1 Division of Molecular Parasitology, Department of Microbiology &
Immunology, Drexel University College of Medicine, Philadelphia, PA 19129,
USA
2 Case Western Reserve University School of Medicine, Cleveland, Ohio 44106,
USA
3 Infectious Diseases Section, Department of Internal Medicine, Yale University
School of Medicine, New Haven, Connecticut 06520-8022, USA
4 Michael Heidelberger Division, Department of Pathology, New York University
School of Medicine, New York, NY 10016, USA
* Author for correspondence (e-mail: lawrence.bergman{at}drexel.edu)
Accepted 26 September 2002
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Summary |
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Key words: Plasmodium, Gliding motility, Cell invasion, Apicomplexa, MyoA
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Introduction |
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Gliding motility and invasion of host cells are inhibited by cytochalasins,
which indicates that the parasites' actin filaments are required for
locomotion (Dobrowolski and Sibley,
1996). In addition, the reversible myosin inhibitor
butane-2,3-monoxime (BDM) blocks locomotion, which suggests that a myosin
motor together with actin provides the underlying force
(Dobrowolski et al., 1997a
;
Matuschewski et al., 2001
;
Pinder et al., 1998
).
Apicomplexan zoites are delimited by a tri-laminar pellicle consisting of a
plasma membrane and two closely aligned inner membranes that form the inner
membrane complex (IMC). The cytoplasmic side of the IMC faces the
subpellicular microtubule system and might be connected to it by
intramembranous particles (IMPs)
(Morrissette et al., 1997).
Myosin and actin localize to the space between the plasma membrane and the
outer membrane of the IMC in Toxoplasma tachyzoites
(Dobrowolski et al., 1997a
) and
Plasmodium merozoites (Pinder et
al., 2000
). Thus the putative actomyosin motors are in the right
position to power gliding motility and invasion.
The most likely candidate myosin motor is myosin A (MyoA), a class XIV
unconventional myosin that is unique to the phylum Apicomplexa
(Heintzelman and Schwartzman,
1997; Pinder et al.,
1998
). It is expressed in all Plasmodium invasive stages
(merozoites, sporozoites and ookinetes)
(Margos et al., 2000
;
Matuschewski et al., 2001
;
Pinder et al., 1998
) and also
in Toxoplasma tachyzoites
(Heintzelman and Schwartzman,
1999
). Recently it was shown that MyoA is indeed essential for
gliding motility and host cell invasion of Toxoplasma tachyzoites
(Meissner et al., 2002
). The
MyoA head domain displays the universally conserved ATP and actin binding
sites. MyoA binds actin and is released from actin in an ATP-dependant fashion
(Heintzelman and Schwartzman,
1999
; Hettmann et al.,
2000
). However, MyoA has unusual features. Its putative short neck
domain has no conserved IQ motifs (consensus IQXXXRGXXXRK) that function as
light chain binding sites in the neck domains of many myosins
(Mooseker and Cheney, 1995
).
In addition, MyoA has a very short C-terminal tail domain.
In this report we attempted to identify proteins that interact with the MyoA neck and tail domains using the yeat two-hybrid system. We reasoned that MyoA-interacting proteins should be components of the apicomplexan motility and host cell invasion apparatus.
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Materials and Methods |
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Plasmid construction
To construct the two hybrid bait vector containing the C-terminal 75 amino
acids of P. yoelii MyoA, a region of P. yoelii yoelii 17XL
genomic DNA was amplified using primers containing a 5'-EcoRI
site and a 3'-PstI site. The resulting fragment was sequenced,
and cloned into the Gal4 DNA binding domain vector pAS2.1 (Clontech). A
deletion of the MyoA, which removed the C-terminal 15 amino acids, was
constructed as described above, except that the 3'-primer introduced a
stop condon after amino acid 902. Plasmids expressing binding domain fusions
containing solely the C-terminal 15 amino acids of MyoA (or mutants within
that region) were constructed using synthetic oligonucleotides directly cloned
in the vector pAS2.1. Deletions of MTIP were made using oligonucleotides
containing either a 5'-EcoRI site or 3'-SalI
site. The resulting fragments were sequenced and subsequently cloned into the
Gal4 activation domain vector pGAD424 (Clontech).
