Figure 1.
Apicomplexan actins differ in sequence and polymerization kinetics.
(A) Model of TgACTI (blue) mapped onto PfACTI (red) highlighting amino acid differences (yellow). (B) Model of PfACTI (red) mapped onto PfACTII (green) highlighting amino acid differences (yellow). (See supplementary Figure S6 for enlargement). (C) Expression of recombinant apicomplexan actins purified from baculovirus, resolved using a 12% SDS-PAGE gel, stained with SYPRO Ruby. (D) Comparison of actin polymerization kinetics. Polymerization of 5 µM actin was induced by the addition of F buffer (arrow) and monitored by light scattering. Insert shows parasite actins on the expanded Y-axis.
Figure 2.
Apicomplexan actins form inherently unstable filaments that are rescued by phalloidin.
(A) In vitro polymerization of parasite actins visualized by fluorescence microscopy of phalloidin stained actin. Parasite actins were incubated at 5 µM with no addition (0) or with equimolar unlabeled phalloidin (5 µM) and visualized by addition of low levels of Alexa-488 labeled phalloidin (0.13 µM or 0.33 µM) and visualized by fluorescence microscopy. The values at the top of the figure panel indicate the amounts of phalloidin used in each reaction: µM unlabeled phalloidin (µM labeled phalloidin). Scale bars, 5 µm. Representative of three or more similar experiments. (B) Quantitation of length of filaments formed during in vitro polymerization of parasite actins. Mean ± S.D. shown by horizontal line. PfACTI and PfACTII formed significantly longer filaments than TgACTI at either of the low doses of phalloidin added for visualization (i.e. 0.13 or 0.33 µM) (Student's t-test, P<0.001). Concentrations of phalloidin used for treatment are indicated on below the graphs as µM unlabeled phalloidin (µM labeled phalloidin).
Figure 3.
Ultrastructural features of parasite actins revealed by electron microscopy.
(A) TgACTI was incubated in G buffer or F buffer with or without equimolar concentration of phalloidin for 1 hr. The reactions were added to grids, negatively stained with uranyl acetate and examined by EM. Identical conditions were used to observe PfACTI (B), PfACTII (C) and ScACT (D). Images are representative of 3 or more experiments. Scale bars, 50 nm.
Figure 4.
Predicted binding site of phalloidin in muscle and parasite actin filaments.
Molecular details of the interaction of phalloidin in the muscle actin filament. (A) Side chains of amino acids within 3.5 Å are explicitly shown and the protein around phalloidin is depicted as a transparent surface. Hydrogens are omitted for clarity. (B) Position of phalloidin (purple) in the filament showing its interaction with three individual protomers. (C) 2D interaction diagrams showing the interaction differences between muscle, TgACTI and PfACTII actin. Hydrogen bonding interaction are depicted by dashed green lines and portions of the molecule that are solvent accessible are highlighted in yellow. Blue-basic residues; red-acidic residues; cyan-polar residues.
Figure 5.
Identification of single substitutions within TgACTI that affect filament stability.
(A) Modeling of S199-D179 hydrogen bond and M269 in the hydrophobic loop of mammalian actin (B) Modeling of loss of hydrogen bond with G200 substitution and reduced hydrophobicity at position 270 in TgACTI. (C) Expression of TgACTI recombinant proteins containing mammalian-like substitutions in baculovirus, resolved using a 12% SDS-PAGE gel, and stained with SYPRO Ruby. (D) Comparison of polymerization kinetics of TgACTI substitutions. F buffer was added at time = 0 sec to induce polymerization of 5 µM actin and polymerization was monitored by light scattering.
Figure 6.
Phylogenetic tree highlighting the diversity of actins.
Actin sequences were retrieved from Genbank and aligned using Clustal. Neighbor joining with PAUP* was used to assemble the sequences into phylogenetic tree based on 1000 bootstrap replicates (bootstrap values indicted in circles at specific nodes). The branches are color coded to show phylogenetic differences. Red represents branches containing a lysine or arginine at position 270, which is conventionally a methionine. Green represents branches that contain the K270 substitution as well as a glycine at position 200, which is conventionally a serine or threonine. Numbering based on the TgACTI sequence.
Figure 7.
Substituted TgACTI alleles demonstrate enhanced in vitro polymerization.
