Skip to main content
SpringerLink
Log in

Cholesterol segregates into submicrometric domains at the living erythrocyte membrane: evidence and regulation

  • Research Article
  • Published:
Cellular and Molecular Life Sciences Aims and scope Submit manuscript

Abstract

Although cholesterol is essential for membrane fluidity and deformability, the level of its lateral heterogeneity at the plasma membrane of living cells is poorly understood due to lack of appropriate probe. We here report on the usefulness of the D4 fragment of Clostridium perfringens toxin fused to mCherry (theta*), as specific, non-toxic, sensitive and quantitative cholesterol-labeling tool, using erythrocyte flat membrane. By confocal microscopy, theta* labels cholesterol-enriched submicrometric domains in coverslip-spread but also gel-suspended (non-stretched) fresh erythrocytes, suggesting in vivo relevance. Cholesterol domains on spread erythrocytes are stable in time and space, restricted by membrane:spectrin anchorage via 4.1R complexes, and depend on temperature and sphingomyelin, indicating combined regulation by extrinsic membrane:cytoskeleton interaction and by intrinsic lipid packing. Cholesterol domains partially co-localize with BODIPY-sphingomyelin-enriched domains. In conclusion, we show that theta* is a useful vital probe to study cholesterol organization and demonstrate that cholesterol forms submicrometric domains in living cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

Abbreviations

CalA:

Calyculin A

DF-BSA:

Defatted-bovine serum albumin

FRAP:

Fluorescence recovery after photobleaching

LatB:

Latrunculin B

mβCD:

Methyl-β-cyclodextrin

MLVs:

Multilamellar vesicles

PC:

Phosphatidylcholine

PKC:

Protein kinase C

PMA:

Phorbol 12-myristate 13-acetate

RBC:

Red blood cell

SEM:

Scanning electron microscopy

SM:

Sphingomyelin

SMase:

Sphingomyelinase

theta*:

His-mCherry-theta-D4

References

  1. Lange Y, Swaisgood MH, Ramos BV, Steck TL (1989) Plasma membranes contain half the phospholipid and 90% of the cholesterol and sphingomyelin in cultured human fibroblasts. J Biol Chem 264:3786–3793

    CAS  PubMed  Google Scholar 

  2. Lingwood D, Simons K (2010) Lipid rafts as a membrane-organizing principle. Science 327:46–50

    Article  CAS  PubMed  Google Scholar 

  3. Pike LJ (2006) Rafts defined: a report on the Keystone Symposium on Lipid Rafts and Cell Function. J Lipid Res 47:1597–1598

    Article  CAS  PubMed  Google Scholar 

  4. Parton RG, Way M, Zorzi N, Stang E (1997) Caveolin-3 associates with developing T-tubules during muscle differentiation. J Cell Biol 136:137–154

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Schroeder F, Nemecz G, Wood WG, Joiner C, Morrot G, Ayraut-Jarrier M, Devaux PF (1991) Transmembrane distribution of sterol in the human erythrocyte. Biochim Biophys Acta 1066:183–192

    Article  CAS  PubMed  Google Scholar 

  6. Lange Y, Slayton JM (1982) Interaction of cholesterol and lysophosphatidylcholine in determining red cell shape. J Lipid Res 23:1121–1127

    CAS  PubMed  Google Scholar 

  7. Sun M, Northup N, Marga F, Huber T, Byfield FJ, Levitan I, Forgacs G (2007) The effect of cellular cholesterol on membrane-cytoskeleton adhesion. J Cell Sci 120:2223–2231

    Article  CAS  PubMed  Google Scholar 

  8. Laurenzana A, Fibbi G, Chilla A, Margheri G, Del Rosso T, Rovida E, Del Rosso M, Margheri F (2015) Lipid rafts: integrated platforms for vascular organization offering therapeutic opportunities. Cell Mol Life Sci 72:1537–1557

    Article  CAS  PubMed  Google Scholar 

  9. Baumann P, Thiele W, Cremers N, Muppala S, Krachulec J, Diefenbacher M, Kassel O, Mudduluru G, Allgayer H, Frame M, Sleeman JP (2012) CD24 interacts with and promotes the activity of c-src within lipid rafts in breast cancer cells, thereby increasing integrin-dependent adhesion. Cell Mol Life Sci 69:435–448

    Article  CAS  PubMed  Google Scholar 

  10. Chichili GR, Rodgers W (2009) Cytoskeleton-membrane interactions in membrane raft structure. Cell Mol Life Sci 66:2319–2328

