Abstract
Members of Apicomplexa are defined by apical cytoskeletal structures and secretory organelles, tailored for motility, invasion and egress. Gliding is powered by actomyosin-dependent rearward translocation of apically secreted transmembrane adhesins. In the human parasite Toxoplasma gondii, the conoid, composed of tubulin fibres and preconoidal rings (PCRs), is a dynamic organelle of undefined function. Here, using ultrastructure expansion microscopy, we established that PCRs serve as a hub for glideosome components including Formin1. We also identified components of the PCRs conserved in Apicomplexa, Pcr4 and Pcr5, that contain B-box zinc-finger domains, assemble in heterodimer and are essential for the formation of the structure. The fitness conferring Pcr6 tethers the PCRs to the cone of tubulin fibres. F-actin produced by Formin1 is used by Myosin H to generate the force for conoid extrusion which directs the flux of F-actin to the pellicular space, serving as gatekeeper to control parasite motility.
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Data availability
All data are available within the paper and its supplementary information. Sequences used in this study have been obtained from EuPathDB. HDX–MS data are available via ProteomeXchange with identifier PXD031816. All biological materials and data are available from the author upon request. Source data are provided with this paper.
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Acknowledgements
We thank for their technical assistance, the team at the Bioimaging Core Facility, R. Visentin at the Protein platform and A. Hainard at the proteomics platform of the faculty of Medicine University of Geneva as well as F. Pojer, L. Abriata and K. Lau from the ‘Protein Production and Structure Core Facility’ at the EPFL. We thank D. Baker for supply of C1. We thank Varsha Mathur and Jan Janouškovec for kindly providing critical sequence data and assemblies used for phylogenetic analysis. This work was supported by the Swiss National Foundation (grant Nos. 310030_185325 to D.S.-F., and BSSGI0_155852 and 310030_208151 to M.B.), the Novartis Foundation (grant No. 19C189 to L.B.) and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 695596 to B.M.).
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D.S-F., O.V., N.D.S.P., L.B. and M.B. conceptualized the project. N.D.S.P., O.V., R.H., L.B., B.M. and N.T. designed the methodology, and N.D.S.P., O.V., R.H., L.B. and N.T. performed the investigations. N.D.S.P., O.V., R.H., L.B., N.T. and B.M. did the formal analysis. N.D.S.P., D.S-F. and O.V. wrote the original draft and N.D.S.P., D.S-F., O.V., R.H., L.B., N.T. and M.B reviewed and edited the manuscript. D.S-F. and M.B. did the funding acquisition, D.S-F., O.V. and M.B. obtained resources and D.S-F., O.V. and M.B. supervised. All authors contributed to the article and approved the submitted version.
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Extended data
Extended Data Fig. 1 U-ExM allows high-resolution localization of apical proteins and conoid extrusion assessment.
a, U-ExM localization of conoidal proteins fused with a mAiD-HA cassette. Scale bar = 2 µm. b, Localization of AKMT and GAC in intracellular vacuoles as seen by U-ExM. Scale bar = 2 µm. c, Representative pictures of some U-ExM samples used for quantification of Fig. 1c. Dash boxes represents crop parasites displayed in Fig. 1b. Scale bar = 5 µm.
Extended Data Fig. 2 Additional phenotyping of Pcr1,4,5,6,7.
a, IFA of Pcr1,4,5,6,7 in non-dividing and dividing parasite showing that Pcr1,4,5,6 are also found in forming daughter cells. White arrowhead = apical pole of mature cells. Cyan arrowhead = apical pole of forming daughter cells. Scale bar = 2 µm. b, Additional U-ExM pictures of Pcr1,4,5,6,7. Scale bar = 2 µm. c, Quantification of the plaque size. The different replicates are displayed in shades of grey. Ten plaques were measures for each condition and replicate. All data are presented as mean ± SD (n = 3). Unpaired two-tailed Student’s t-tests where non-significant (n.s.) if P > 5E-02.
Extended Data Fig. 3 Conservation of Pcr4 and Pcr5 across Alveolata.
a, Conservation of Pcr4 and Pcr5 in the Alveolate superphylum. Presence (coloured) or absence (white) of homologues identified across alveolate predicted proteomes following. b, Results of a maximum likelihood inference based on an alignment of sequences retrieved for homologues of Pcr4 and Pcr5. Numbers beside nodes indicate bootstrap support (1000 ultrafast bootstrap replicates).
Extended Data Fig. 4 Protein downregulation and microneme secretion assays.
a, Regulation of Pcr4 and Pcr5 on intracellular parasites. Time of auxin incubations are indicated in hour. Actin is used as loading control. Molecular weight in kDa. A small scheme of the experimental timeline is presented. b, Regulation of Pcr4 and Pcr5 on extracellular parasites. Time of auxin incubations are indicated in hour. Molecular weight in kDa. Actin is used as loading control. A small scheme of the experimental timeline is presented. c, Stability of Pcr4,5,6 and FRM1 fused with a mAiD-HA cassette, after short IAA treatment. ISP1 is used as a reference marker appearing early during daughter cells biogenesis. White arrowhead = apical signal at the apical pole of mature cells. Cyan arrowhead = apical signal at the apical pole of forming daughter cells. Black filled arrowhead = absence of signal at the apical pole. d, Representative pictures of microneme secretion quantified in Fig. 2d. Molecular weight in kDa. White arrowhead = secreted MIC2. Black arrowhead = secreted MIC2 trimmed by SUB1 protease. GRA1 is used as loading control while catalase is used as a lysis control.
