, parasites were
fixed as described above and incubated for 30 min with
FITC-DNAse I (Molecular Probes Inc., Eugene, OR)
diluted in PBS. Slides were mounted in 10% glycerolPBS or ProLong anti-fade media (Molecular Probes),
examined and photographed using a Zeiss Axioscope
microscope.
Immuno-EM
Extracellular T. gondii tachyzoites were fixed for 1
hr at 4°C with 2.5% formalin, 0.5% glutaraldehyde,
0.02% saponin in actin-stabilization buffer. Samples were
then embedded in 10% gelatin, infiltrated with sucrose/
polyvinylpyrrolidone, and frozen in liquid N2. Ultrathin
cryosections were blocked in 0.12 mM glycine and
PBS-10% FBS, then incubated in primary antibodies
diluted in PBS-1% FBS followed by secondary antibodies, goat anti-rabbit or goat anti-mouse IgG coupled to
18-nm colloidal gold (Jackson Laboratories, Bar Harbor,
ME), diluted in PBS-1% FBS. Sections were counterstained with 0.15 M oxalic acid/2% uranyl acetate and
stained with a 1:2 mixture of 2% methylcellulose and 4%
uranyl acetate and examined using a Zeiss EM902
microscope.
Saponin Permeabilization and Cell Fractionation
The RH strain transformant 2F, which constitutively expresses b-galactosidase (b-Gal) [Dobrowolski
and Sibley, 1996] was used to examine the relative
amounts of filamentous and globular actin in detergentpermeabilized cells. Freshly harvested tachyzoites were
resuspended in PBS, actin stabilization buffer, or MKEI
buffer (100 mM KCl, 1 mM EGTA, 2 mM MgCl2, 20 mM
imidazole [Karpova et al., 1995] containing 0.1% saponin
(Sigma Chemical, Inc.) in the absence or presence of 50
255
nM phalloidin (Molecular Probes). As a positive control,
HFF cells were processed in parallel. Samples were
incubated on ice for 30 min and then centrifuged at
100,000 3 g for 30 min at 4°C. The supernatant and pellet
fractions were separated on SDS-PAGE gels, transferred
to nitrocellulose, and western-blotted. b-Gal was detected
with mAb 40-1a (kindly provided by J. Sanes, Washington University).
RESULTS
Genomic Characterization and Expression
of ACT1
The T. gondii ACT1 gene was isolated by screening
a cDNA library [Burg et al., 1988] with a chicken b-actin
probe [Cleveland et al., 1980], hybridized at moderate
stringency (63 SSPE, 23 Denhardt’s, 50% formamide at
37°C overnight followed by final washing at 32°C, 0.23
SSPE, for 30 min). A series of overlapping clones were
sequenced to generate a composite cDNA sequence
spanning from 83 bp upstream of the ATG to 385 bp
downstream of the stop codon. Neither the 58 end of the
mature message nor the polyadenylation site were identified. However, both presumably lie within the corresponding genomic locus that was also sequenced and which
contains a 420-bp intron interrupting the coding region
and an additional 560 bp of upstream sequence. This
entire 2,586-bp nucleotide sequence was been submitted
to the GenBanky data base with the accession number
U10429.
Southern blot analysis revealed that ACT1 is likely
present as a single-copy gene in T. gondii based on the
similar restriction maps of cloned fragments and total
genomic DNAs. In the example shown in Figure 1A, the
probe pTact-1 hybridized to two fragments in genomic
HindIII and PstI digests but to only a single fragment in
genomic EcoRI digests of T. gondii. This result is
consistent with the presence of a single restriction site
each for HindIII and PstI and the absence of an EcoRI site
in the probe. Additional mapping studies and the transfection of a cytochalasin-resistant allele of actin are also
consistent with the presence of a single copy of the ACT1
gene in T. gondii (Dobrowolski and Sibley, 1996). Low
stringency hybridization (hybridized: 26°C, 63 SSPE,
23 Denhardt’s, 50% formamide, washed: 0.23 SSPE,
26°C) did not reveal additional alleles or closely related
actin-like genes (data not shown). To determine the
genomic location of the ACT1 gene, Southern blot
analysis was performed on chromosomes separated by
CHEF gel electrophoresis (Fig. 1B). Hybridization with
pTact-1 showed that ACT1 is located on the smallest
band, which contains chromosomes IA and IB (Fig. 1B).
pTact-1 did not cross-hybridize to any of the P. falciparum chromosomes, presumably because of the high A/T
256
Dobrowolski et al.