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Veiga MP, Arrondo JL, Goni FM, Alonso A, Marsh D (2001) Interaction of cholesterol with sphingomyelin in mixed membranes containing phosphatidylcholine, studied by spin-label ESR and IR spectroscopies. A possible stabilization of gel-phase sphingolipid domains by cholesterol. Biochemistry 40:2614–2622

    Article  CAS  PubMed  Google Scholar 

  12. Fidorra M, Heimburg T, Bagatolli LA (2009) Direct visualization of the lateral structure of porcine brain cerebrosides/POPC mixtures in presence and absence of cholesterol. Biophys J 97:142–154

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Bali R, Savino L, Ramirez DA, Tsvetkova NM, Bagatolli L, Tablin F, Crowe JH, Leidy C (2009) Macroscopic domain formation during cooling in the platelet plasma membrane: an issue of low cholesterol content. Biochim Biophys Acta 1788:1229–1237

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Grossmann G, Opekarova M, Malinsky J, Weig-Meckl I, Tanner W (2007) Membrane potential governs lateral segregation of plasma membrane proteins and lipids in yeast. EMBO J 26:1–8

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Malinsky J, Opekarova M, Grossmann G, Tanner W (2013) Membrane microdomains, rafts, and detergent-resistant membranes in plants and fungi. Annu Rev Plant Biol 64:501–529

    Article  CAS  PubMed  Google Scholar 

  16. Hamilton-Miller JM (1973) Chemistry and biology of the polyene macrolide antibiotics. Bacteriol Rev 37:166–196

    CAS  Google Scholar 

  17. Gimpl G, Gehrig-Burger K (2011) Probes for studying cholesterol binding and cell biology. Steroids 76:216–231

    Article  CAS  PubMed  Google Scholar 

  18. Waheed AA, Shimada Y, Heijnen HF, Nakamura M, Inomata M, Hayashi M, Iwashita S, Slot JW, Ohno-Iwashita Y (2001) Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts). Proc Natl Acad Sci USA 98:4926–4931

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Shimada Y, Maruya M, Iwashita S, Ohno-Iwashita Y (2002) The C-terminal domain of perfringolysin O is an essential cholesterol-binding unit targeting to cholesterol-rich microdomains. Eur J Biochem 269:6195–6203

    Article  CAS  PubMed  Google Scholar 

  20. Bavdek A, Gekara NO, Priselac D, Gutierrez Aguirre I, Darji A, Chakraborty T, Macek P, Lakey JH, Weiss S, Anderluh G (2007) Sterol and pH interdependence in the binding, oligomerization, and pore formation of Listeriolysin O. Biochemistry 46:4425–4437

    Article  CAS  PubMed  Google Scholar 

  21. Farrand AJ, LaChapelle S, Hotze EM, Johnson AE, Tweten RK (2010) Only two amino acids are essential for cytolytic toxin recognition of cholesterol at the membrane surface. Proc Natl Acad Sci USA 107:4341–4346

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Nelson LD, Johnson AE, London E (2008) How interaction of perfringolysin O with membranes is controlled by sterol structure, lipid structure, and physiological low pH: insights into the origin of perfringolysin O-lipid raft interaction. J Biol Chem 283:4632–4642

    Article  CAS  PubMed  Google Scholar 

  23. Rossjohn J, Feil SC, McKinstry WJ, Tweten RK, Parker MW (1997) Structure of a cholesterol-binding, thiol-activated cytolysin and a model of its membrane form. Cell 89:685–692

    Article  CAS  PubMed  Google Scholar 

  24. Das A, Brown MS, Anderson DD, Goldstein JL, Radhakrishnan A (2014) Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis. Elife 3:e02882

    Article  PubMed Central  Google Scholar 

  25. Das A, Goldstein JL, Anderson DD, Brown MS, Radhakrishnan A (2013) Use of mutant 125I-perfringolysin O to probe transport and organization of cholesterol in membranes of animal cells. Proc Natl Acad Sci USA 110:10580–10585

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Ohno-Iwashita Y, Shimada Y, Waheed AA, Hayashi M, Inomata M, Nakamura M, Maruya M, Iwashita S (2004) Perfringolysin O, a cholesterol-binding cytolysin, as a probe for lipid rafts. Anaerobe 10:125–134

    Article  CAS  PubMed  Google Scholar 

  27. Mizuno H, Abe M, Dedecker P, Makino A, Rocha S, Ohno-Iwashita Y, Hofkens J, Kobayashi T, Miyawaki A (2011) Fluorescent probes for superresolution imaging of lipid domains on the plasma membrane. Chem Sci 2:1548–1553

