Toxoplasma gondii myosin F, an essential motor for centrosomes positioning and apicoplast inheritance

Abstract

Members of the Apicomplexa phylum possess an organelle surrounded by four membranes, originating from the secondary endosymbiosis of a red alga. This so-called apicoplast hosts essential metabolic pathways. We report here that apicoplast inheritance is an actin-based process. Concordantly, parasites depleted in either profilin or actin depolymerizing factor, or parasites overexpressing the FH2 domain of formin 2, result in loss of the apicoplast. The class XXII myosin F (MyoF) is conserved across the phylum and localizes in the vicinity of the Toxoplasma gondii apicoplast during division. Conditional knockdown of TgMyoF severely affects apicoplast turnover, leading to parasite death. This recapitulates the phenotype observed upon perturbation of actin dynamics that led to the accumulation of the apicoplast and secretory organelles in enlarged residual bodies. To further dissect the mode of action of this motor, we conditionally stabilized the tail of MyoF, which forms an inactive heterodimer with endogenous TgMyoF. This dominant negative mutant reveals a central role of this motor in the positioning of the two centrosomes prior to daughter cell formation and in apicoplast segregation.

Figure 1

Schematic model of apicoplast division in T. gondii. (A) Apical secretory organelles, rhoptries (Rh) and micronemes (Mc); Ap, apicoplast surrounded by four membranes; Ct, centrosome; Cc, centrocone; Mt, mitochondrion; ER, endoplasmic reticulum; IMC, inner membrane complex; MTs, microtubules; N, nucleus. (B) Schematic representation of T. gondii tachyzoite division. Endodyogeny starts with duplication of the centrosomes and fission of the Golgi apparatus. Meanwhile, the apicoplast elongates with both extremities closely associated with the centrosomes. During nuclear division, the daughter cells emerge while the apicoplast remains associated to the centrosomes and adopts a U-shaped structure. Following constriction, the apicoplast segregates in two. Rhoptries and micronemes are made de novo and at the end of endodyogeny. Following division, residual mother cell constituents are disposed off in the residual body. ER, Mt, Cc and MTs are not represented.

Figure 2

Actin dynamics are implicated in apicoplast inheritance. (A) Indirect immunofluorescence analysis (IFA) of T. gondii RH parasites (wild type) showed abnormal segregation of the apicoplast-associated thioredoxin family protein (ATrx1) after 14 h±0.2 μM CD treatment. The Gliding-Associated Protein 45 (GAP45) was used to stain the periphery of the parasites. Scale bar, 2 μm. (B) Western blot analysis of RH parasites treated 14 h±0.2 μM CD showed accumulation of the Cpn60 precursor and the concomitant reduction of the processed (mature) form. Detection of T. gondii actin (TgACT1) was used as loading control. The percentage of Cpn60 precursor was quantified. Data are mean values±s.d. from three independent experiments. A representative western blot is presented. pCpn60, Cpn60 precursor; mCpn60, Cpn60 mature; *nonspecific binding. (C) RH parasites treated 14 h±0.2 μM CD showed accumulation of rhoptries (ROP2-4), micronemes (MIC3) and mitochondria (HSP70) in residual bodies (arrows). The micronemes and mitochondrion were only slightly affected. Scale bar, 2 μm. (D) Live fluorescence microscopy of a synchronized P. falciparum strain expressing PfSOD2-GFP treated from trophozoite to schizont stage (12 h)±0.3 μM CD. In the presence of CD, apicoplast inheritance was impaired in newly formed schizonts and parasites were blocked in egress. Scale bar, 8 μm. (E) Live fluorescence microscopy of P. berghei expressing PbACP-GFP treated 14 h±0.3 μM CD in vitro. Interference with actin polymerization resulted in aberrant plastid segregation. Accumulation of apicoplast staining was observed in close proximity to the food vacuole. Scale bar, 5 μm.

Figure 3

Regulators of actin dynamics are essential for apicoplast inheritance in T. gondii. (A) Long-term depletion of TgPRF leads to abnormal apicoplast (Cpn60) inheritance. In the absence of ATc, PRF-iKO parasites showed normal plastid segregation. Following 48h depletion of PRF, the apicoplast adopted an elongated structure (arrowhead) and is lost in some parasites. After 80 h of ATc treatment, most of the vacuoles contained parasites lacking an apicoplast and Cpn60 staining was often found outside of the parasites (arrows), presumably in residual bodies. Functionally complemented parasites expressing wild-type PRF (PRF-iKO+WTc) showed no phenotype after 80 h of ATc treatment. (B) ADF-iKO parasites treated for 48 h with ATc loss the apicoplast as shown by anti-ATrx1 staining that accumulated outside of the parasites (arrow). (C) Apicoplast inheritance was affected 36 h after stabilization of DDFH2/2. (D–F) Western blot analysis revealed the accumulation of the Cpn60 precursor form and reduction of the mature form after 80h depletion of TgPRF, 48h depletion of ADF or after 36h stabilization of DDFH2/2. Complemented and parental strain as well as untreated parasites showed proper processing of Cpn60. Regulation of Myc-tagged inducible copy of PRF in PRF-iKO and the presence of the Ty-tagged complemented wild-type copy (PRF-iKO+WTc) was analysed using the corresponding antibodies. Anti-HA was used to assess the regulation of ADF-iKO. Detection of TgACT1 was used as loading control. The percentage of Cpn60 precursor was quantified. Data are mean values±s.d. from three independent experiments. A representative western blot is presented. pCpn60, Cpn60 precursor; mCpn60, Cpn60 mature; *nonspecific binding. Scale bar, 2 μm.

