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Three F-actin assembly centers regulate organelle inheritance, cell-cell communication and motility in Toxoplasma gondii

Research Article
Cite this article as: eLife 2019;8:e42669 doi: 10.7554/eLife.42669
9 figures, 7 videos and 2 additional files

Figures

Schematic representations of T. gondii division, motility and invasion.

(A) Intracellular growth development of T. gondii consists of the synchronous geometric expansion of two daughter cells within a mother cell. Apicoplast inheritance is coupled to cell division. All parasites are connected by their basal pole to the central residual body (RB) that allows rapid diffusion of materials between parasites of the same parasitophorous vacuole (PV). The PV contains a network of elongated nanotubules that form connections with the PV membrane. (B) Schematic representation of a gliding parasite. The parasite plasma membrane (PPM) and the inner membrane complex (IMC, a system of flattened membranous sacs called alveoli that directly underlies the PPM) compose the pellicle. Transmembrane adhesins (MICs) are secreted apically by the micronemes and will interact with host cell ligands. Within the pellicle MICs bind to GAC with the latter connecting the complex to F-actin. The rearward translocation of the GAC-adhesin complexes by the successive action of the MyoH and MyoA glideosomes will result in parasite forward motion. (C) During invasion, rhoptry organelles secrete the rhoptry neck proteins (RONs) in the host plasma membrane. This parasite-derived receptor will interact with the micronemal apical membrane antigen 1 (AMA1) to form the moving junction (MJ). The rearward translocation of this junction by MyoH and MyoA will result in host cell invasion. Invagination of the host plasma membrane leads to the formation of the PV. APR: apical polar ring.

https://doi.org/10.7554/eLife.42669.002
Figure 2 with 2 supplements
FRM2 localizes in a juxtanuclear region and participates in apicoplast inheritance.

(A) Expression of Cb-GFPTy in RH parasites showed a strong staining in the RB (asterisk) and in a juxtanuclear region (arrowhead) often overlapping the apicoplast (α-ATrx). α-IMC1 antibodies stain the pellicle. (B) FRM2-Ty is mainly confined above the nucleus and co-localizes with the apical juxtanuclear staining of Cb-GFP. α-GAP45 antibodies stain the pellicle. (C) FRM2-Ty localizes at the proximity of the Golgi (transiently transfected with GRASP-YFP). Triple colocalization of FRM2-Ty, GRASP-YFP and apicoplast (α-Cpn60) showed a constant association of FRM2-Ty with the Golgi, but not with the apicoplast. (D) During daughter cells development, FRM2-Ty accumulates on top of the dividing apicoplast. (E) MyoF (Myc-MyoF-iKD) partially co-localizes with FRM2-Ty and its conditional depletion did not impact on FRM2-Ty localization. (F) Parasites lacking FRM2 are impaired in apicoplast inheritance and showed abnormal daughter cell orientation (arrow: up/up; asterisk: up/down, arrowhead: down/down). (G) Quantification of apicoplast inheritance defects. (H) Growth competition assay reveals a significant defect confirmed by (I) intracellular growth assay. Data are presented as mean ±SD. Significance was assessed using a parametric paired t-test and the two-tailed p-values are written on the graphs. Dashed lines outline parasites periphery. Scale bars: 2 µm.

https://doi.org/10.7554/eLife.42669.003
Figure 2—figure supplement 1
FRM2 localization.

(A) Treatment with high concentration of anhydrous tetracycline (ATc) resulted in apicoplast (α-Cpn60) loss but did not affect localization of FRM2-Ty. (B) FRM2-Ty localizes on top of divided apicoplast and in close proximity of the Golgi. Dashed lines highlight parasites periphery. Scale bar: 2 µm.

https://doi.org/10.7554/eLife.42669.004
Figure 2—figure supplement 2
Characterization of FRM2-KO.