The full length MTIP gene region was amplified from P. yoelii
yoelii 17XL genomic DNA by PCR using oligonucleotides containing a
5'-EcoRI site and a 3'-PstI site. Following
restriction endonuclease digestion, the insert was directionally cloned into
the pGEX-4T-1 expression vector (Pharmacia). DNA sequencing of the insert and
vector junctions confirmed an in-frame joining with the 3' end of the
S. japonicum GST gene. E. coli cells (BL21-CodonPlus-RIL,
Stratagene) were transformed with the expression vector and clones expressing
the fusion protein, designated GST-MTIP, were identified by immunoblot using
anti-GST serum. GST-MTIP was prepared by affinity chromatography using
glutathioneagarose (Sigma) (Smith
and Johnson, 1988).
Yeast two-hybrid screen
The pAS2.1-MyoA vector was used to transform strain PJ69-4a and
subsequently transformed with the P. yoelii blood stage
cDNA-activation domain library essentially as previously described
(Daly et al., 2001). Activation
domain plasmids were rescued into E. coli from His+,
Ade+ yeast colonies. Seventy-six plasmids were positive upon
re-screening. The nature of these plasmids was determined either by DNA
sequencing using a 5'-vector-specific activation domain primer (15
plasmids) or by PCR analysis using a primer derived from MTIP and a
3'-vector-specific primer (61 plasmids). Yeast cells containing the MyoA
and MTIP constructs shown in Fig.
2 were grown in synthetic media lacking leucine and tryptophan to
an OD600=0.7. Cell extracts were prepared by glass bead lysis and
assays performed as previously described
(Bergman, 1986
). The activity
was normalized to the protein concentration of the extract and was expressed
as nanomoles of O-nitrophenyl-ß-D-galactoside cleaved per minute
per milligram of protein.
|
Experimental animals and antisera
One hundred micrograms of GST-MTIP fusion protein, suspended in phosphate
buffered saline, pH 7.4 (PBS), with RAS (Ribi Adjuvant System, Ribi
Immunochemical Research Laboratories) was injected subcutaneously into male
BALB/cByJ mice and rabbits at 3-week intervals. Two weeks after the second
boost, blood was collected and serum was isolated. Polyclonal mouse anti-GST
antiserum was prepared similarly. Normal mouse serum (NMS) was taken from a
non-immunized BALB/cByJ mouse. The rabbit anti-MyoA antiserum was raised
against a synthetic peptide (FMQLVISHEGGIRYG) corresponding to amino acids
251-265 of MyoA (Pinder et al.,
1998).
Immunoblotting
Sporozoite and schizont proteins were solubilized in sample buffer and
separated on 10% polyacrylamide gels. Molecular weights were verified using a
prestained molecular weight marker (Biorad). Proteins were transferred to
nitrocellulose (BioRad) membranes by electroblotting. Following transfer,
membranes were blocked and then incubated with a 1:500 dilution of anti-MTIP
antibody. Membranes were washed and then incubated for 1 hour at room
temperature with a horseradish-peroxidase-conjugated secondary antibody.
Membranes were washed again and immunostained proteins were visualized with
enhanced chemi-luminescence detection (Pierce).
Immunofluorescence
Infected erythrocytes were spread in PBS/gelatin, fixed with
methanol/acetone at -20°C and air-dried. Salivary gland sporozoites were
permeabilized with 0.05% saponin and fixed with 2% paraformaldehyde. MTIP was
detected with the polyclonal anti-MTIP-GST fusion protein antibody (1:1000)
and FITC-conjugated goat anti-mouse IgG (1:100). For immunofluorescence
staining of hepatic stages the hepatoma cell line HepG2 was infected with
P. berghei salivary gland sporozoites and infected cells were
cultured for 24 and 48 hours. Cells were permeabilized with 0.05% saponin,
fixed with 2% paraformaldehyde and stained with an anti-HSP70 monoclonal
antibody (Tsuji et al., 1994)
and the rabbit polyclonal anti-MTIP-GST fusion protein antibody. Bound
antibodies were detected using Alexa-Fluor-488- and Alexa-Fluor-594 (Molecular
Probes)-conjugated anti-mouse and anti-rabbit secondary antibodies.