Fig. 1. A: Genomic Southern analysis of ACT1 in T. gondii. pTact-1
hybridizes specifically to bands in genomic digests of T. gondii (lanes
1–3) but not HFF cells (lane 4) used to propagate T. gondii. Positive
control plasmid pTact-1 was digested with EcoRI and loaded at
approximate molar equivalents of 1 and 10 genomic copies, respectively. B: Chromosome separation of T. gondii (Tg) strain RH and P.
falciparum (Pf) strain HB3 by CHEF gel electrophoresis. Left,
ethidium bromide-stained gel. Right, Southern blot probed with
pTact-1 that hybridized to chromosomes IA and IB. Molecular weights
were estimated based on P. falciparum and Sacchromyces cerevisiae
strain YNN295 (not shown) [Sibley and Boothroyd, 1992]. C: Northern blot analysis of actin mRNA in T. gondii. Left, ethidium bromidestained gel. Right, Northern blot probed with pTact-1 that hybridized to
a single band approximately 2.1 kb in size.
content of the P. falciparum genome. A single message of
2.1 kb was detected in a Northern blot of total RNA from
T. gondii tachyzoites probed with pTact-1 (Fig. 1C).
lie on the external surface of the molecule [Kabsch et al.,
1990].
Generation of T. gondii Actin-Specific Antibodies
Similarity of T. gondii ACT1 to Other Actins
A search of the Swiss-protein data base was performed using the FastA algorithm [Pearson and Lipman,
1988]. The top 20 matches with the predicted amino acid
sequence were all actins, and T. gondii ACT1 was most
similar to Pfact-1 from Plasmodium falciparum [Wesseling et al., 1988a] and Cryptosporidium parvum actin
[Kim et al., 1992]. T. gondii ACT1 was only 83%
identical to the human g- and b-actin isoforms [NakajimaIijima et al., 1985; Erba et al., 1988]. An alignment of the
predicted amino acid sequences of T. gondii, P. falciparum, C. parvum, and human g and b actin isoforms is
shown in Figure 2. The three parasite actins from T.
gondii, P. falciparum and C. parvum contain an extra
N-terminal amino acid in their sequence when compared
to vertebrate actin isoforms. The ACT1 sequence was
most divergent from related actins at amino acids 269–
281, 223–225, and 27–46 which, based on the crystal
structure of rabbit skeletal actin complexed to DNAse I,
To provide reagents for immunodetection, rabbit
antibodies were generated to recombinant ACT1 and
compared in specificity by SDS-PAGE and western
blotting to the mAb C4 which reacts to a diverse array of
actins [Lessard, 1988]. The mAb C4 recognized the
expected 43-kD band comprising actin isoforms in HFF
cells (Fig. 3, lane 4). The mAb C4 recognized a slightly
higher band (44 kD) in lysates of T. gondii tachyzoites
(Fig. 3, lane 2) and also specifically recognized the
ACT1-GST fusion protein produced in E. coli (data not
shown). Affinity-purified anti-ACT1 antibodies reacted to
the same 44-kD tachyzoite protein recognized by mAb
C4 (Fig. 3, lane 3) but did not react with actin from HFF
host cells (Fig. 3, lane 5). Serum from a rabbit immunized
with GST alone did not recognize any proteins in
tachyzoites (Fig. 3, lane 1). Collectively these results
demonstrate that rabbit anti-ACT1 antibodies specifically
recognize a single isoform of actin in T. gondii.
Characterization of Toxoplasma Actin
Fig. 2. Amino acid sequence alignment of the actin sequences from T. gondii (this report), P. falciparum
Pfact-1 [Wesseling et al., 1988a], C. parvum actin [Kim et al., 1992], and human g- and b-actins
[Nakajima-Iijima et al., 1985; Erba et al., 1988]. Amino acids identical to those of T. gondii ACT1 are
indicated by periods.
257
258
Dobrowolski et al.
Fig. 3. Western blot of T. gondii tachyzoites (lanes 1–3) and HFF cell
lysates (lanes 4–5). Lane 1: whole serum from rabbit immunized with
GST alone (a-GST). Lanes 2 and 4: C4 monoclonal anti-actin
antibody [Lessard, 1988]. Lanes 3 and 5, affinity-purified rabbit
anti-ACT1 antibodies (a-ACT1).
Localization of Actin in T. gondii
Actin was localized in T. gondii by indirect IF
staining of extracellular tachyzoites, that were allowed to
glide on serum coated glass before fixation [Dobrowolski
and Sibley, 1996], or intracellular parasites grown within
HFF cells. Extracellular parasites stained with anti-GST
antibodies exhibited minimal background staining (Fig.
