Dense granule trafficking in Toxoplasma gondii requires a unique class 27 myosin and actin filaments

Abstract

The survival of Toxoplasma gondii within its host cell requires protein release from secretory vesicles, called dense granules, to maintain the parasite's intracellular replicative niche. Despite the importance of DGs, nothing is known about the mechanisms underlying their transport. In higher eukaryotes, secretory vesicles are transported to the plasma membrane by molecular motors moving on their respective cytoskeletal tracks (i.e., microtubules and actin). Because the organization of these cytoskeletal structures differs substantially in T. gondii, the molecular motor dependence of DG trafficking is far from certain. By imaging the motions of green fluorescent protein-tagged DGs in intracellular parasites with high temporal and spatial resolution, we show through a combination of molecular genetics and chemical perturbations that directed DG transport is independent of microtubules and presumably their kinesin/dynein motors. However, directed DG transport is dependent on filamentous actin and a unique class 27 myosin, TgMyoF, which has structural similarity to myosin V, the prototypical cargo transporter. Actomyosin DG transport was unexpected, since filamentous parasite actin has yet to be visualized in vivo due in part to the prevailing model that parasite actin forms short, unstable filaments. Thus our data uncover new critical roles for these essential proteins in the lytic cycle of this devastating pathogen.

FIGURE 1:

Characterization of dense granule motions in T. gondii. (A) Cartoon showing four T. gondii parasites growing inside a PV in a host cell. Inset shows cytoskeletal structures and organelles in T. gondii referred to in the text. Parasites contain 23 MTs (orange) that run two-thirds the length of the parasite, a single Golgi stack (yellow), and dense granules (green) distributed throughout the parasite. The inner membrane complex (IMC; green inset) is a series of flattened vesicles that are sutured together and underlie the parasite’s plasma membrane. The apical end of the parasite is indicated with an arrowhead. (B) Image of a four-parasite PV (yellow circle) with eGFP-labeled dense granules. eGFP accumulates in the PV after dense granule secretion. The apical end of each parasite is indicated with a white arrowhead. (C) Frequency distribution of MSD alpha values in control parasites. Trajectories with an α < 0.5 are classified as stationary (red), trajectories with an α between 0.5 and 1.4 are classified as diffusive-like (blue), and trajectories with an α > 1.4 are classified as directed (green). (D) Outline of parasites shown in B, with dense granule trajectories overlaid. Directed granule trajectories are indicated in green, diffusive-like trajectories are indicated in blue, and stationary trajectories are indicated in red. (E) Outline of parasites shown in B with directed dense granule trajectories in D depicted as arrows to highlight directionality. In D and E, black arrowheads indicate the parasites’ apical ends.

FIGURE 2:

Parasite actin but not MTs are necessary for directed dense granule transport. (A–D) Top, fluorescence images of control and oryzalin-, cytochalasin D–, and jasplakinolide-treated parasites. Green: mCherryFP-tubulin; yellow: SAG1-ΔGPI-eGFP to identify dense granules; pink: anti-actin. (A–D) Bottom, left, generic parasite outline with dense granule trajectories from 10 parasites overlaid; trajectories from five vacuoles were overlaid for oryzalin. Images were overlaid based on the x,y coordinates of the apical and basal ends of each parasite. Arrowhead indicates parasite’s apical end. Right, bar chart showing the percentage of granules exhibiting stationary (red), diffusive-like (blue), and directed (green) motion in each condition. See Supplemental Table S1 for trajectory specifics.

FIGURE 3:

Loss of TgACT1 perturbs directed dense granule motions. (A and B) Top, fluorescence images of control and rapamycin-treated LoxP-Actin parasites. Yellow: SAG1-ΔGPI-eGFP to identify dense granules; pink: anti-actin; green: YFP. (A and B) Bottom, left, parasite outline with dense granule trajectories from 10 parasites overlaid. Images were overlaid based on the x,y coordinates of the apical and basal ends of each parasite. Arrowhead indicates parasite’s apical end. Right, bar chart showing the percentage of granules exhibiting stationary (red), diffusive-like (blue), and directed (green) motion in each condition. See Supplemental Table S1 for trajectory specifics.

FIGURE 4:

Comparison of TgMyoF structure with myosin Va and localization of TgMyoF. (A) Depiction of myosin Va and putative TgMyoF structure. (B) Fluorescence image of a two-parasite vacuole showing TgMyoF localization in the parasite’s cytosol. TgMyoF is excluded from the parasite’s nucleus (N). Images are maximum-intensity projections of deconvolved images. Green: endogenous TgMyoF-emeraldFP; yellow: SAG1-ΔGPI-mCherryFP to identify dense granules; pink: anti-IMC1.

FIGURE 5:

TgMyoF is required for directed dense granule motion. (A) Strategy for creating LoxP-TgMyoF and TgMyoF-ΔCT parasite lines. See the text for details. (B) Relative expression levels of full-length TgMyoF in parental and untreated LoxP-TgMyoF parasites compared with the expression level of TgMyoF-ΔCT in rapamycin-treated LoxP-TgMyoF parasites assessed using qPCR. (C) Growth of parental and LoxP-TgMyoF parasite lines as assessed by plaque assay with and without rapamycin treatment. (D and E) Top, fluorescence image of control and rapamycin-treated LoxP-TgMyoF parasites indicating aberrant apicoplast inheritance in TgMyoF-deficient parasites. Green: TgMyoF-emeraldFP; yellow: TgFNR-RFP highlighting the apicoplast; pink: anti-IMC1. (D and E) Bottom, left, parasite outline with dense granule trajectories from 10 parasites overlaid. Images were overlaid based on the x,y coordinates of the apical and basal ends of each parasite. Arrowhead indicates parasite’s apical end. Right, bar chart showing the percentage of granules exhibiting stationary (red), diffusive-like (blue), and directed (green) motion in each condition. See Supplemental Table S1 for trajectory specifics.

FIGURE 6:

Model of dense granule trafficking. (A) Teams of TgMyoF motors interact with the granule surface via their WD40 domains and can move processively (i.e., take consecutive steps without dissociating) along actin tracks. (B) Model of TgMyoF and actin-based dense granule transport and sites of secretion: 1) Dense granules (green) formed at the Golgi (yellow) are transported (green trajectory) by TgMyoF motors along actin tracks to the parasite’s periphery. 2) Once at the periphery, diffusive-like motion (blue squiggle) provides a means to rapidly probe a small area of the IMC for a potential IMC gap and access to a docking site. 3) Dense granules that have yet to dock on the plasma membrane (PM) can once again undergo TgMyoF-directed transport (green trajectory) on filamentous actin parallel to the IMC. Once directed motion terminates, the granule is again free to move in a diffusive manner (blue squiggle) in search of a plasma membrane docking site. 4) An encounter with an IMC gap and access to the plasma membrane allows dense granule docking and secretion. Red arrows indicate the direction of directed granule motion.