United States, Switzerland, European Union, 4 items
Organization
Grant number
Country
David and Lucile Packard Foundation
United States
Swiss National Science Foundation
310030_185325
Switzerland
European Research Council (ERC)
695596
European Union
Burroughs Wellcome Fund
1022785
United States
Citation
Journal: Nat Commun / Year: 2024 Title: Sustained rhoptry docking and discharge requires Toxoplasma gondii intraconoidal microtubule-associated proteins. Authors: Nicolas Dos Santos Pacheco / Albert Tell I Puig / Amandine Guérin / Matthew Martinez / Bohumil Maco / Nicolò Tosetti / Estefanía Delgado-Betancourt / Matteo Lunghi / Boris Striepen / Yi- ...Authors: Nicolas Dos Santos Pacheco / Albert Tell I Puig / Amandine Guérin / Matthew Martinez / Bohumil Maco / Nicolò Tosetti / Estefanía Delgado-Betancourt / Matteo Lunghi / Boris Striepen / Yi-Wei Chang / Dominique Soldati-Favre / Abstract: In Apicomplexa, rhoptry discharge is essential for invasion and involves an apical vesicle (AV) docking one or two rhoptries to a macromolecular secretory apparatus. Toxoplasma gondii is armed with ...In Apicomplexa, rhoptry discharge is essential for invasion and involves an apical vesicle (AV) docking one or two rhoptries to a macromolecular secretory apparatus. Toxoplasma gondii is armed with 10-12 rhoptries and 5-6 microtubule-associated vesicles (MVs) presumably for iterative rhoptry discharge. Here, we have addressed the localization and functional significance of two intraconoidal microtubule (ICMT)-associated proteins instrumental for invasion. Mechanistically, depletion of ICMAP2 leads to a dissociation of the ICMTs, their detachment from the conoid and dispersion of MVs and rhoptries. ICMAP3 exists in two isoforms that contribute to the control of the ICMTs length and the docking of the two rhoptries at the AV, respectively. This study illuminates the central role ICMTs play in scaffolding the discharge of multiple rhoptries. This process is instrumental for virulence in the mouse model of infection and in addition promotes sterile protection against T. gondii via the release of key effectors inducing immunity.
Download / File: emd_42119.map.gz / Format: CCP4 / Size: 2.2 GB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
Annotation
Toxoplasma gondii apical end with ICMAP2 conditionally knocked down
Voxel size
X=Y=Z: 10.6 Å
Density
Minimum - Maximum
-1673.0 - 576.0
Average (Standard dev.)
59.427044000000002 (±29.385764999999999)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
-209
209
-401
Dimensions
1440
1023
800
Spacing
1023
1440
800
Cell
A: 10843.801 Å / B: 15264.001 Å / C: 8480.0 Å α=β=γ: 90.0 °
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Supplemental data
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Sample components
-
Entire : Toxoplasma Gondii tachyzoite apical complex with ICMAP2 condition...
Entire
Name: Toxoplasma Gondii tachyzoite apical complex with ICMAP2 conditionally knocked down
Components
Cell: Toxoplasma Gondii tachyzoite apical complex with ICMAP2 conditionally knocked down
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Supramolecule #1: Toxoplasma Gondii tachyzoite apical complex with ICMAP2 condition...
Supramolecule
Name: Toxoplasma Gondii tachyzoite apical complex with ICMAP2 conditionally knocked down type: cell / ID: 1 / Parent: 0
Source (natural)
Organism: Toxoplasma gondii (eukaryote)
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Experimental details
-
Structure determination
Method
cryo EM
Processing
electron tomography
Aggregation state
cell
-
Sample preparation
Buffer
pH: 7.4 Details: DMEM supplemented with 5% fetal bovine serum and 2 mM glutamine
Grid
Model: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
Vitrification
Cryogen name: ETHANE-PROPANE / Chamber humidity: 95 % / Chamber temperature: 310 K / Instrument: LEICA EM GP / Details: Front blot for 4 seconds before plunging.
Details
Egressed tachyzoites from a culture of confluent HFF cells. ICMAP2 knockdown induced by treatment with 50 nM rapamycin for 48 hours prior to egress.
Sectioning
Other: NO SECTIONING
Fiducial marker
Manufacturer: Ted Pella, Inc / Diameter: 10 nm
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Phase plate: VOLTA PHASE PLATE / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Software
Name: SerialEM (ver. 3.8)
Image recording
Film or detector model: GATAN K3 (6k x 4k) / Average exposure time: 0.4 sec. / Average electron dose: 2.3 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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