Extended Data Fig. 5 Integrity of apical markers and PCR structure.
a, IFA showing that the depletion of Pcr6 did not induce the loss of Pcr5. Scale bars = 2 µm. b, IFA showing that the depletion of FRM1 did not induce the loss of Pcr4 and Pcr5. Scale bars = 2 µm. c, IFA showing that the depletion of Pcr5 does not affect conoid (Cam2) and APR (RNG1/KinA) markers. d, Depletion of Pcr5 leads to the loss of the PCRs signal of AKMT in intracellular parasite by U-ExM. Scale bars = 2 µm. e, Depletion of Pcr5 leads to the loss of the PCRs signal of GAC in intracellular parasite by U-ExM. Scale bars = 2 µm. f, Gallery of additional EM images of Pcr4 and Pcr5 knockdown strains. Scale bar = 200 nm. g, Gallery of additional EM images of Pcr6 knockdown strains. Scale bar = 200 nm. h, Gallery of EM images of FRM1 knockdown strains. Scale bar = 200 nm.
Extended Data Fig. 6 Additional data on Pcr4,5 biochemical properties and interactions.
a, Schematic of Pcr5 two B-Box domains. The amino acid sequences of the four versions of Pcr5 used for complementation are presented. Cysteine and histidine residues (violet), known to chelate zinc ions, were mutated to alanine to create mutated versions of the B-Box domains. b, Representative Coomassie-stained gel of the solubility assay for the individual proteins quantified in Fig. 4a. L = Whole cell lysate. S = Soluble fraction. c, Representative Coomassie-stained gel of a solubility assay when Pcr4 is co-expressed with the four versions of Pcr5, quantified in Fig. 4b. d, Representative Coomassie-stained gels used for solubility quantification and corresponding quantified Western Blot by band densitometry. e, Mass photometry of the Pcr4–Pcr5 complex allowed the measure of the complex mass. f, SEC–MALS coupled with size-exclusion chromatography for the Pcr4–Pcr5 complex. g, Representative Coomassie-stained gel of pull-down assays performed with Pcr4 co-expressed with the four versions of Pcr5. Pull-down was performed using anti-Strep column (Pcr5 pull-down). h, HDX–MS analysis highlight a different dynamic of Pcr5 when mutated in the first B-Box domain (Pcr5MUT1) i, Immunofluorescence of the four complemented strains. The endogenous copy is presented in green (HA) and the second copy is presented in magenta (Ty). White arrowhead = presence of apical signal. Black arrowhead = no apical signal. Scale bars = 2 µm.
Extended Data Fig. 7 Generation of Plasmodium berghei strains and additional phenotyping.
a, Schematics of the promoter-swap strategy to obtain the Pclag9Pcr4 and Pclag9Pcr5 strains. On the right, integration PCR are presented. b, Quantification of microgametocyte exflagellation for the Pclag9Pcr4 and Pclag9Pcr5 strains. Data are presented as mean ± SD (individual biological replicates are also presented). c, Quantification of ookinete conversion for the Pclag9Pcr4 and Pclag9Pcr5 strains. All three states of the conversion (round, retort and ookinete) are presented. Data are presented as mean ± SD (individual biological replicates are also presented). For all statistical analysis, an unpaired two-tailed Student’s t-tests where non-significant (n.s.) if P>5E-02 was used. All data are presented as mean ± SD (n=3).
Supplementary information
Supplementary Information
Supplementary Text and Discussion, and HDX–MS peptide map.
Supplementary Table 1
HDX–MS raw data.
Supplementary Table 2
List of strains, primers, plasmids, antibodies and supplies used in this study.
Supplementary Video 1
Representative live-imaging video of wild-type ookinete gliding in Matrigel. Several videos were used to calculate gliding speed and track individual parasites. Acquisition time, 10 min. Acquisition rate, three frames a min. Scale bar, 25 µm.
Supplementary Video 2
Representative live-imaging video of Pclag9Pcr4 ookinete gliding in Matrigel. Several videos were used to calculate gliding speed and track individual parasites. Acquisition time, 10 min. Acquisition rate, three frames a min. Scale bar, 25 µm
Supplementary Video 3
Representative live-imaging video of Pclag9Pcr5 ookinete gliding in Matrigel. Several videos were used to calculate gliding speed and track individual parasites. Acquisition time, 10 min. Acquisition rate, three frames a min. Scale bar, 25 µm
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Dos Santos Pacheco, N., Brusini, L., Haase, R. et al. Conoid extrusion regulates glideosome assembly to control motility and invasion in Apicomplexa. Nat Microbiol 7, 1777–1790 (2022). https://doi.org/10.1038/s41564-022-01212-x
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DOI: https://doi.org/10.1038/s41564-022-01212-x
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