(A) CRISPR/Cas9 strategy to knockout the entire FRM2 locus with a double gRNAs. Thunderbolts show gRNA target sites. Integration was confirmed by PCR analysis. (B) Golgi (GRASP-YFP) division and inheritance was not affected in FRM2-KO parasites while the apicoplast (α-Cpn60) was affected. (C) Quantification of the orientation of the daughter cells in FRM2-KO parasites (Related to Figure 2F: arrow: up/up; asterisk: up/down, arrowhead: down/down). (D) No major defects were observed in other organelles (micronemes stained with α-MIC2; mitochondria stained with α-HSP70); however, in addition to the apicoplast, some rhoptries (α-ROP2-4; asterisk) were present in the RB. Data are presented as mean ±SD. Significance was assessed using a parametric paired t-test and the two-tailed p-values are written on the graphs. Scale bar: 2 µm.

https://doi.org/10.7554/eLife.42669.005
Figure 3 with 4 supplements
FRM3 localizes to the basal pole and residual body and participates in cell-cell communication.

(A) FRM3-Ty accumulates at the basal pole and in the residual body (asterisks). FRM3-Ty is also located in the apical region of growing daughter cells (arrowhead). (B) FRM3-KO parasites were unable to form rosettes and divided asynchronously (asterisks). IMC Sub-compartment Proteins 1 (ISP1) stains the apical cap. (C) Quantification of asynchronous division within a vacuole in FRM3-KO parasites. (D–E) Time-lapse images of FRAP experiments in wt RHΔKu80 and FRM3-KO parasites. A flash indicates the bleached area and fluorescence recovery quantifications were recorded in the areas delimited with colors. (F) Quantification of cell-cell communication in wt and FRM3-KO. (G) In FRM3-KO parasites, MyoI is no longer present in the RB. (H) No difference in MyoJ localization or basal pole (arrowheads) constriction was observed in the absence of FRM3. Arrow highlight the basal pole of forming daughter cells. Data are presented as mean ±SD. Significance was assessed using a parametric paired t-test and the two-tailed p-values are written on the graphs. Scale bars: 2 µm.

https://doi.org/10.7554/eLife.42669.007
Figure 3—figure supplement 1
Localization of FRM3.

(A) In addition to its localization at the basal end and in the residual body (asterisks), FRM3-Ty is also found in the apical regions of the forming daughter cells (arrowhead). (B) Co-localization with Golgi (GRASP-YFP) and apicoplast (α-Cpn60) proved that FRM3-Ty is restricted to the apical part of nascent daughter cells but in a localization distinct from FRM2. Dashed lines outline parasite periphery. Scale bar: 2 µm.

https://doi.org/10.7554/eLife.42669.008
Figure 3—figure supplement 2
Generation and characterization of FRM3-KO.

(A) FRM3-KO was obtained by double homologous recombination of an HXGPRT cassette in the FRM3 locus. Integration was confirmed by PCR analysis. (B) Plaque assay conducted over 7 days showed no difference in plaque size between wt and FRM3-KO parasites. (C) Competition assay did not reveal any significant defect. FRM3-KO parasites were mixed with a wild-type GFP expressing strain at an 80/20 ratio. Ratios were followed over six passages. An RHΔKu80 was used as control using the same settings.

https://doi.org/10.7554/eLife.42669.009
Figure 3—figure supplement 3
FRAP experiments in FRM2-KO and FRM3-KO.

(A) To rule out possible de novo synthesis of GFP in the FRAP experiments, an entire vacuole was bleached in wt parasites. No recovery of the fluorescence was observed even after 3 min. The bleached area is indicated by a flash. (B) FRM2-KO parasites have no defect in cell-cell communication. (C) Quantification of (B). (D) Impaired cell-cell communication in FRM3-KO. In few vacuoles, some parasites partially recovered staining suggesting some residual connections (arrow). Data are presented as mean ±SD. Scale bar: 2 µm.

https://doi.org/10.7554/eLife.42669.010
Figure 3—figure supplement 4
Characterization of FRM3-KO.

(A) and (B) The rhoptries, apicoplast, micronemes, mitochondria or Golgi were not affected by FRM3-KO. Scale bar: 2 µm.

https://doi.org/10.7554/eLife.42669.011
Figure 4 with 3 supplements
FRM1 is localized at the apical tip of parasites to sustain gliding motility, egress and invasion.

