The unusual dynamics of parasite actin result from isodesmic polymerization

Abstract

Previous reports have indicated that parasite actins are short and inherently unstable, despite being required for motility. Here we re-examine the polymerization properties of actin in Toxoplasma gondii, unexpectedly finding that it exhibits isodesmic polymerization in contrast to the conventional nucleation-elongation process of all previously studied actins from both eukaryotes and bacteria. Polymerization kinetics of actin in T. gondii lacks both a lag phase and critical concentration, normally characteristic of actins. Unique among actins, the kinetics of assembly can be fit with a single set of rate constants for all subunit interactions, without need for separate nucleation and elongation rates. This isodesmic model accurately predicts the assembly, disassembly and the size distribution of actin filaments in T. gondii in vitro, providing a mechanistic explanation for actin dynamics in vivo. Our findings expand the repertoire of mechanisms by which actin polymerization is governed and offer clues about the evolution of self-assembling, stabilized protein polymers.

Figure 1. Concentration dependent polymerization of TgACTI vs ScACT

(A) Light scattering (arbitrary units, A.U.) vs. concentrations (µM) of TgACTI. Plot at right shows the initial time course expanded to highlight the lack of lag phase. (B) Light scattering (arbitrary units, A.U.) vs. concentrations (µM) of ScACT. Initial plots show evidence of lag (expanded plot at right), and plateau at later time points. (C) Fluorescence microscopy of polymerized TgACTI visualized with 0.33 µM Alexa 488-phalloidin ± equimolar amounts of unlabeled phalloidin (1:1). Scale bar = 5 µm. (D) Quick-freeze, deep-etch electron micrographs of ScACT (5 µM) vs. TgACTI (40 µM) polymerized in F buffer. Scale bars = 100 nm. (E) Lengths of actin filaments measured in vitro for ScACT and TgACTI as in D, or for TgACTI in vivo as reported previously. Left y-axis, ScACT, right y-axis, TgACTI. Means ± S.D. (F) Circular dichroism measurements of purified ScACT vs. TgACTI, 0.4 µM. All panels are representative experiments of three or more similar experiments.

Figure 2. TgACTI forms small heterogeneous polymers

(A) TgACTI (5 µM top vs. 30 µM middle) was polymerized in F buffer for 1 h at room temperature ± equimolar phalloidin (Ph) and centrifuged at 100,000g or 350,000g for 1 h at room temperature. Pellet (P) or precipitated supernatant (S) fractions were resolved on a 12% SDS-PAGE gel, stained with SYPRO Ruby, and quantified by phosphorimager analysis. ScACT (5 µM, bottom) was analyzed in parallel. Mean ± S.D., n = 3 or more experiments combined in graphs; representative gels are shown. (B) Sedimentation of TgACTI (5 µM top, 30 µM middle) in G buffer (G, blue), F buffer (F, red) or F buffer with equimolar phalloidin (F+, green) by sucrose density centrifugation. ScACT (5 µM) was analyzed in a parallel gradient. Dashed and solid black lines correspond to the proportion of TgACTI that would be found in the 100,000g and 350,000g pellets under standard sedimentation conditions as in A. Size standards denoted with black arrows. Representative of 3 or more similar experiments.

Figure 3. TgACTI polymerizes by an isodesmic process

(A) Polymerization of TgACTI in F buffer pH 8.0 for 1 h followed by centrifugation at 100,000g for 1 h at room temperature. The points are experimental results for the concentration of protein in the pellet (blue) or supernatant (red), plotted versus initial total concentration. The solid-line curves are the results of simulations using the isodesmic model. Inset shows the concentration range from 0.5 – 20 µM. (B) Similar sedimentation analysis performed as in (A) except KMEI (see methods) pH 7.2. (C) Polymerization of yeast actin (ScACT) in F buffer pH 8.0 for 1 h followed by centrifugation at 100,000g for 1 h at room temperature. Inset shows linear regression plot of lower concentrations (0.5 – 5 µM) used to estimate the Cc. (D) Polymerization of TgACTI in F-buffer at steady state (i.e. 20 h), as monitored by light scattering. Linear regression (blue) or nonlinear (red) fit curves. Dotted line indicates 95% confidence interval. Mean, n ≥ 3 experiments. Lower concentrations are expanded in inset. (E) Sedimentation of TgACTI polymerized to steady state in F buffer for 20 h. Centrifugation at 100,000g for 1 h at room temperature. The points are experimental data, and the solid curve is the result of a simulation with the isodesmic model. (F) Dilution-induced depolymerization of TgACTI vs. ScACT. Steady-state samples polymerized for 20 h were diluted 4-fold in F buffer, and depolymerization was monitored by light scattering. The red curve is the prediction from the isodesmic model for TgACTI and from a nucleation-elongation model for ScACT. Unless otherwise noted, all panels are representative experiments of three or more similar experiments.

Figure 4. Modeling filament polymerization and dynamics

(A, B) Standard nucleation-elongation mechanism vs. isodesmic model. Under isodesmic polymerization the on rate (k₊) and off rate (k₋) are the same for every monomer addition, independent of length. (C) Molecular dynamics simulation reflects a propensity for weaker lateral interactions in Toxoplasma vs. muscle actin filaments (conventional). (D) Key amino acid differences that were predicted to alter the lateral interaction between protofilaments are highlighted in yellow and green, while the two strands of the filament are colored blue and pink.