4A,A8). Staining of extracellular parasites with antiACT1 antibodies revealed a diffuse cytoplasmic labeling
that was more intense at the perimeter (Fig. 4B,B8). Both
the apical end, including the conoid, and the posterior end
of the parasite were prominently stained by the antiACT1 antibodies (Fig. 4B,B8). Similar results were
observed with the mAb C4 (data not shown). Anti-ACT1
antibodies exhibited minimal staining of host cell actin,
but strongly labeled intracellular parasites in a circumferential pattern that was less intense at the apical end (Fig.
4C,C8). To examine the distribution of globular actin,
extracellular parasites were stained with FITC-DNAse I
which revealed a punctate pattern of staining throughout
the cytoplasm of the cell (Fig. 5A,A8). Intracellular T.
gondii stained with DNAse I exhibited a punctate staining
pattern that was concentrated primarily at the apical end
of the parasite (Fig. 5B,B8).
The distribution of actin in extracellular T. gondii
tachyzoites was also examined by immuno-EM. Minimal
background staining was observed in tachyzoites stained
with rabbit anti-GST (Fig. 6A). In cryosections stained
with anti-ACT1 antibodies, actin was localized beneath
the parasite plasma membrane, closely associated with
Fig. 4. Immunofluorescence labeling of actin in T. gondii tachyzoites.
(A,A8) Background fluorescence staining of extracellular parasites
incubated with rabbit anti-GST sera. (B,B8) Extracellular parasites
stained with anti-ACT1 antibodies showing a diffuse intracellular
labeling and intense staining at both the apical (top) and posterior ends
(arrowhead). C,C8: Intracellular rosettes stained with anti-ACT1
antibodies. Pattern is primarily circumferential with less intense
staining of the outward-pointing, apical ends. Bar 5 5 µm.
the inner membrane complex and in scattered clusters in
the cytosol (Fig. 6B and data not shown). Actin was not
restricted to the apical end of the parasite, but was present
beneath the plasma membrane at both the apical (Fig. 6C)
and posterior (Fig. 6D) ends of the parasite. In cryosections stained with mAb C4, actin was also detected
beneath the parasite plasma membrane and in discreet
clusters scattered throughout the cytoplasm (data not
shown).
Separation of Globular and Filamentous Actin
To analyze the physical state of actin in T. gondii
tachyzoites, cells were gently permeabilized with saponin
and centrifuged to separate the detergent soluble (globular) and insoluble (filamentous) actin. HFF host cells
Characterization of Toxoplasma Actin
Fig. 5. Direct fluorescence labeling with FITC-DNAse I. Extracellular
T. gondii tachyzoites stained with DNAse I (A,A8). Punctate staining is
seen scattered throughout the cell. Intracellular parasites stained with
DNAse I (B,B8 ). Staining is primarily apical with some diffuse
cytoplasmic staining. Bar 5 5 µm.
contained both filamentous actin and globular actin that
were present in the high speed pellet (P) and supernatant
(S), respectively (Fig. 7). In contrast, all of the actin
detected in T. gondii preparations, regardless of the buffer
conditions used, was in the soluble fraction indicating it
was primarily globular (Fig. 7). b-Gal was also exclusively found in the soluble fraction confirming the
parasites were completely permeabilized (Fig. 7). The
addition of excess phalloidin, which normally stabilizes
actin filaments in eukaryotic cells including yeast, did not
alter the distribution of actin in T. gondii which remained
predominantly globular (Fig. 7, 1ph). Because phalloidin
does not stain T. gondii [Cintra and DeSousa, 1985], this
result may indicate a failure of phalloidin to bind to T.
gondii actin rather than an absence of filaments. However, the addition of other agents known to stabilize
filaments also had no effect on the pool of globular actin
in T. gondii including addition of: purified rabbit cytoskeletal actin (10 µg/ml), MgCl2 (50 mM), spermine (5 mM),
or PIP2 (1 mM) (data not shown). Collectively, these
studies indicate that the majority of actin in T. gondii cell
lysates exists in a globular form.
DISCUSSION
We have characterized the gene encoding a conventional actin isoform from Toxoplasma gondii. ACT1 is
259
apparently present as a single-copy gene in T. gondii that
is abundantly transcribed and translated into a single actin
isoform of 44 kD. The closely related parasite, Plasmodium falciparum, has two actin isoforms, Pfact-1 and
Pfact-2, which are expressed in different stages of the life
cycle [Wesseling et al., 1988, 1989]. We have not
examined actin expression in different life cycle stages of
T. gondii, but genomic Southern analysis failed to detect
other closely related actin genes.