(A) FRM1-KO resulted in extremely small plaques formed after 7 days compared to RH parasites. Reverted FRM1-KO cl.4 parasites formed plaques comparable to wt parasites. (B) FRM1-mAID-HA localized at the apical tip (arrowheads) and was tightly regulated by IAA. (C) Depletion of FRM1-mAID-HA resulted in no plaques formation after 7 day of IAA treatment. TIR1 represents the parental strain. (D–F) In absence of FRM1 (+IAA), parasites were unable to glide on gelatin-coated glass (trails labelled with α-SAG1) and were severely impaired in both egress and invasion. (G) Fluorescent beads capping assay revealed a complete block of capping in absence of FRM1 with a large increase of bound parasites. Conditional depletion of GAC resulted with a block of capping and an accumulation of bound/capped parasites. (H) Localization of FRM1-Ty is restricted to the apical tip of mature parasites (asterisks) and forming daughter cells (arrows). FRM1-Ty localization is not affected upon treatment with CytD and JAS, an actin polymerization enhancer resulting in actin projections (arrowhead). Data are presented as mean ±SD. Significance was assessed using a parametric paired t-test and the two-tailed p-values are written on the graphs. Dashed lines highlight parasites periphery. Scale bars: 2 µm.

https://doi.org/10.7554/eLife.42669.013
Figure 4—figure supplement 1
Generation and characterization of FRM1-KO.

(A) Schematic representations of the strategies used to generate a direct knockout of FRM1. CRISPR/Cas9 approaches with either a single gRNA or double gRNA were attempted. (B) Two independent clones containing deletions causing out of frame mutations using a single gRNA were obtained. (C–E) FRM1-KO clones were severely impacted in all aspects of motility. (F) Revertants emerged from the initially clonal FRM1-KO population, revealed spontaneous mutations leading to the correction of the out of frame mutation. Data are presented as mean ±SD. Significance was assessed using a parametric paired t-test and the two-tailed p-values are written on the graphs. Scale bar: 2 µm.

https://doi.org/10.7554/eLife.42669.014
Figure 4—figure supplement 2
Generation and characterization of FRM1-mAID-HA.

(A) Schematic representations of the strategy used to obtain the FRM1-mAID-HA. (B) FRM1 was not involved in apicoplast inheritance (α-ATrx). (C) and (D) FRM1 was not involved in cell-cell communication. (E) Representative pictures of the parasites scored in the fluorescent beads capping assay. From left to right: unbound, bound, bound/capped, and capped. α-MIC2 antibodies were used to label the apical end. Asterisks label the basal end. Data are presented as mean ±SD.

https://doi.org/10.7554/eLife.42669.015
Figure 4—figure supplement 3
FRM1 associates with the apical end early during division.

(A) FRM1 emerged very early during the division process. The centrosomes were stained with α-Centrin1. Dashed lines outline parasites periphery. Scale bars: 2 µm.

https://doi.org/10.7554/eLife.42669.016
Figure 5 with 5 supplements
The FRMs have no overlapping functions and FRM2 and FRM2 generate the two specific subpopulations of F-actin observed in intracellular parasites.

(A) Selective disruptions of F-actin staining in the different FRMs knockout. FRM2 is linked to the juxtanuclear Cb-GFPTy staining (arrowhead) while FRM3 generates the F-actin in the RB (asterisks). Conditional depletion of FRM1 was not affecting Cb-GFPTy staining. Absence of both FRM2 and 3 resulted with a diffuse Cb-GFPTy staining. Cb-GFPTy was stably expressed in RH and FRM2/3-KO and transiently transfected in FRM2-KO, FRM1-mAID-HA and FRM3-KO. (B–C) Basal pole (arrowheads) constriction is not affected upon deletion of FRM3 or FRM2/3. The EF-hand-containing protein centrin 2 (CEN2) was used as marker of the basal pole and C-terminally YFP tagged at the endogenous locus. CEN2-YFP localizes not only to the basal pole but also to the apical end and annuli (arrows), and to the centrosome (asterisks). Data are presented as mean ±SD. Scale bars: 2 µm.

https://doi.org/10.7554/eLife.42669.018
Figure 5—figure supplement 1
Supplementary images of Cb-GFPTy.

