Abstract
Glycosylphosphatidylinositol (GPI)-anchored proteins typically localise to lipid rafts. GPI-anchored protein microdomains may be present in the plasma membrane; however, they have been studied using heterogeneously expressed GPI-anchored proteins, and the two-dimensional distributions of endogenous molecules in the plasma membrane are difficult to determine at the nanometre scale. Here, we used immunoelectron microscopy using a quick-freezing and freeze-fracture labelling (QF-FRL) method to examine the distribution of the endogenous GPI-anchored protein SAG1 in Toxoplasma gondii at the nanoscale. QF-FRL physically immobilised molecules in situ, minimising the possibility of artefactual perturbation. SAG1 labelling was observed in the exoplasmic, but not cytoplasmic, leaflets of T. gondii plasma membrane, whereas none was detected in any leaflet of the inner membrane complex. Point pattern analysis of SAG1 immunogold labelling revealed mostly random distribution in T. gondii plasma membrane. The present method obtains information on the molecular distribution of natively expressed GPI-anchored proteins and demonstrates that SAG1 in T. gondii does not form significant microdomains in the plasma membrane.
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Abbreviations
- CSR:
-
Complete spatial randomness
- DMEM:
-
Dulbecco’s modified Eagle’s medium
- EGF:
-
Epithelial growth factor
- EM:
-
Electron microscopy
- FRET:
-
Fluorescence resonance energy transfer
- FRAP:
-
Fluorescence recovery after photobleaching
- FCS:
-
Fluorescence correlation spectroscopy
- GPI:
-
Glycosylphosphatidylinositol
- IMC:
-
Inner membrane complex
- NGF:
-
Nerve growth factor
- PBS:
-
Phosphate-buffered saline
- PC-PALM:
-
Photo-activation localisation microscopy
- PDGF:
-
Platelet-derived growth factor
- SDS:
-
Sodium dodecyl sulphate
- SPT:
-
Single particle tracking
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Acknowledgements
We thank Dr. Toyoshi Fujimoto (Nagoya University) for the kind gift of mouse fibroblast cell line.
Funding
This work was supported by JSPS KAKENHI Grant Number JP17H03935 and JP16K15056, and Cooperation Research Grant of National Research Center for Protozoan Diseases in Obihiro University of Agriculture and Veterinary Medicine, research grants from Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering, Takeda Science Foundation, The Naito Foundation, ONO Medical Research Foundation and The NOVARTIS Foundation (Japan) for the Promotion of Science (to A.F.).
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AF and XX provided funding; AF, YK and TM conceived the idea; AF supervised the study and designed experiments; AF, YK, TM and KT performed experiments; AF, YK and RK analysed data; AF wrote the manuscript; YK and TM made manuscript revisions.
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Kurokawa, Y., Masatani, T., Konishi, R. et al. Nanoscale analysis reveals no domain formation of glycosylphosphatidylinositol-anchored protein SAG1 in the plasma membrane of living Toxoplasma gondii. Histochem Cell Biol 152, 365–375 (2019). https://doi.org/10.1007/s00418-019-01814-3
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DOI: https://doi.org/10.1007/s00418-019-01814-3