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


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.


Lipid Electron microscopy Freeze-fracture GPI-anchored protein Laft 



Complete spatial randomness


Dulbecco’s modified Eagle’s medium


Epithelial growth factor


Electron microscopy


Fluorescence resonance energy transfer


Fluorescence recovery after photobleaching


Fluorescence correlation spectroscopy




Inner membrane complex


Nerve growth factor


Phosphate-buffered saline


Photo-activation localisation microscopy


Platelet-derived growth factor


Sodium dodecyl sulphate


Single particle tracking



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.


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

418_2019_1814_MOESM1_ESM.docx (20 kb)
Supplementary material 1 (DOCX 20 kb)
418_2019_1814_MOESM2_ESM.pdf (6 mb)
Supplementary material 2 (PDF 6144 kb)
418_2019_1814_MOESM3_ESM.pdf (935 kb)
Supplementary material 3 (PDF 934 kb)
418_2019_1814_MOESM4_ESM.pdf (779 kb)
Supplementary material 4 (PDF 778 kb)
418_2019_1814_MOESM5_ESM.pdf (7 mb)
Supplementary material 5 (PDF 7212 kb)
418_2019_1814_MOESM6_ESM.pdf (126 kb)
Supplementary material 6 (PDF 125 kb)
418_2019_1814_MOESM7_ESM.pdf (850 kb)
Supplementary material 7 (PDF 849 kb)
418_2019_1814_MOESM8_ESM.pdf (1.2 mb)
Supplementary material 8 (PDF 1237 kb)