(A) Supplementary images of Cb-GFPTy in FRM2-KO, FRM3-KO and FRM2/3-KO. (B) Transient expression of Cb-GFPTy in wild type parasites sometimes resulted with an apicoplast (arrow) inheritance defect. Scale bar: 2 µm.

https://doi.org/10.7554/eLife.42669.019
Figure 5—figure supplement 2
Generation and characterization of FRM2/3-KO.

(A) A second FRM2-KO was generated using a single gRNA approach to disrupt the FRM2-Ty locus in ΔKu80 strain. Knockout of FRM2 was assessed by PCR and immunofluorescence assay using α-Ty antibodies. Disruption of the FRM3 locus was generated in this background as described previously to generate the FRM2/3-KO. PCR analyses were used to assess integration. (B) Plaque assays conducted over 7 days did not reveal any significant difference between singles and double KO of FRM2 and FRM3, (C) which was confirmed by competition assay. (D) Apicoplast inheritance defect was not aggravated upon deletion of FRM3 in FRM2-KO. (E) Gliding motility assay and scoring of upright twirling, circular gliding, and helical rotation. Absence of FRM1 resulted in no gliding, while FRM2/3-KO parasites behaved like wild type. Data are presented as mean ±SD except for (E) where parasite movements were scored by pooling motile parasites from three independent experiments.

https://doi.org/10.7554/eLife.42669.020
Figure 5—figure supplement 3
Absence of FRM2 and 3 is not affecting dense granule proteins localizations.

(A) PV and PVM localizations of the dense granule proteins (GRAs) GRA1, GRA2 and GRA3 in FRM2-KO, FRM3-KO and FRM2/3-KO. No alteration of the signal was observed for the different knockouts. 30 min with PFA/GA fixation allows efficient labelling of the GRAs present at the PVM and in the PV while the staining from the dense granule is weak. (B) 10 min PFA fixation allows in contrast an efficient labelling of the GRAs within the dense granules, while the PV and PVM staining are sometime lost. No alteration of the GRA1 and GRA3 signals were observed in these conditions. (C) Transient transfection of GRA16-Myc was used to assess the export of proteins beyond the PVM. In wt condition, GRA16 is exported in the host nucleus. FRM2, FRM3 or FRM2/3 deletions did not affect the export of GRA16 to the host nucleus. Scale bar: 2 µm.

https://doi.org/10.7554/eLife.42669.021
Figure 5—figure supplement 4
Absence of FRM2 and 3 is not affecting the nanotubular network.

(A) The PV contains a network of elongated nanotubules forming connections with the PVM. The GRAs decorate this intravacuolar network after invasion. No alteration of this nanotubular network (arrows) was observed by electron microscopy in FRM2-KO, FRM3-KO and FRM2/3-KO.

https://doi.org/10.7554/eLife.42669.022
Figure 5—figure supplement 5
Generations and characterizations of FRM1/2 and FRM1/3 mutants.

(A) FRM2-KO, in the FRM1-mAID-HA strain, was generated using a single gRNA approach to disrupt the FRM2 locus. The same strategy was used for FRM3 in the FRM1-mAID-HA strain. (B) Knockouts of both FRM2 and FRM3 displayed the previously reported phenotypes; loss of apicoplast in absence of FRM2 and loss of the Cb-GFPTy staining in the RB in absence of FRM3. (C) Absence of FRM1 in FRM2-KO did not aggravate the apicoplast inheritance defect. (D–E) Absence of FRM1 in FRM3-KO did not affect basal constriction. MyoC was used as marker of the basal pole (arrowheads) and C-terminally tagged at the endogenous locus in the different strains. Data are presented as mean ±SD. Scale bar: 2 µm.

https://doi.org/10.7554/eLife.42669.023
Apically generated F-actin by FRM1 accumulates at the basal pole.