Electron microscopy
For transmission electron microscopy salivary glands of P.
yoelii-infected mosquitoes were fixed with 2.5% glutaraldehyde in 0.05 M
phosphate buffer, pH 7.4, with 4% sucrose for 2 hours and then postfixed in 1%
osmium tetroxide for 1 hour. After a 30 minute en bloc stain with 1% aqueous
uranyl acetate, the cells were dehydrated in ascending concentrations of
ethanol and embedded in Epon 812. Ultrathin sections were stained with 2%
uranyl acetate in 50% methanol and with lead citrate. For immunoelectron
microscopy salivary glands of the P. yoelii infected mosquitoes were
fixed for 30 minutes at 4°C with 1% formaldehyde, 0.5% glutaraldehyde in
0.1 M phosphate buffer, pH 7.4. Fixed samples were washed, dehydrated and
embedded in LR White resin (Polysciences, Warrington, PA). Thin sections were
treated with PBS-glycine, then blocked in PBS containing 1% w/v bovine serum
albumin (BSA) and 0.01% v/v Tween 20 (PBTB). Grids were then incubated with
primary antibodies (anti-MTIP or anti-MyoA) diluted 1:50 1:500 in PBTB
for 2 hours at room temperature. Negative controls included normal Mouse or
rabbit serum and PBTM applied as the primary antibody. After washing, grids
were incubated for 1 hour in the gold-conjugated goat anti-mouse or -rabbit
IgG (Amersham Life Sciences, Arlington, IL) diluted 1:20 in PBTB, rinsed with
PBTB, and fixed with glutaraldehyde to stabilize the gold particles. Samples
were stained with uranyl acetate and lead citrate, and then examined in a
Zeiss CEM902 electron microscope. For cryo-immunoelectron microscopy salivary
glands of the P. yoelii infected mosquitoes were fixed for 2 days at
4°C with 8% paraformaldehyde. Infected glands were infiltrated, frozen,
sectioned and labeled as described (Folsch
et al., 2001
) with the difference that rabbit anti-MTIP polyclonal
antibody was used (1:200 in PBS/1% fish skin gelatin), followed directly by 10
nm protein A gold.
Membrane extraction
P. yoelii salivary gland sporozoites (6x105) were
suspended in PBS containing 1% Triton X-100 (TX-100) and incubated on ice for
30 minutes. The preparation was centrifuged at 13,800 g for 20
minutes at 4°C to separate the TX-100 soluble (plasma membrane) from the
insoluble (IMC) fraction. The pellet was washed three times in PBS. Untreated
sporozoites or the total pellet and total supernatant of TX-100-treated
sporozoites were suspended in sample buffer and separated on 10%
polyacrylamide gels. Proteins were transferred to nitrocellulose (BioRad)
membranes by electroblotting. Following transfer, membranes were blocked and
then incubated with a 1:1000 dilution of anti-MTIP antibody or a 1:1000
dilution of a monoclonal anti-P. yoelii CS antibody. Membranes were
washed and then incubated for 1 hour at room temperature with a
horseradish-peroxidase-conjugated secondary antibody. Membranes were washed
again and immunostained proteins were visualized with enhanced
chemiluminescence detection (Pierce).
For immunofluorescence, sporozoites were treated for 30 minutes on ice with 1% TX-100 in PBS, washed three times in PBS and fixed for 10 minutes in 2% of paraformaldehyde in PBS at room temperature. Treated and untreated sporozoites were incubated for 1 hour with anti-CS and anti-MTIP both diluted in 1:1000 in PBS-1% FCS. Bound antibodies were detected using Alexa Fluor 488 and Alexa Fluor 594 (Molecular Probes) conjugated anti-mouse and anti-rabbit secondary antibodies. Relative fluorescence intensities were quantified using the MetaMorph imaging system (Universal Imaging Corporation). Measurements were taken from 50 TX-100-treated and 50 untreated sporozoites. Exposure times were kept constant between individual measurements.
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Results |
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MTIP binds to the tail domain of MyoA
To further map the part of MyoA that is critical for binding to MTIP we
constructed DNA binding domain vectors that contain solely the presumptive
-helical neck domain of MyoA (residues 842-902 of the full length
molecule) or the 15 amino acid tail domain (residues 903-917). Surprisingly,
not the neck domain but the 15 amino acid tail domain was necessary and
sufficient for the MyoA interaction as measured qualitatively using growth in
the absence of adenine or histidine or quantitatively measuring
interaction-dependent expression of an integrated GAL1-LacZ fusion
(Fig. 2A).
