Three F-actin assembly centers regulate organelle inheritance, cell-cell communication and motility in Toxoplasma gondii
- University of Geneva, Switzerland
Figures
Figure 1
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 …
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 …
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Figure 2—source data 1
Numerical data of the graphs presented in Figure 2G, H and I and Figure 2—figure supplement 2C.
- https://doi.org/10.7554/eLife.42669.006
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 …
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 …
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 …
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Figure 3—source data 1
Numerical data of the graphs presented in Figures 3C and 2F, and Figure 3—figure supplement 2C and D.
- https://doi.org/10.7554/eLife.42669.012
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 …
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 …
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 …
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.
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 …
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Figure 4—source data 1
Numerical data of the graphs presented in Figure 4E, F and G and Figure 4—figure supplement 1D and E.
- https://doi.org/10.7554/eLife.42669.017
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 …
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 …
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.
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). …
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Figure 5—source data 1
Numerical data of the graphs presented in Figure 5C and Figure 5—figure supplements 2C, D and E, 5C and E.
- https://doi.org/10.7554/eLife.42669.024
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. …
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. …
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 …
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 …
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 …
Figure 6
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 …
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 …
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Figure 7—source data 1
Numerical data of the graphs presented in Figure 7F and H and Figure 7—figure supplement 1B.
- https://doi.org/10.7554/eLife.42669.030
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.
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 …
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Figure 8—source data 1
Numerical data of the graphs presented in Figure 8A, B, C, D, E, F and G and Figure 8—figure supplement 1B.
- https://doi.org/10.7554/eLife.42669.041
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. …
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 …
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 …
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.
Figure 9
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 …
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.
Video 6
RICM of circular gliding parasite.
Parasites were induced with BIPPO.
Video 7
RICM of helical gliding parasite.
Parasites were induced with BIPPO.
Additional files
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Supplementary file 1
Primers listed in the Materials and methods.
- https://doi.org/10.7554/eLife.42669.043
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Transparent reporting form
- https://doi.org/10.7554/eLife.42669.044