(A–B) Snapshots of egressing RH and FRM2/3-KO parasites expressing Cb-GFPTy after stimulation with BIPPO. Asterisks represent the accumulation of F-actin at the basal pole. The arrow shows the RB left behind after egress. (C) In invading parasites, a ring of F-actin (arrowheads) translocates from the apical to the basal end of the parasites. (D) Accumulation of F-actin at the basal end (asterisks) was observed prior to parasites movement or (E) even in absence of gliding in extracellular parasites on gelatin coated cover slips. Parasites were either stimulated with BIPPO (responsible for the background change in fluorescence) or incubated in extracellular buffer (EC). Dashed lines highlight parasites periphery. Scale bars: 2 µm.

https://doi.org/10.7554/eLife.42669.025
Figure 7 with 1 supplement
An apico-basal F-actin flux is generated by FRM1 and depends on myosins.

(A) Colocalization of Cb-GFPTy and RON4 at the MJ of invading parasites in wt and FRM2/3-KO parasites (arrowheads). Some F-actin staining can be observed within the pellicle posterior to the MJ (arrows). Asterisks represent the accumulation of F-actin at the basal pole. (B) Extracellular wt parasites stimulated with BIPPO, showed a robust accumulation of F-actin by immunofluorescence assays at the basal end with a single basal dot of Cb-GFPTy. α-AMA1 antibodies (arrowheads) label the apical end while asterisks show the basal ends. (C) Quantification of basal accumulation of F-actin in (B). (D–F) Contributions of FRM1 and MyoH to the basal accumulation of F-actin in extracellular parasites stimulated with BIPPO. In the absence of MyoH or FRM1, F-actin basal accumulation is abrogated (asterisk). α-AMA1 antibodies label the apical end. (G–H) In absence of MyoA, F-actin accumulates at the start of the IMC (arrowhead). (I) RICM analysis of BIPPO stimulated extracellular parasites. Circular gliding parasites (arrows) were attached on their entire length on the surface resulting in a continuous signal while (J) helical gliding parasites first attached on the surface with their apical end (arrowhead), followed by translocation of the adhesion site backward with a concomitant detachment of the apical end. A second cycle was generated apically (yellow arrowhead) once the adhesion site reached the basal end (asterisk). Data are presented as mean ±SD. Scale bars: 2 µm.

https://doi.org/10.7554/eLife.42669.028
Figure 7—figure supplement 1
Basal accumulation of F-actin is independent of COR.

(A) Conditional knockdown of COR is not affecting basal accumulation of F-actin. α-AMA1 antibodies (arrowheads) label the apical end while asterisks show the basal ends.

https://doi.org/10.7554/eLife.42669.029
Figure 8 with 4 supplements
Activation of the apico-basal flux of F-actin relies on calcium signaling and AKMT.

(A) Absence of microneme secretion, abolished by depletion of TFP1, did not affect the apico-basal flux of F-actin. Extracellular parasites were stimulated with BIPPO. (B) F-actin flux is blocked by C1 (PKG inhibitor) and can only be by-passed with the calcium ionophore A23187. (C) CDPK3 is not involved in F-actin flux. Here, parasites were incubated in intracellular buffer and stimulated with A23187. (D) The CDPK1-specific inhibitor 3MB-PP1 blocked the apico-basal flux of F-actin in extracellular parasites stimulated with BIPPO. (E) Conditional depletion of CDPK1 using the same stimulation, resulted in no F-actin flux and (F) in a severe defect in conoid protrusion (α-GAC arrowheads). (G) AKMT is critical for the establishment of the apico-basal flux, while GAC is dispensable. Scale bar: 2 µm.

https://doi.org/10.7554/eLife.42669.036
Figure 8—figure supplement 1
BAPTA-AM inhibits microneme secretion and F-actin flux.

(A) Pretreatment with the calcium chelator BAPTA-AM resulted in inhibition of microneme secretion. Pellets and supernatants (SN) were analyzed for secretion by western blot using α-MIC2 antibodies. α-catalase (CAT) and α-dense granule 1 (GRA1) antibodies were used to assess parasite lysis and constitutive secretion respectively. (B) Pretreatment with BAPTA-AM abolishes the flux of F-actin.

https://doi.org/10.7554/eLife.42669.037
Figure 8—figure supplement 2
CDPK3 regulates microneme secretion in intracellular conditions.

