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Histochemistry and Cell Biology

, Volume 152, Issue 5, pp 365–375 | Cite as

Nanoscale analysis reveals no domain formation of glycosylphosphatidylinositol-anchored protein SAG1 in the plasma membrane of living Toxoplasma gondii

  • Yuna Kurokawa
  • Tatsunori Masatani
  • Rikako Konishi
  • Kanna Tomioku
  • Xuenan Xuan
  • Akikazu FujitaEmail author
Original Paper

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.

Keywords

Lipid Electron microscopy Freeze-fracture GPI-anchored protein Laft 

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

Notes

Acknowledgements

We thank Dr. Toyoshi Fujimoto (Nagoya University) for the kind gift of mouse fibroblast cell line.

Author contributions

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.

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.).

Compliance with ethical standards

Conflict of interest

The authors declare no potential conflict of interests.

Supplementary material

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Supplementary material 1 (DOCX 20 kb)
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Authors and Affiliations

  1. 1.Department of Molecular Cell Biology and Biochemistry, Basic Veterinary Medicine, Faculty of Veterinary MedicineKagoshima UniversityKagoshimaJapan
  2. 2.Transboundary Animal Diseases Research Center, Joint Faculty of Veterinary MedicineKagoshima UniversityKagoshimaJapan
  3. 3.National Research Center for Protozoan DiseasesObihiro University of Agriculture and Veterinary MedicineObihiroJapan

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