Next we asked which region of MTIP is involved in the interaction with the
MyoA tail domain. The minimal interacting region of MTIP isolated from the
original library screen encompassed residues 80-204
(Fig. 2B). We removed
additional 15 amino acids from the N-terminal end of the minimal interacting
region (residues 80-94) and this deletion eliminated the ability of MTIP to
interact with the MyoA tail domain. Removal of 15 C-terminal amino acids
(residues 190-204) also eliminated the interaction. Therefore the entire
79-204 MTIP region may be required for the interaction with MyoA.
Alternatively, the binding site could be composed of two separate regions
(residues 80-94 and 190-204) in MTIP. A previous study implicated the tail
domain of T. gondii MyoA (TgMyoA) in its targeting to the tachyzoite
periphery, presumably the plasma membrane. Mutagenesis of a dibasic motif
within the tail of TgMyoA abolished localization to the periphery
(Hettmann et al., 2000). We
made the analogous mutations in the tail of PyMyoA and found that these
changes abolished the interaction of the PyMyoA tail domain with MTIP
(Fig. 2C).
MTIP is expressed in invasive stages of Plasmodium
We examined the expression pattern of transcripts encoding MTIP using cDNA
blots and reverse transcriptase-PCR. MTIP was expressed in blood
stage parasites and sporozoites of P. yoelii (data not shown). To
localize MTIP, we expressed and purified a GST-MTIP fusion protein. Both a
polyclonal mouse antiserum and a polyclonal rabbit antiserum directed against
the fusion protein specifically recognized MTIP released by thrombin cleavage
of the GST-MTIP fusion in western blots, whereas a mouse polyclonal anti-GST
antiserum recognized only the GST moiety (data not shown). Western analysis of
total protein isolated from sporozoites or a schizont enriched fraction of
blood stage parasites revealed a band of approximately 25 kDa
(Fig. 3A) in both parasite
stages. The protein ran as a closely migrating doublet, indicating that MTIP
may be subject to proteolytic processing or post-translational modifications.
MTIP contains multiple consensus serine and threonine phosphorylation sites
making it possible that MTIP function is regulated by phosphorylation.
|
Indirect immunofluorescence assay (IFA) showed that MTIP is concentrated around the periphery of merozoites during the late stages of schizogony (Fig. 3B). In sporozoites (Fig. 3C) MTIP showed strong peripheral staining of the sporozoite with varying intensity.
MTIP localizes to the inner membrane complex of Plasmodium
sporozoites
In transmission electron microscopy P. yoelii sporozoites showed
the typical tri-laminar pellicle consisting of a plasma membrane and two inner
membranes that form the IMC (Fig.
4A). We investigated the localization of MTIP in sporozoites by
immunoelectron microscopy (Fig.
4B-D). Labeling of sections with anti-MTIP (15 nm gold particles)
localized the protein to the periphery of sporozoites. Almost no labeling was
observed in the internal cytoplasm (Fig.
4B). The gold particles decorated a circumferential electron dense
structure that was positioned 15 nm underneath the outer boundary of the
sporozoite, the presumed plasma membrane. We interpreted this electron dense
structure as the IMC, which extends
15 nm underneath the plasma membrane
(Fig. 4A,B). Double labeling of
sections with anti-MyoA (5 nm gold particles) and anti-MTIP (15 nm gold
particles) localized both proteins to the periphery of sporozoites
(Fig. 4C,D). The MyoA
localization was similar to that previously described for Toxoplasma
tachyzoites and Plasmodium merozoites
(Dobrowolski et al., 1997a
;
Pinder et al., 1998
).
Frequently MyoA was found clustered with MTIP
(Fig. 4C,D). Next we used cryo
sections of salivary gland sporozoites to assign MTIP more clearly to a
particular membrane compartment. Labeling with anti-MTIP (10 nm gold
particles) localized MTIP to the IMC and also to the cortical space between
the IMC and the plasma membrane (Fig.
