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. 2002 May 1;21(9):2149-58.
doi: 10.1093/emboj/21.9.2149.

Toxoplasma gondii myosin A and its light chain: a fast, single-headed, plus-end-directed motor

Angelika Herm-Götz  1 , Stefan Weiss, Rolf Stratmann, Setsuko Fujita-Becker, Christine Ruff, Edgar Meyhöfer, Thierry Soldati, Dietmar J Manstein, Michael A Geeves, Dominique Soldati
Affiliations

Toxoplasma gondii myosin A and its light chain: a fast, single-headed, plus-end-directed motor

Angelika Herm-Götz et al. EMBO J. .

Abstract

Successful host cell invasion is a prerequisite for survival of the obligate intracellular apicomplexan parasites and establishment of infection. Toxoplasma gondii penetrates host cells by an active process involving its own actomyosin system and which is distinct from induced phagocytosis. Toxoplasma gondii myosin A (TgMyoA) is presumed to achieve power gliding motion and host cell penetration by the capping of apically released adhesins towards the rear of the parasite. We report here an extensive biochemical characterization of the functional TgMyoA motor complex. TgMyoA is anchored at the plasma membrane and binds a novel type of myosin light chain (TgMLC1). Despite some unusual features, the kinetic and mechanical properties of TgMyoA are unexpectedly similar to those of fast skeletal muscle myosins. Microneedle-laser trap and sliding velocity assays established that TgMyoA moves in unitary steps of 5.3 nm with a velocity of 5.2 microm/s towards the plus end of actin filaments. TgMyoA is the first fast, single-headed myosin and fulfils all the requirements for power parasite gliding.