  1. Anderson RG, Jacobson K (2002) A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains. Science 296(5574):1821–1825. CrossRefPubMedGoogle Scholar
  2. Assossou O, Besson F, Rouault JP, Persat F, Brisson C, Duret L, Ferrandiz J, Mayencon M, Peyron F, Picot S (2003) Subcellular localization of 14-3-3 proteins in Toxoplasma gondii tachyzoites and evidence for a lipid raft-associated form. FEMS Microbiol Lett 224(2):161–168. CrossRefPubMedGoogle Scholar
  3. Bremer EG, Schlessinger J, Hakomori S (1986) Ganglioside-mediated modulation of cell growth. Specific effects of GM3 on tyrosine phosphorylation of the epidermal growth factor receptor. J Biol Chem 261(5):2434–2440PubMedGoogle Scholar
  4. Brown DA, London E (2000) Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J Biol Chem 275(23):17221–17224. CrossRefPubMedGoogle Scholar
  5. Burg JL, Perelman D, Kasper LH, Ware PL, Boothroyd JC (1988) Molecular analysis of the gene encoding the major surface antigen of Toxoplasma gondii. J Immunol 141(10):3584–3591PubMedGoogle Scholar
  6. Chen X, Resh MD (2002) Cholesterol depletion from the plasma membrane triggers ligand-independent activation of the epidermal growth factor receptor. J Biol Chem 277(51):49631–49637CrossRefGoogle Scholar
  7. Cheng J, Fujita A, Yamamoto H, Tatematsu T, Kakuta S, Obara K, Ohsumi Y, Fujimoto T (2014) Yeast and mammalian autophagosomes exhibit distinct phosphatidylinositol 3-phosphate asymmetries. Nat Commun 5:3207. CrossRefPubMedGoogle Scholar
  8. Dubremetz JF, Torpier G (1978) Freeze fracture study of the pellicle of an eimerian sporozoite (Protozoa, Coccidia). J Ultrastruct Res 62(2):94–109CrossRefGoogle Scholar
  9. Eggeling C, Ringemann C, Medda R, Schwarzmann G, Sandhoff K, Polyakova S, Belov VN, Hein B, von Middendorff C, Schonle A, Hell SW (2009) Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457(7233):1159–1162. CrossRefPubMedGoogle Scholar
  10. Fujimoto T (1996) GPI-anchored proteins, glycosphingolipids, and sphingomyelin are sequestered to caveolae only after crosslinking. J Histochem Cytochem 44(8):929–941CrossRefGoogle Scholar
  11. Fujimoto T, Fujimoto K (1997) Metal sandwich method to quick-freeze monolayer cultured cells for freeze-fracture. J Histochem Cytochem 45(4):595–598CrossRefGoogle Scholar
  12. Fujita A, Cheng J, Hirakawa M, Furukawa K, Kusunoki S, Fujimoto T (2007) Gangliosides GM1 and GM3 in the living cell membrane form clusters susceptible to cholesterol depletion and chilling. Mol Biol Cell 18(6):2112–2122. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Fujita A, Cheng J, Fujimoto T (2009a) Segregation of GM1 and GM3 clusters in the cell membrane depends on the intact actin cytoskeleton. Biochim Biophys Acta 1791(5):388–396CrossRefGoogle Scholar
  14. Fujita A, Cheng J, Tauchi-Sato K, Takenawa T, Fujimoto T (2009b) A distinct pool of phosphatidylinositol 4,5-bisphosphate in caveolae revealed by a nanoscale labeling technique. Proc Natl Acad Sci USA 106(23):9256–9261. CrossRefPubMedGoogle Scholar
  15. Fujita A, Cheng J, Fujimoto T (2010) Quantitative electron microscopy for the nanoscale analysis of membrane lipid distribution. Nat Protoc 5(4):661–669. CrossRefPubMedGoogle Scholar
  16. Gautier I, Tramier M, Durieux C, Coppey J, Pansu RB, Nicolas JC, Kemnitz K, Coppey-Moisan M (2001) Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins. Biophys J 80(6):3000–3008. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Goswami D, Gowrishankar K, Bilgrami S, Ghosh S, Raghupathy R, Chadda R, Vishwakarma R, Rao M, Mayor S (2008) Nanoclusters of GPI-anchored proteins are formed by cortical actin-driven activity. Cell 135(6):1085–1097. CrossRefPubMedGoogle Scholar
  18. Grimwood J, Smith JE (1992) Toxoplasma gondii: the role of a 30-kDa surface protein in host cell invasion. Exp Parasitol 74(1):106–111CrossRefGoogle Scholar
  19. Harder T, Simons K (1997) Caveolae, DIGs, and the dynamics of sphingolipid-cholesterol microdomains. Curr Opin Cell Biol 9(4):534–542CrossRefGoogle Scholar
  20. Heerklotz H (2002) Triton promotes domain formation in lipid raft mixtures. Biophys J 83(5):2693–2701CrossRefGoogle Scholar
  21. Howard MF, Murakami Y, Pagnamenta AT, Daumer-Haas C, Fischer B, Hecht J, Keays DA, Knight SJ, Kolsch U, Kruger U, Leiz S, Maeda Y, Mitchell D, Mundlos S, Phillips JA 3rd, Robinson PN, Kini U, Taylor JC, Horn D, Kinoshita T, Krawitz PM (2014) Mutations in PGAP3 impair GPI-anchor maturation, causing a subtype of hyperphosphatasia with mental retardation. Am J Hum Genet 94(2):278–287. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Jacobson K, Dietrich C (1999) Looking at lipid rafts? Trends Cell Biol 9(3):87–91CrossRefGoogle Scholar
  23. Johnson AM, McDonald PJ, Neoh SH (1983) Monoclonal antibodies to Toxoplasma cell membrane surface antigens protect mice from toxoplasmosis. J Protozool 30(2):351–356CrossRefGoogle Scholar