4E,F). The IMC of apicomplexan zoites terminates at the apical
prominence, leaving the extreme apical prominence covered by the plasma
membrane only. No MTIP labeling was found at the extreme prominence of
sporozoites (Fig. 4F,
inset).
|
To confirm the potential IMC localization of MTIP we made use of the
differential solubility characteristics of the apicomplexan plasma membrane
and IMC. Treatment with the non-ionic detergent Triton X-100 (TX-100) removes
the plasma membrane and its associated proteins of Toxoplasma
tachyzoites quantitatively; however, the IMC and associated proteins are
relatively resistant to TX-100 treatment
(Mann and Beckers, 2001). We
subjected sporozoites to the same treatment and then used the rabbit anti-MTIP
antisera for detection by IFA and immunoblots. As a control for plasma
membrane localization we used a monoclonal antibody specific for the P.
yoelii circumsporozoite (CS) protein
(Ak et al., 1993
). CS is the
major surface protein of sporozoites presumably linked to the plasma membrane
by a glycosylphosphatydilinositol (GPI) anchor
(Nussenzweig and Nussenzweig,
1985
). Untreated sporozoites showed uniform peripheral
distribution of MTIP and CS with a notable absence of MTIP staining at one
pole of the sporozoite (Fig.
5A). TX-100-treated sporozoites showed no significant changes in
the linear, circumferential MTIP staining when compared with untreated
sporozoites. However, TX-100-treated sporozoites showed complete loss of the
peripheral CS staining. This finding was substantiated by quantitative
analysis of fluorescent intensities showing no significant loss of MTIP
fluorescence in TX-100 treated sporozoites but a 90% decrease of CS
fluorescence (Fig. 5B).
Immunoblot analysis confirmed the IFA results
(Fig. 5C). TX-100-treated
sporozoites retained most MTIP in the insoluble pellet fraction after
high-speed centrifugation, while CS showed an inverse distribution with most
protein found in the soluble supernatant fraction.
|
Sporozoites invade hepatocytes and transform into hepatic trophozoites. We
followed MTIP localization in intracellular parasites by IFA using the
hepatoma cell line HepG2 as host cells
(Fig. 6). The transformation of
intracellular sporozoites into hepatic trophozoites is marked by the
development of a bulbous enlargement and an increase in heat shock protein 70
(HSP70) expression (Fig. 6A).
In early trophozoites MTIP localized to the parasites' periphery showing an
apparently uninterrupted, circumferential pattern
(Fig. 6B). MTIP signal was
progressively lost in later stage parasites
(Fig. 6C,D) and was not
detectable at 48 hours post invasion. The pattern we observed for MTIP loss in
hepatic stages reflected closely the progressive disassembly of the IMC that
was previously described using electron microscopy
(Meis et al., 1985).
|
Taken together, the IEM localization, the TX-100 solubility characteristics and the pattern of loss in hepatic stages strongly support an association of MTIP with the sporozoite IMC and not the plasma membrane.
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Discussion |
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However, neither the MyoA cargo nor accessory molecules that regulate its
activity, such as light chains, had been identified. In many myosins, the
cargo interacts with the tail domain and light chains bind to the IQ motifs in
the neck domain (Mermall et al.,
1998). These neck and tail domains of MyoA are strikingly
distinct. Similar to other myosins, the putative neck domain of MyoA is
predicted to form an extended
-helix but it lacks consensus IQ motifs.
We did not identify any molecule that interacts with the neck domain in the
yeast two-hybrid screen. The MyoA tail is unusually short, apparently formed
by the C-terminal 15 amino acids. Our results show that it is the tail of MyoA
that specifically interacts with MTIP. Therefore, MTIP is integral to the
motor complex. MTIP is distantly related to myosin light chains and might
function as a regulatory or essential light chain for MyoA. However, its
single EF-hand motif is unusual since EF-hands almost always occur in pairs,
presumably to stabilize the protein conformation and to act cooperatively in
the binding of Ca2+ (Nelson and
Chazin, 1998
). We cannot exclude the possibility that MTIP
dimerizes bringing two EF-hands together. EF-hands consist of a
helix-loop-helix structure where the loop coordinates the metal. The calcium
ion is coordinated in a pentagonal bipyramidal configuration. The six residues
involved in the binding are in positions 1, 3, 5, 7, 9 and 12 of the loop with
an invariant glutamate or aspartate at position 12
(Lewit-Bentley and Rety,
2000
). Secondary structure prediction indicated that the EF-hand
motif of MTIP forms a helix-loop-helix structure; however, the residue at
position 12 is a glutamine, making it unlikely that this motif coordinates
Ca2+ at physiological concentrations. However, EF-hand-containing
proteins can also exhibit Ca2+-independent binding to targets. Thus
the functional properties of the MTIP EF-hand motif remain to be elucidated by
site-directed mutagenesis and Ca2+-binding assays.