(A) In vitro polymerization of recombinant TgACTI alleles substituted with mammalian actin residues. Actins (5 µM) were visualized by fluorescence microscopy using 0.33 µM Alexa 488-phalloidin and different molar ratios of unlabeled phalloidin to actin. Scale bars, 5 µm. Representative of three or more similar experiments. The values at the top of the figure panel indicate the amounts of phalloidin used: µM unlabeled phalloidin (µM labeled phalloidin). (B) Quantitation of filaments formed during in vitro polymerization of substituted TgACTI alleles. Mean ± S.D. shown by horizontal line. Concentrations of phalloidin used for treatment are indicated below the graphs as µM unlabeled phalloidin (µM labeled phalloidin). In the presence of only low levels of labeled phalloidin (i.e. 0.33 µM), filaments formed by TgACTI-K270M (P<0.01), TgACTI-G200S (P<0.001), and TgACTI-G200S/K270M (P<0.001) were significantly longer that wild type TgACTI (Student's t-test).
Figure 8.
Expression of degradation domain (DD)-tagged TgACTI alleles in Toxoplasma.
(A) Expression of DD-TgACTI fusion proteins following treatment ± Shield-1 for 40 hr and detected by Western blot with anti-TgACTI antibody. All strains express the endogenous TgACTI while the fusion proteins (DD-TgACTI) were only expressed by the transfected strains in the presence of Shield-1. (B) Expression of DD-tagged TgACTI alleles following treatment ± Shield-1 for 24 hr and stained for immunofluorescence with anti-SAG1 (surface antigen 1, red) and anti-c-myc (green) to detect the DD-TgACTI fusion protein. (C) Effects of DD-TgACTI allele expression on plaque formation. HFF monolayers were infected with untransfected parasites or those expressing DD-TgACTI alleles ± Shield-1 for 7 days and visualized by crystal violet staining. Representative of three or more similar experiments.
Figure 9.
Stabilized actin alleles are more sensitive to JAS-stabilization than endogenous TgACTI in Toxoplasma.
(A) Localization of c-myc-tagged DD-TgACTI alleles in parasites treated with Shield-1 for 40 hr as visualized by immunofluorescence with anti-c-myc antibody (green) and SAG1 (red). Treatment with low levels of JAS (i.e. 0.25 µM) induced spiral patterns of filaments in parasites expressing stabilized actin alleles (right). Apical end noted with arrowhead. Scale bar, 5 µm. (B) Images from (A) shown as z-slices (∼0.3 µm). Actin spirals in JAS-treated parasites were visualized by staining with anti-c-myc antibody. Apical end noted with arrowhead. Scale bar, 5 µm. (C) Quantitation of length of actin puncta and spirals formed in individual parasites treated with ±0.25 µM JAS. Mean ± S.D. * P<0.05 ** P<0.005 (Student's t-test) vs. DD-wild type with same JAS treatment. (D) Sedimentation analysis of F actin in parasites expressing DD-TgACTI fusions and treated ± Shield-1 for 40 hr. Cell lysates were prepared ±0.5 µM JAS, sedimented for 1 hr at 350,000g and analyzed by SDS-PAGE and quantitative Western blotting. Mean ± S.D., n = 3 experiments. * P<0.05 (Student's t-test) vs. DD-wild type.
Figure 10.
Parasites expressing stabilized actin undergo aberrant gliding motility.
(A) Representative composite videos of normal gliding motility by parasites expressing DD-wild type TgACTI. (B,C) Representative composite videos of gliding by parasites expressing stabilized actin mutants revealed examples of stalled, incomplete or off-track circular patterns and disrupted helical patterns that were classified as aberrant. For helical gliding, the number of complete turns made during the time-lapse sequence are numbered. Images are composite frames from 60 sec of video recording. Scale bars, 5 µm. (D) Quantification of number of parasites undergoing each category of gliding motility following treatment ± Shield-1 for 40 hr. The percentage of parasites undergoing aberrant gliding (as defined in B,C) increased in the presence of Shield-1 in the mutant actins as compared to wild type, * P<0.05 ** P<0.01, (Student's t-test). Mean ± S.D. (E) Comparison of radii of circular tracks formed during normal gliding by DD- wild type expressing parasites vs. parasites expressing mutant actins that formed aberrant circular tracks (i.e. stalled or off-track). * P<0.001 ** P<0.0001 (Mann-Whitney test). Mean shown by horizontal line. (F) Comparison of parasite curvature during normal gliding by DD-wild type expressing parasites vs. parasites expressing mutant actins that formed aberrant circular tracks (i.e. stalled or off-track). Data shown are the average curvature radii of individual parasites during gliding motility. (G) Quantification of number of parasites undergoing each category of gliding motility following treatment ± Shield-1 for 6 hr. The percentage of parasites undergoing aberrant gliding (as defined in B, C) increased in the presence of Shield-1 in the mutant actins as compared to wild type, * P<0.001, (Student's t-test). Mean ± S.D.
Table 1.
Quantification of T. gondii gliding motility rates from video microscopy.