  24. Kenworthy AK, Edidin M (1998) Distribution of a glycosylphosphatidylinositol-anchored protein at the apical surface of MDCK cells examined at a resolution of < 100 A using imaging fluorescence resonance energy transfer. J Cell Biol 142(1):69–84CrossRefGoogle Scholar
  25. Kenworthy AK, Petranova N, Edidin M (2000) High-resolution FRET microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma membranes. Mol Biol Cell 11(5):1645–1655. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Kenworthy AK, Nichols BJ, Remmert CL, Hendrix GM, Kumar M, Zimmerberg J, Lippincott-Schwartz J (2004) Dynamics of putative raft-associated proteins at the cell surface. J Cell Biol 165(5):735–746. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Kim K, Bulow R, Kampmeier J, Boothroyd JC (1994) Conformationally appropriate expression of the Toxoplasma antigen SAG1 (p30) in CHO cells. Infect Immun 62(1):203–209PubMedPubMedCentralGoogle Scholar
  28. Kusumi A, Koyama-Honda I, Suzuki K (2004) Molecular dynamics and interactions for creation of stimulation-induced stabilized rafts from small unstable steady-state rafts. Traffic 5(4):213–230. CrossRefPubMedGoogle Scholar
  29. Kusunoki S, Shimizu J, Chiba A, Ugawa Y, Hitoshi S, Kanazawa I (1996) Experimental sensory neuropathy induced by sensitization with ganglioside GD1b. Ann Neurol 39(4):424–431. CrossRefPubMedGoogle Scholar
  30. Leidich SD, Drapp DA, Orlean P (1994) A conditionally lethal yeast mutant blocked at the first step in glycosyl phosphatidylinositol anchor synthesis. J Biol Chem 269(14):10193–10196PubMedGoogle Scholar
  31. Lenne PF, Wawrezinieck L, Conchonaud F, Wurtz O, Boned A, Guo XJ, Rigneault H, He HT, Marguet D (2006) Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork. EMBO J 25(14):3245–3256. CrossRefPubMedPubMedCentralGoogle Scholar
  32. Luft BJ, Remington JS (1992) Toxoplasmic encephalitis in AIDS. Clin Infect Dis 15(2):211–222CrossRefGoogle Scholar
  33. Marquardt T, Shirasaki R, Ghosh S, Andrews SE, Carter N, Hunter T, Pfaff SL (2005) Coexpressed EphA receptors and ephrin-A ligands mediate opposing actions on growth cone navigation from distinct membrane domains. Cell 121(1):127–139. CrossRefPubMedGoogle Scholar
  34. Mayor S, Rothberg KG, Maxfield FR (1994) Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking. Science 264(5167):1948–1951CrossRefGoogle Scholar
  35. Meuillet EJ, Kroes R, Yamamoto H, Warner TG, Ferrari J, Mania-Farnell B, George D, Rebbaa A, Moskal JR, Bremer EG (1999) Sialidase gene transfection enhances epidermal growth factor receptor activity in an epidermoid carcinoma cell line, A431. Cancer Res 59(1):234–240PubMedGoogle Scholar
  36. Meuillet EJ, Mania-Farnell B, George D, Inokuchi JI, Bremer EG (2000) Modulation of EGF receptor activity by changes in the GM3 content in a human epidermoid carcinoma cell line, A431. Exp Cell Res 256(1):74–82. CrossRefPubMedGoogle Scholar
  37. Mineo JR, McLeod R, Mack D, Smith J, Khan IA, Ely KH, Kasper LH (1993) Antibodies to Toxoplasma gondii major surface protein (SAG-1, P30) inhibit infection of host cells and are produced in murine intestine after peroral infection. J Immunol 150(9):3951–3964PubMedGoogle Scholar
  38. Moran P, Caras IW (1994) Requirements for glycosylphosphatidylinositol attachment are similar but not identical in mammalian cells and parasitic protozoa. J Cell Biol 125(2):333–343CrossRefGoogle Scholar
  39. Morrissette NS, Murray JM, Roos DS (1997) Subpellicular microtubules associate with an intramembranous particle lattice in the protozoan parasite Toxoplasma gondii. J Cell Sci 110(Pt 1):35–42PubMedGoogle Scholar
  40. Murata D, Nomura KH, Dejima K, Mizuguchi S, Kawasaki N, Matsuishi-Nakajima Y, Ito S, Gengyo-Ando K, Kage-Nakadai E, Mitani S, Nomura K (2012) GPI-anchor synthesis is indispensable for the germline development of the nematode Caenorhabditis elegans. Mol Biol Cell 23(6):982–995. CrossRefPubMedPubMedCentralGoogle Scholar
  41. Mutoh T, Tokuda A, Miyadai T, Hamaguchi M, Fujiki N (1995) Ganglioside GM1 binds to the Trk protein and regulates receptor function. Proc Natl Acad Sci USA 92(11):5087–5091CrossRefGoogle Scholar
  42. Nagamune K, Nozaki T, Maeda Y, Ohishi K, Fukuma T, Hara T, Schwarz RT, Sutterlin C, Brun R, Riezman H, Kinoshita T (2000) Critical roles of glycosylphosphatidylinositol for Trypanosoma brucei. Proc Natl Acad Sci USA 97(19):10336–10341. CrossRefPubMedGoogle Scholar
  43. Philimonenko AA, Janacek J, Hozak P (2000) Statistical evaluation of colocalization patterns in immunogold labeling experiments. J Struct Biol 132(3):201–210. CrossRefPubMedGoogle Scholar
  44. Porchet E, Torpier G (1977) Freeze fracture study of Toxoplasma and Sarcocystis infective stages (author’s transl). Z Parasitenkd 54(2):101–124CrossRefGoogle Scholar