Figure 4

T. gondii myosin F is associated with the apicoplast during parasite division. (A) TgMyoF (DQ131541) contains six IQ motifs, a coiled-coil domain and seven WD40 domains. All domains were predicted using SMART EMBL. (B) TgMyoF-3Ty is found at the expected molecular mass by western blot (216 kDa). Detection of T. gondii catalase was used as loading control. (C) MyoF-3Ty concentrates at the extremities of the apicoplast during division (arrow, upper panel) and to the newly formed daughter cells after apicoplast fission (arrowhead, upper and middle panels). In nondividing parasites (lower panel), TgMyoF is concentrated in the apical region, on top of the nucleus and at the pellicle. Scale bar, 2 μm.

Figure 5

Knockdown of TgMyoF is deleterious for parasite survival and affects apicoplast inheritance. (A) Inducible MyoF (iMycMyoF) migrates at the predicted molecular size (216 kDa) by western blot and is downregulated following 48 h of ATc treatment. Detection of T. gondii catalase was used as a loading control. (B) The iMycMyoF localized in the vicinity of the apicoplast and accumulated at the extremities of the dividing organelle (Cpn60; arrow). After apicoplast fission, iMycMyoF localized in the nascent daughter cells (arrowhead). (C) IFA showed abnormal apicoplast inheritance in parasites treated 32 h with ATc. The apicoplast was found in residual bodies (arrow). (D) Accumulation of Cpn60 precursor and the concomitant decrease in processed Cpn60 were observed upon 48 h of ATc treatment. Parental Ku80-KO strain and MyoF-iKO parasites grown in the absence of ATc showed proper maturation of Cpn60. The percentage of Cpn60 precursor was quantified. Data are mean values±s.d. from three independent experiments. A representative western blot is presented. pCpn60, Cpn60 precursor; mCpn60, Cpn60 mature; *nonspecific binding. (E) Except for a few cases, the mitochondrion stained with anti-HSP70 was not affected by TgMyoF depletion and no or very little accumulation in residual bodies was observed. In contrast, some rhoptry and microneme contents stained with anti-ROP2-4 and anti-MIC3 antibodies respectively were accumulated in residual bodies in TgMyoF-depleted parasites. The inheritance of the Golgi (*) was unaffected. Division was visualized using anti-ISP1 antibodies that stained the apical cap of the mother cell (arrow) and the nascent daughter cells (arrowhead). Parasites were treated 32 h±ATc. (F) Intracellular growth of MyoF-iKO and Ku80-KO grown 78 h±ATc. Parasites per vacuole were counted 30 h after inoculation of new host cells. TgMyoF-depleted parasites were blocked at 2–4 parasites. Ku80-KO strain and nontreated MyoF-iKO displayed normal growth. Data are mean values±s.d. from three biological independent experiments where 200 vacuoles were counted for each condition. Scale bar, 2 μm.

Figure 6

Overexpression of TgMyoF tail acts as a dominant negative mutant. (A) Schematic representation of the FKBP destabilization domain (DD) constructs and dominant negative effect of DDMyoF-tail caused by the formation of an inactive heterodimer that poisons the endogenous TgMyoF. (B) Western blot analysis using anti-Myc showed the stabilization of DDMyoF-tail at the predicted size (118 kDa) after Shld-1 treatment for 48 h. Catalase serves as loading control. (C) IFA (anti-Myc) detected DDMyoF-tail in the cytosol and in enlarged residual bodies (arrowhead). (D) The 24h stabilization of DDMyoF-tail led to a defect in apicoplast (anti-ATrx1) inheritance with the majority found outside the parasites (arrowhead). (E) Golgi (*) was not affected by stabilization of DDMyoF-tail. Dividing parasite were visualized with anti-ISP1. Arrow, mother cell; arrowhead, nascent daughter cell. In the presence of Shld-1, few mitochondrial fragments stained with anti-F1-ATPase (5F4) were observed in residual bodies (arrowhead). Microneme (anti-MIC3) and rhoptry contents (anti-ROP7) were found accumulating in residual bodies (arrowhead) and some rhoptries were dispersed in the cytosol (arrow). (F, left) Intracellular growth assay was performed on parental (RH) and DDMyoF-tail parasites grown for 30 h±Shld-1 before fixation and no alteration of growth was observed. (F, right) RH and DDMyoF-tail parasites were pretreated±Shld-1 during 20 h. Host cells were inoculated and the pretreated parasites were grown for 30 h±Shld-1 before fixation. Stabilization of DDMyoF-tail led to a severe arrest at 2–4 parasites per vacuole. Data are mean values±s.d. from three independent experiments. (G) The 48h stabilization of DDMyoF-tail led to the accumulation of Cpn60 precursor and decrease of the processed Cpn60 by western blot. TgACT1 was used as a loading control. The percentage of Cpn60 precursor was quantified. Data are mean values±s.d. from three independent experiments. pCpn60, Cpn60 precursor; mCpn60, Cpn60 mature; *nonspecific binding. (H) DDGFPMyoF-tail was stably expressed in MyoF-3Ty parasites. Immunoprecipitation (IP) was performed with anti-GFP coupled beads. Bound fractions were analysed by western blot and revealed the presence of MyoF-3Ty only when DDGFPMyoF-tail is stabilized (+Shld-1). Scale bar, 2 μm.

