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.  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]. 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