The predicted amino acid sequence of T. gondii
ACT1 showed greatest sequence identity to P. falciparum
Pfact-1 (93.1%) and C. parvum actin (88.1%). All three of
these organisms belong to the phylum Apicomplexa that
is unified by the presence of specialized apical structures
involved in host cell invasion. All three contain forms of
actin that include an additional acidic residue at the
N-terminus relative to conventional vertebrate actins. The
N-terminal region of actin is important in controlling
polymerization through interactions with actin binding
proteins. In yeast, Dictyostelium, and vertebrates, actins
are typically post-translationally processed to remove 1
or 2 N-terminal amino acids followed by acetylation of
the new N-terminus [Rubenstein, 1990; Cook et al.,
1991]. It is not known if T. gondii actin or those of other
apicomplexans are post-translationally processed in an
analogous fashion.
We also generated ACT1-specific antibodies and
used them to localize actin in T. gondii by immunofluorescence microscopy and immuno-EM. In extracellular
parasites, actin was localized as diffuse cytoplasmic
staining that was more intense at the perimeter and
included both ends of the organism. Immuno-EM staining
revealed that actin is primarily located beneath the
plasma membrane in a pattern that included both the
apical and posterior ends of the parasite. Similar results
were obtained using mAb C4, which recognizes all
known actin isoforms [Lessard, 1988] including recombinant ACT1 from T. gondii. Clusters of globular actin,
located primarily in the apical half of the cell, were also
revealed by DNAseI staining. These pools of globular
actin may play a role in the regulation of actin assembly
and/or cell locomotion by providing a local concentration
of actin subunits for structural assembly as has been
suggested for mammalian cells [Cao et al., 1993]. Differences in the intensity of apical staining of actin between
nonmotile, intracellular parasites and actively gliding,
extracellular parasites may reflect a redistribution in the
cytoskeleton that is associated with motility.
Our results differ from previous reports that suggested actin was primarily concentrated at the apical end
of the parasite [Cintra and DeSousa, 1985; Endo et al.,
1988; Yasuda et al., 1988]. While there are several
possible explanations for these differences, they are
260
Dobrowolski et al.
Fig. 6. Immuno-EM localization of actin in extracellular T. gondii tachyzoites. Actin is localized beneath
the parasite plasma membrane at both the apical end (C) and posterior end (D) and scattered throughout the
cytoplasm. A: Stained with rabbit anti-GST; B–D: stained with rabbit anti-ACT1 antibodies and goat
anti-rabbit IgG conjugated to 12 nm gold. Bar 5 0.1 µm: c, conoid; n, nucleus; d, dense granule; r, rhoptry.
unlikely to result from differences in fixation conditions
as we have observed similar circumferential actin staining in aldehyde, methanol of acetone-treated parasites
(Dobrowolski and Sibley unpublished). Instead, the use
of antisera to heterologous actins in these previous
studies may have resulted in incomplete detection of T.
gondii actin thereby enhancing the apparent concentration in the apical end of the organism. Whether this
reflects a true difference in the concentration of actin or
simply the relative accessibility of cross-reactive epitopes
is uncertain. Differences in patterns of actin staining may
also reflect the activity level of the cell prior to fixation:
Characterization of Toxoplasma Actin
Fig. 7. Detection of filamentous (P, pellet) and globular (S, supernatant) actin in saponin permeabilized cells resuspended in MKEI buffer.
Actin in HFF cells was found in both the S and P fractions (detected
with mAb C4). Actin in T. gondii was exclusively in the S fraction
either with (1ph) or without (2ph) the addition of phalloidin (detected
with rabbit a-ACT1). Similar fractions of clone 2F were immunoblotted with mAb 40-1a recognizing b-Gal to ensure complete permeabilization of the parasites.
dramatic changes in the cytoskeleton are known to occur
in vertebrate cells undergoing attachment, motility and
cell division. Consistent with this, the one other previous
study that also used adherent parasites (poly-L-lysine
instead of serum-coated glass used here) also reported
posterior staining in 25% of cells [Cintra and DeSouza,
1985].