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Figures

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Fig. 1. Co-purification of TgMLC1 with TgMyoA as a 160 kDa complex. (A) Purified TgMyoA was fractionated through 10–30% glycerol gradients. Aliquots of each fraction were subjected to SDS–PAGE and immunoblotting using antibodies against TgMLC1 and TgMyoA. The two proteins co-fractionated and peaked at ∼160 kDa (fractions 11–13). A control gradient with size makers was run in parallel and analysed by SDS–PAGE followed by Coomassie Blue staining. TgMyoAΔtail was analysed on an identical gradient and peaked at ∼100 kDa (fractions 15–16). (B) Coomassie Blue-stained SDS–polyacrylamide gel of the purified TgMyoA under native conditions (left panel). Western blot analysis of several preparations of TgMyoA and TgMyoAΔtail purified on Ni-NTA columns under native (n) and denaturing (d) conditions using anti-MyoA (middle panel) and anti-MLC1 antibodies (right panel). TgMLC1 co-purified only with intact TgMyoA under native conditions. (C) Western blot analysis of purified native preparations of TgMyoA and TgMyoAΔtail using anti-TgCaM antibodies. TgCaM corresponds to bacterially expressed recombinant protein. The specificity of the anti-TgCaM antibodies was controlled on the left panel by analysing total lysates prepared from host cells (HFF, human foreskin fibroblast) and wild-type parasites (RH). (D) Western blot analysis of total RH lysates and purified fraction of HisGFP and HisGFPAtail (indicated by an asterisk on the gel) after elution from Ni-NTA resin. TgMLC1 is present in parasites’ total lysate and co-purified with GFPAtail but not with GFP alone.
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Fig. 2. Subcellular distribution of TgMLC1 during parasite penetration of host cells. Double indirect immunofluorescence using differential permeabilization was analysed by confocal microscopy. The major surface antigen (SAG1) is detected prior to permeabilization to label the extracellular portion of the penetrating parasites preferentially, while TgMLC1 was visualized after permeabilization (Triton X-100, 0.2%). (A) Partial penetration of parasites attached to the host surface. The arrow indicates the site of the moving junction. (B) Less than 10 min after invasion, intracellular parasites are not stainable with anti-SAG1 without permeabilization. The arrows point to the posterior end of the parasites. (C) At 24 h post-invasion, a double immunofluorescence with anti-MLC1 and anti-SAG1 under permeabilized conditions showed co-localization of both proteins. Scale bar: 1 µm.
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Fig. 3. Sequence alignments of apicomplexan myosin light and heavy chains. (A) Myosin light chains from P.falciparum, Eimeria tenella, Neospora caninum and T.gondii. TgMLC1 accession No. AY048862. The position of the four proposed degenerate EF hand domains is indicated by a bar. (B) Alignment of the C-terminus of class XIV myosin heavy chains from T.gondii, Plasmodium spp., E.tenella and Theileira parva. (C) Alignment of myosin sequences around the converter domain and the lever arm helix of selected myosins of different classes that have been characterized biochemically, except PfMyoA. Myosin VI is the only minus-end-directed myosin characterized so far. The alignment starts at position 650 of D.discoideum myosin II and ends at residue 821. The arrowhead indicates where the TgMyoAΔtail was truncated. The structural domains are indicated at the top of the alignment, as described by Geeves and Holmes (1999). The asterisk indicates two cysteines (SH1, SH2) conserved in class II myosins; the hash sign indicates three glycines conserved among most myosin classes. The first glycine is the proposed ‘pivot point’ corresponding to Gly699 of GgFSk (Kinose et al., 1996), and is absolutely conserved except in a plant myosin (HaMyok3) and a Caenorhabditis elegans class XII myosin. Conservation between class XIV myosins and others is highest upstream of and including the actin-binding domain. Residues conserved in at least six of eight sequences are in red; residues conserved in at least four of the five non-class XIV myosins are in green; and the C-terminus highly conserved among class XIV myosins is in blue. The myosin VI-specific insertion at the end of the converter domain is cut out of the alignment and presented below in pink. Dictyostelium discoideum myosin II (DdII, accession No. P08799); Gallus gallus (chicken) fast skeletal myosin II (GgFsk, P13538); G.gallus brush border myosin I (GgBb, U04049); G.gallus p190 myosin V (Ggp190V, Z11718); Sus scrofa domestica myosin VI (SsVI, A54818); P.falciparum myosin A (PfMyoA, AAD21242); T.gondii myosin D (TgMyoD, AF105118); T.gondii myosin A (TgMyoA, AAC47724).
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Fig. 4. Transient kinetics of TgMyoA ATPase. ATP induced dissociation of actin from TgMyoAΔtail and full-length TgMyoA in flash photolysis. (A) Light scattering signals from a 20 µl sample containing 0.5 µM actin, 1 µM TgMyoAΔtail, 0.5 mM ATP and apyrase in a multiple flash experiment. The best fit to a single exponential decay of the light scattering decrease is shown superimposed. (B) The observed rate constants of ATP-induced dissociation, kobs, of TgMyoAΔtail (filled squares) and full-length TgMyoA (open circles) are plotted versus the concentration of ATP released. The slope of a linear fit to each set of data from one isoform gives a second-order rate constant (K1k+2) of 0.46 ± 0.01/µM/s with an intercept of 3.8 ± 0.4/s for TgMyoAΔtail. For full-length TgMyoA, the linear fit gives a second-order rate constant (K1k+2) of 0.28 ± 0.005/µM/s with an intercept of 0.17 ± 0.2/s. Other conditions: experimental buffer containing 130 mM KCl, 20 mM MOPS, 5 mM MgCl2 and 10 mM fresh DTT at pH 7.0 and 22°C. (C) ADP affinity for acto·TgMyoAΔtail and full-length acto·TgMyoA. The inhibition of the kobs for ATP-induced dissociation of actomyosin is plotted versus the concentration of ADP for TgMyoAΔtail (filled squares) and full-length TgMyoA (open circles). For easier comparison between the two data sets, all kobs values were divided by k0, the value of kobs at 0 µM [ADP]. For each series of measurements, a fit to Equation 1 is superimposed, giving KAD values of 840 ± 60 µM for TgMyoAΔtail and 720 ± 90 µM for full-length TgMyoA.
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Fig. 5. Sliding velocity of actin filaments. (A) Histogram of the distribution of velocities of actin filaments sliding in the in vitro motility assay over TgMyoA. Only filaments with a minimal run length of 10 µm were scored. N, number of filaments; v, velocity; sd, standard deviation. (B) The directionality of TgMyoA movement was determined by the sliding of polarity-labelled actin filaments over a TgMyoA-decorated surface.
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Fig. 6. Determination of the step size. The frequency distribution of displacements resulting from 598 interactions of single TgMyoA molecules with single actin filaments. To obtain a sufficiently large number, events were pooled from three independent recordings with single actin filaments that allowed unambiguous determination of the direction of the events. The shift in the fitted Gaussian distribution (solid line) was interpreted as the step size (Ruff et al., 2001). The estimated step size of TgMyoA was 5.26 ± 0.45 nm.
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