Figure 7

Electron microscopy analysis of parasites expressing DDMyoF-tail. (A) In the absence of Shld-1, DDMyoF-tail parasites show a typical morphology, with the apicoplast positioned near the nucleus and the rhoptries and micronemes at the apical pole. Scale bar, 1 μm. (B) A vacuole showing a residual body devoid of organelles and surrounded by parasites. Scale bar, 1 μm. (C) Stabilization of DDMyoF-tail led to an accumulation of intact micronemes, rhoptries and mitochondrial fragments in enlarged residual bodies. An intact apicoplast surrounded by four membranes was also detected. Some rhoptries and micronemes were also found correctly localized to the parasite apical pole. Scale bar, 1 μm. (D) A vacuole showing a residual body containing rhoptries and micronemes and undefined membranous material. Scale bar, 2 μm. Ap, apicoplast; C, conoid; N, nucleus; Mc, micronemes; Mt, mitochondria; RB, residual body; Pl, pellicle; Rh, rhoptries.

Figure 8

MyoF contributes to the close positioning of centrosomes during division. (A) Scoring of daughter cell orientation during parasite division by IFA using anti-ISP1 antibodies. Interference with TgMyoF function led to up/down topology whereas wild-type or untreated parasites adopted an up topology. Loss of apicoplast by antibiotic treatment (RH+ATc 4 μg/ml) did not affect the orientation of division. Data are mean values±s.d. from three independent experiments where 200 vacuoles were counted for each condition. (B) IFA of representative parasites scored in (A). Daughter cells (ISP1) are marked with *. (C) Time-lapse video microscopy images of DDMyoF-tail transiently transfected with ACP-DsRed (apicoplast) and GFP-GAP45Ct (daughter cells, IMC). In the presence of Shld-1, daughter cells (*) emerged in an up/down orientation and the apicoplast failed to be encapsulated in contrast with nontreated parasites that divided in up orientation. (D–E) Regardless of MyoF functional impairment, the centrosomes (centrin1, *) were always found in association with the forming daughter cell. Arrow, mother cell; arrowhead, nascent daughter cell. DDMyoF-tail parasites were treated 24 h±Shld-1 and MyoF-iKO 48 h±ATc. (F) Wild-type parasites treated with 4 μg/ml ATc for 14 h lost the apicoplast but are still able to form rosette. Scale bar, 2 μm.

Figure 9

Contribution of TgMyoF to apicoplast inheritance. (A) Schematic representation of TgMyoF localization (grey area) during parasite division. In the early steps, TgMyoF localizes along the apicoplast and accumulates at the extremities of the organelle. After apicoplast fission, TgMyoF localizes inside the growing daughter cells (DCs). PR, posterior ring; N, nucleus; Ct centrosome; SM, spindle microtubules; Ap, apicoplast; Rh, rhoptries; Mc, micronemes. (B) In the wild-type situation, division starts with the duplication of the centrosomes, fission of the Golgi and elongation of the apicoplast, which associates with both centrosomes. Slightly before the end of DNA replication, daughter cells start to emerge and engulf the apicoplast. During daughter bud extension the apical organelles (rhoptries and micronemes) are made de novo and anchored at the apical pole. Meanwhile, the mother apical organelles are degraded. The large majority of daughter cells were found to bud in the same orientation; towards the apical end of the mother cell. (C) Alteration of MyoF function does not affect the duplication of the centrosomes or fission of the Golgi. However, daughter cells emerge in opposite or random orientations and fail to encapsulate the apicoplast. MyoF participates in the correct positioning of the daughter cell likely by maintaining the centrosomes in close proximity. Not only the apicoplast but also rhoptries and micronemes accumulate in the residual bodies either as a result of alteration of centrosome positioning or another function of TgMyoF.