Despite evidence that filamentous actin plays a
critical role in gliding motility and host cell invasion by T.
gondii [Dobrowolski and Sibley, 1996], it has thus far not
been possible to visualize actin filaments in any apicomplexan parasite using conventional methods [Russell and
Sinden, 1982; Cintra and DeSouza, 1985]. Consistent
with this, filamentous actin was not observed in detergent
permeabilized T. gondii tachyzoites examined by cellfractionation, SDS-PAGE, and western blotting. These
extraction and precipitation procedures were capable of
preserving actin filaments, as both filamentous and globular actin were detected in detergent preparations of HFF
host cells. The abundance of globular actin in T. gondii
was also not changed by addition of agents that normally
stabilize filaments during the extraction procedure. These
data indicate that the majority of actin in T. gondii
tachyzoites is in a globular form or is rapidly destabilized
following permeabilization of the cell. Improved methods
to stabilize and visualize actin filaments in these organisms will be important for further characterization of their
dynamics. One development that offers promise is the
capture of actin-like filaments beneath the plasma membrane of gliding tachyzoites by rapid, freeze-etch electron
microscopy (J.E. Heuser and L.D. Sibley unpublished).
It has been suggested by Karpova et al. [1995] that
the relative ratio of globular to filamentous actin in a
given organism is a reflection of its activity level. Thus,
highly motile organisms, such as Dictyostelium, have
high concentrations on monomeric actin and very dynamic actin filaments that undergo rapid polymerization
261
and depolymerization for motility [Karpova et al., 1996].
Less motile organisms, such as yeast, require lower
concentrations of actin monomers and consequently have
a greater percentage of filamentous actin [Karpova et al.,
1995]. The highly motile nature of T. gondii tachyzoites is
consistent with their high content of globular actin.
Combined with evidence on the essential role that actin
filaments play in invasion, the predominance of globular
actin in T. gondii implies that filament turnover is
dynamic and likely involved in parasite motility and host
cell invasion.
ACKNOWLEDGMENTS
We thank John Cooper and Paul Bridgeman for
valuable advice and Jim Lessard for the generous gift of
C4 antibody.
REFERENCES
Burg, J.L., Perlman, D., Kasper, L.H., Ware, P.L., and Boothroyd, J.C.
(1988): Molecular analysis of the gene encoding the major
surface antigen of Toxoplasma gondii. J. Immunol. 141:3584–
3591.
Cao, L., Fishkind, D.J., and Wang, Y. (1993): Localization and
dynamics of nonfilamentous actin in cultured cells. J. Cell Biol.
123:173–181.
Cintra, W.M., and De Souza, W. (1985): Immunocytochemical localization of cytoskeletal proteins and electron microscopy of detergent extracted tachyzoites of Toxoplasma gondii. J. Submicrosc.
Cytol. 17:503–508.
Cleveland, D.W., Lopata, M.A., MacDonald, R.J., Cowan, N.J., Rutter,
W.J., and Kirshner, M.W. (1980): Number and evolutionary
conservation of alpha- and beta-tubulin and cytoplasmic betaand gamma-actin genes using specific cloned DNA probes. Cell
20:95–105.
Cook, R.K., Scheff, D.R., and Rubenstein, P.A. (1991): Unusual
metabolism of the yeast actin amino terminus. J. Biochem.
266:16825–16833.
Cooper, J.A. (1987): Effects of cytochalasin and phalloidin on actin. J.
Cell Biol. 105:1473–1478.
Dobrowolski, J.M., and Sibley, L.D. (1996): Toxoplasma invasion of
mammalian cells is powered by the actin cytoskeleton of the
parasite. Cell 84:933–939.
Endo, T., Yagita, K., Yasuda, T., and Nakamura, T. (1988): Detection
and localization of actin in Toxoplasma gondii. Parasitol. Res.
75:102–106.
Erba, H.P., Eddy, R., Shows, Y., Kedes, L., and Gunning, P. (1988):
Structure, chromosome location and expression of the human
g-actin gene: Differential evolution, location and expression of
the cytoskeletal b- and g-actin genes. Mol. Cell. Biol. 8:1775–
1789.
Kabsch, W., Mannherz, H.G., D., S., Pai, E.F., and Holmes, K.C.
(1990): Atomic structure of actin: DNAseI complex. Nature
347:37–43.
Karpova, T.S., Tatchell, K., and Cooper, J.A. (1995): Actin filaments in
yeast are unstable in the absence of capping protein or fimbrin.
J. Cell Biol. 131:1483–1493.
Kim, K., Gooze, L., Petersen, C., Gut, J., and Nelson, R.G. (1992):
Isolation, sequence and molecular karyotype analysis of the
262
Dobrowolski et al.
actin gene of Cryptosporidum parvum. Molec. Biochem. Parasitol. 50:105–114.