In a previous study it was shown that the tail domain of TgMyoA was
essential for targeting the protein to the tachyzoite periphery
(Hettmann et al., 2000) and
that mutagenesis of a conserved dibasic motif within the tail abolished
correct localization. Here we showed that mutagenesis of the dibasic motif in
the Plasmodium MyoA tail domain abolished interaction with MTIP. It
is therefore likely that, in the Toxoplasma study, it was the T.
gondii MTIP ortholog that localized MyoA to the tachyzoite periphery.
Indeed, concurrent with our findings, the MTIP ortholog of Toxoplasma
(TgMLC1) was identified (Herm-Gotz et al.,
2002
). This study also demonstrated an association of TgMLC1 with
TgMyoA using biochemical assays. Taken together our results and previous
studies thus indicate that MTIP/MLC1 acts as a cargo for MyoA and suggest that
their interactions determine IMC localization of MyoA. MTIP might also
regulate MyoA motor activity, but no available experimental data support such
a function.
By immunoelectron microscopy MTIP showed a distribution consistent with its
residence in the IMC. IMC association of MTIP was also supported by its
solubility characteristics in TX-100-treated sporozoites. In addition, MTIP is
lost during development of hepatic stages in a spatio-temporal pattern that
was consistent with its IMC association. Finally, MTIP is absent at the
extreme apical prominence of sporozoites, which reflects the absence of IMC at
this position. This has also been shown for TgMLC1/MTIP
(Herm-Gotz et al., 2002).
Since the MTIP amino acid sequence does not predict transmembrane domains or
lipid attachment sites it might bind to a yet unidentified protein anchored in
the IMC.
The localization of MTIP has important implications for the topology of the
linear actomyosin motor that powers invasion and motility in apicomplexan
parasites. The prevailing model (Fig.
7A) of the motor complex assumed that a transmembrane protein (now
known to be a protein of the TRAP family) is linked by its cytoplasmic domain
directly or indirectly with the myosin motor
(King, 1988;
Pinder et al., 1998
). In this
model filamentous actin remains stationary, tethered to the outer membrane of
the IMC by a hypothetical protein and the myosin/TRAP complex moves along
those filaments. Our results favor an opposing model of motor complex
configuration that is also mentioned by Pinder et al.
(Fig. 7B). The localization of
MTIP to the IMC suggests that it is the MTIP/MyoA complex that remains
stationary tethered to the outer membrane of the IMC by an unidentified
protein. This model predicts that actin, directly or indirectly connected to
the cytoplasmic domain of TRAP, is then moved along this complex. Indeed,
recent work identified aldolase as a protein that interacts with the T.
gondii MIC2 cytoplasmic domain and P. falciparum TRAP
cytoplasmic domain (D. Sibley, personal communication). It is well established
that aldolase of mammalian cells interacts with the actin cytoskeleton
(Kusakabe et al., 1997
;
Schindler et al., 2001
).
Hence, aldolase is probably the bridge linking the cytoplasmic domains of
TRAP-like proteins to the parasites' actin filaments.
|
The model also predicts that short actin filaments that are nucleated at
the anterior end of the zoite form a complex with the cytoplasmic domain of
TRAP and, after redistribution, this complex is disassembled at the posterior
end. In agreement with this prediction, only 5% of total actin was
assembled in filaments in T. gondii tachyzoites
(Dobrowolski et al., 1997b
;
Poupel and Tardieux, 1999
).
Furthermore, when tachyzoites are treated with jasplakinolide, an
actin-polymerizing and filament-stabilizing drug, actin filaments are mainly
observed at the apical end (Shaw and
Tilney, 1999
), which might indicate the preferential localization
of parasite actin polymerization factors in this region.
Whatever the topology of the motor complex, the MyoA/MTIP interaction could be essential for the function of the parasite motor. The yeast system is well poised to approach the isolation of molecules that may disrupt the interaction between these two molecules and thus may block the motility and/or invasion of malarial and other apicomplexan parasites.
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Acknowledgments |
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