(A) To generate CDPK3-KO, a CRISPR/Cas9-assisted double homologous recombination of an HXGPRT cassette in the CDPK3 locus was performed. Integration was confirmed by PCR analysis. (B) Microneme secretion is impaired in CDPK3-KO parasites stimulated with A23187. Parasites were kept in intracellular buffer during the entire experiment. SN: supernatant.

https://doi.org/10.7554/eLife.42669.038
Figure 8—figure supplement 3
Generation and characterization of CDPK1-iKD.

(A) CDPK1-iKD was obtained by replacing the endogenous promoter with a TetO7 inducible promoter. The inducible vector also encodes for the transactivator TATi-1. Double homologous recombination was assisted by CRISPR/Cas9 (B) CDPK1 (α-Myc) was tightly regulated after 48 hr of ATc treatment. (C) Depletion of CDPK1 resulted in no plaque formation after 7 days of ATc treatment. (D) Depletion of CDPK1 impaired microneme secretion. SN: supernatant.

https://doi.org/10.7554/eLife.42669.039
Figure 8—figure supplement 4
Validation of GAC antibodies.

(A) Western blot confirmed the specificity of the antibodies raised against full length TgGAC. α-GAC antibodies were used as a marker of conoid protrusion.

https://doi.org/10.7554/eLife.42669.040
Schematic models.

(A) Schematic representation of the contribution of FRM2 in apicoplast inheritance and FRM3 in synchronous division and rosette formation. (B) During invasion, a ring of F-actin translocates with the MJ to the rear of the parasite. Small filaments are likely present within the pellicle and translocated by the actomyosin system to the basal pole, contributing to the F-actin accumulation. (C) Schematic summary of the distribution of F-actin produced at the apical end by FRM1 under the different conditions tested in this study. F-actin either accumulated at the basal end (left), did not show any accumulation (middle), accumulated at the junction between MyoH and MyoA (right). (D) Schematic summary highlighting the essential roles of myosins, AKMT and calcium signaling in controlling the apico-basal flux of F-actin. The events leading to parasite egress and motility are initiated by the activity of the cGMP-dependent protein kinase (PKG) which activates both the calcium and the lipid branches of the signaling pathway. APH, located on the microneme surface, binds to PA and mediates microneme exocytosis. CDPK1, activated by the calcium release, controls microneme secretion, F-actin apico-basal flux and conoid protrusion. CDPK1 possibly activates AKMT that is also essential for the flux. The exact molecular effectors of both enzymes are however not known. FRM1, localized at the apical end, generates the actin filament that will be further stabilized by GAC in complex with the secreted adhesins. The entire complex will then be translocated to the rear of the parasite, by the successive actions of MyoH and MyoA, generating forward motion.

https://doi.org/10.7554/eLife.42669.042

Videos

Video 1
Progressive basal accumulation of F-actin in RH egressing parasites.
https://doi.org/10.7554/eLife.42669.026
Video 2
Progressive basal accumulation of F-actin in FRM2/3-KO egressing parasites.
https://doi.org/10.7554/eLife.42669.027
Video 3
Ring of F-actin in a moving FRM2/3-KO parasite.
https://doi.org/10.7554/eLife.42669.031
Video 4
After BIPPO induction, basal accumulation of F-actin was observed even before parasite movement.
https://doi.org/10.7554/eLife.42669.032
Video 5
Basal accumulation of F-actin was observed even without parasite movement.

Here extracellular parasites were incubated with extracellular buffer.

https://doi.org/10.7554/eLife.42669.033
Video 6
RICM of circular gliding parasite.

Parasites were induced with BIPPO.

https://doi.org/10.7554/eLife.42669.034
Video 7
RICM of helical gliding parasite.

Parasites were induced with BIPPO.

https://doi.org/10.7554/eLife.42669.035

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