Laemmli, U.K. (1970): Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680–685.
Lessard, J.L. (1988): Two monoclonal antibodies to actin: One muscle
selective and one generally reactive. Cell Motil. Cytoskel.
10:349–362.
Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982): ‘‘Molecular
Cloning, A Laboratory Manual.’’ Cold Spring Harbor, New
York: Cold Spring Harbor Laboratory Press.
Miller, L.H., Aikawa, M., Johnson, J.G. and Shiroishi, T. (1979):
Interaction between cytochalasin B-treated malarial parasites
and erythrocytes. J. Exp. Med. 149:172–184.
Morisaki, J.H., Heuser, J.E., and Sibley, L.D. (1995): Invasion of
Toxoplasma gondii occurs by active penetration of the host cell.
J. Cell Sci. 108:2457–2464.
Nakajima-Iijima, S., Hamada, H., Reddy, P., and Kakunaga, T. (1985):
Molecular structure of the human cytoplasmic b-actin gene:
Interspecies homology of sequences in the introns. Proc. Natl.
Acad. Sci., USA 82:6133–6137.
Nichols, B.A., and O’Connor, R.G. (1981): Penetration of mouse
peritoneal macrophages by the protozoan Toxoplasma gondii.
Lab. Invest. 44:324–335.
Pearson, W.R., and Lipman, D.J. (1988): Improved tools for biological
sequence comparison. Proc. Natl. Acad. Sci. USA 85:2444–2448.
Pfefferkorn, E.R., and Pfefferkorn, L.C. (1976): Arabinosyl nucleosides inhibit Toxoplasma gondii and allow the selection of
resistant mutants. J. Parasitol. 62:993–999.
Rubenstein, P.A. (1990): The functional importance of multiple actin
isoforms. BioEssays 12:309–315.
Russell, D.G. (1983): Host cell invasion by Apicomplexa: An expression of the parasite’s contractile system? Parasitology 87:199–
209.
Russell, D.G., and Sinden, R.E. (1981): The role of the cytoskeleton in
the motility of coccidian sporozoites. J. Cell Sci. 50:345–359.
Russell, D.G., and Sinden, R.E. (1982): Three-dimensional study of the
intact cytoskeleton of coccidian sporozoites. Int. J. Parasitlol.
12:221–226.
Ryning, F.W., and Remington, J.S. (1978): Effect of cytochalasin D on
Toxoplasma gondii cell entry. Infect. Immun. 20:739–743.
Schwartzman, J.D., and Pfefferkorn, E.R. (1983): Immunofluorescent
localization of myosin at the anterior pole of the coccidian,
Toxoplasma gondii. J. Protozool. 30:657–661.
Sibley, L.D., and Boothroyd, J.C. (1992): Construction of a molecular
karyotype for Toxoplasma gondii. Molec. Biochem. Parasitol.
51:291–300.
Silva, S.R.L., Meirelles, S.S.L., and De Souza, W. (1982): Mechanism
of entry of Toxoplasma gondii into vertebrate cells. J. Submicrosc. Cytol. 14:471–482.
Smith, D.B., and Johnson, K.S. (1988): Single-step purification of
polypeptides expressed in Escherichia coli as fusions with
glutathione S-transferase. Gene 67:31–40.
Werk, R. (1985): How does Toxoplasma gondii enter host cells? Rev.
Infect. Dis. 7:449–457.
Wesseling, J.G., de Ree, J.M., Ponnudurai, T., Smits, M.A. and
Schoenmakers, J.G.G. (1988a): Nucleotide sequence and deduced amino acid sequence of a Plasmodium falciparum actin
gene. Molec. Biochem. Parsitol. 27:313–320.
Wesseling, J.G., Smits, M.A., and Schoenmakers (1988b): Extremely
diverged actin proteins in Plasmodium falciparum. Molec.
Biochem. Parsitol. 30:143–154.
Wesseling, J.G., Snijders, P.J.F., van Someren, P., Jansen, J., Smits,
M.A., and Schoenmakers, J.G.G. (1989): Stage-specific expression and genomic organization of the actin genes of the malaria
parasite Plasmodium falciparum. Molec. Biochem. Parisitol.
35:167–176.
Yasuda, T., Tagita, K., Nakamura, T. and Endo, T. (1988): Immunocytochemical localization of actin in Toxoplasma gondii. Parasitol.
Res. 75:107–113.
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