  45. Prior IA, Muncke C, Parton RG, Hancock JF (2003) Direct visualization of Ras proteins in spatially distinct cell surface microdomains. J Cell Biol 160(2):165–170CrossRefGoogle Scholar
  46. Rebbaa A, Hurh J, Yamamoto H, Kersey DS, Bremer EG (1996) Ganglioside GM3 inhibition of EGF receptor mediated signal transduction. Glycobiology 6(4):399–406CrossRefGoogle Scholar
  47. Ringerike T, Blystad FD, Levy FO, Madshus IH, Stang E (2002) Cholesterol is important in control of EGF receptor kinase activity but EGF receptors are not concentrated in caveolae. J Cell Sci 115(Pt 6):1331–1340PubMedGoogle Scholar
  48. Ripley BD (1977) Modeling spatial patterns. J R Stat Soc Ser B 39:172–212Google Scholar
  49. Ripley BD (1979) Tests of randomness for spatial point patterns. J R Stat Soc Ser B 41:368–374Google Scholar
  50. Sengupta P, Jovanovic-Talisman T, Skoko D, Renz M, Veatch SL, Lippincott-Schwartz J (2011) Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis. Nat Methods 8(11):969–975. CrossRefPubMedPubMedCentralGoogle Scholar
  51. Sharma P, Varma R, Sarasij RC, Ira GK, Krishnamoorthy G, Rao M, Mayor S (2004) Nanoscale organization of multiple GPI-anchored proteins in living cell membranes. Cell 116(4):577–589CrossRefGoogle Scholar
  52. Simons K, Ikonen E (1997) Functional rafts in cell membranes. Nature 387(6633):569–572. CrossRefPubMedGoogle Scholar
  53. Simons K, Toomre D (2000) Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1(1):31–39CrossRefGoogle Scholar
  54. Suarez Pestana E, Greiser U, Sanchez B, Fernandez LE, Lage A, Perez R, Bohmer FD (1997) Growth inhibition of human lung adenocarcinoma cells by antibodies against epidermal growth factor receptor and by ganglioside GM3: involvement of receptor-directed protein tyrosine phosphatase(s). Br J Cancer 75(2):213–220CrossRefGoogle Scholar
  55. Suzuki KG, Fujiwara TK, Edidin M, Kusumi A (2007a) Dynamic recruitment of phospholipase C gamma at transiently immobilized GPI-anchored receptor clusters induces IP3-Ca2+ signaling: single-molecule tracking study 2. J Cell Biol 177(4):731–742. CrossRefPubMedPubMedCentralGoogle Scholar
  56. Suzuki KG, Fujiwara TK, Sanematsu F, Iino R, Edidin M, Kusumi A (2007b) GPI-anchored receptor clusters transiently recruit Lyn and G alpha for temporary cluster immobilization and Lyn activation: single-molecule tracking study 1. J Cell Biol 177(4):717–730. CrossRefPubMedPubMedCentralGoogle Scholar
  57. Takatori S, Mesman R, Fujimoto T (2014) Microscopic methods to observe the distribution of lipids in the cellular membrane. Biochemistry 53(4):639–653CrossRefGoogle Scholar
  58. Tanaka KA, Suzuki KG, Shirai YM, Shibutani ST, Miyahara MS, Tsuboi H, Yahara M, Yoshimura A, Mayor S, Fujiwara TK, Kusumi A (2011) Membrane molecules mobile even after chemical fixation. Nat Methods 7(11):865–866CrossRefGoogle Scholar
  59. Tansey MG, Baloh RH, Milbrandt J, Johnson EM Jr (2000) GFRalpha-mediated localization of RET to lipid rafts is required for effective downstream signaling, differentiation, and neuronal survival. Neuron 25(3):611–623CrossRefGoogle Scholar
  60. Varma R, Mayor S (1998) GPI-anchored proteins are organized in submicron domains at the cell surface. Nature 394:798–801CrossRefGoogle Scholar
  61. Wenger J, Conchonaud F, Dintinger J, Wawrezinieck L, Ebbesen TW, Rigneault H, Marguet D, Lenne PF (2007) Diffusion analysis within single nanometric apertures reveals the ultrafine cell membrane organization. Biophys J 92(3):913–919. CrossRefPubMedGoogle Scholar
  62. Wong SY, Remington JS (1993) Biology of Toxoplasma gondii. AIDS 7(3):299–316CrossRefGoogle Scholar
  63. Yates AJ, VanBrocklyn J, Saqr HE, Guan Z, Stokes BT, O’Dorisio MS (1993) Mechanisms through which gangliosides inhibit PDGF-stimulated mitogenesis in intact Swiss 3T3 cells: receptor tyrosine phosphorylation, intracellular calcium, and receptor binding. Exp Cell Res 204(1):38–45. CrossRefPubMedGoogle Scholar
  64. Yoshida A, Shigekuni M, Tanabe K, Fujita A (2016) Nanoscale analysis reveals agonist-sensitive and heterogeneous pools of phosphatidylinositol 4-phosphate in the plasma membrane. Biochim Biophys Acta 185(6):1298–1305. CrossRefGoogle Scholar
  65. Zhang F, Crise B, Su B, Hou Y, Rose JK, Bothwell A, Jacobson K (1991) Lateral diffusion of membrane-spanning and glycosylphosphatidylinositol-linked proteins: toward establishing rules governing the lateral mobility of membrane proteins. J Cell Biol 115(1):75–84CrossRefGoogle Scholar
  66. Zhang F, Lee GM, Jacobson K (1993) Protein lateral mobility as a reflection of membrane microstructure. BioEssays 15(9):579–588. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

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

Personalised recommendations