    Article  CAS  Google Scholar 

  28. Frisz JF, Klitzing HA, Lou K, Hutcheon ID, Weber PK, Zimmerberg J, Kraft ML (2013) Sphingolipid domains in the plasma membranes of fibroblasts are not enriched with cholesterol. J Biol Chem 288:16855–16861

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Frisz JF, Lou K, Klitzing HA, Hanafin WP, Lizunov V, Wilson RL, Carpenter KJ, Kim R, Hutcheon ID, Zimmerberg J, Weber PK, Kraft ML (2013) Direct chemical evidence for sphingolipid domains in the plasma membranes of fibroblasts. Proc Natl Acad Sci USA 110:E613–E622

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Ziomek CA, Schulman S, Edidin M (1980) Redistribution of membrane proteins in isolated mouse intestinal epithelial cells. J Cell Biol 86:849–857

    Article  CAS  PubMed  Google Scholar 

  31. Grimmer S, van Deurs B, Sandvig K (2002) Membrane ruffling and macropinocytosis in A431 cells require cholesterol. J Cell Sci 115:2953–2962

    CAS  PubMed  Google Scholar 

  32. Carquin M, Pollet H, Veiga-da-Cunha M, Cominelli A, Van Der Smissen P, N’Kuli F, Emonard H, Henriet P, Mizuno H, Courtoy PJ, Tyteca D (2014) Endogenous sphingomyelin segregates into submicrometric domains in the living erythrocyte membrane. J Lipid Res 55:1331–1342

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. D’Auria L, Fenaux M, Aleksandrowicz P, Van Der Smissen P, Chantrain C, Vermylen C, Vikkula M, Courtoy PJ, Tyteca D (2013) Micrometric segregation of fluorescent membrane lipids: relevance for endogenous lipids and biogenesis in erythrocytes. J Lipid Res 54:1066–1076

    Article  PubMed Central  PubMed  Google Scholar 

  34. D’Auria L, Van Der Smissen P, Bruyneel F, Courtoy PJ, Tyteca D (2011) Segregation of fluorescent membrane lipids into distinct micrometric domains: evidence for phase compartmentation of natural lipids? PLoS One 6:e17021

    Article  PubMed Central  PubMed  Google Scholar 

  35. Tyteca D, D’Auria L, Van Der Smissen P, Medts T, Carpentier S, Monbaliu JC, de Diesbach P, Courtoy PJ (2010) Three unrelated sphingomyelin analogs spontaneously cluster into plasma membrane micrometric domains. Biochim Biophys Acta 1798:909–927

    Article  CAS  PubMed  Google Scholar 

  36. Zachowski A (1993) Phospholipids in animal eukaryotic membranes: transverse asymmetry and movement. Biochem J 294(Pt 1):1–14

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  37. Goodman SR, Kurdia A, Ammann L, Kakhniashvili D, Daescu O (2007) The human red blood cell proteome and interactome. Exp Biol Med (Maywood) 232:1391–1408

    Article  CAS  Google Scholar 

  38. Salomao M, Zhang X, Yang Y, Lee S, Hartwig JH, Chasis JA, Mohandas N, An X (2008) Protein 4.1R-dependent multiprotein complex: new insights into the structural organization of the red blood cell membrane. Proc Natl Acad Sci USA 105:8026–8031

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  39. Cooper RA (1978) Influence of increased membrane cholesterol on membrane fluidity and cell function in human red blood cells. J Supramol Struct 8:413–430

    Article  CAS  PubMed  Google Scholar 

  40. Cornwell DG, Heikkila RE, Bar RS, Biagi GL (1967) Red blood cell lipids and the plasma membrane. JAOCS 45:297–304

    Google Scholar 

  41. Ohno-Iwashita Y, Iwamoto M, Mitsui K, Ando S, Nagai Y (1988) Protease-nicked theta-toxin of Clostridium perfringens, a new membrane probe with no cytolytic effect, reveals two classes of cholesterol as toxin-binding sites on sheep erythrocytes. Eur J Biochem 176:95–101

    Article  CAS  PubMed  Google Scholar 

  42. Betz T, Lenz M, Joanny JF, Sykes C (2009) ATP-dependent mechanics of red blood cells. Proc Natl Acad Sci USA 106:15320–15325

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  43. Wustner D (2007) Plasma membrane sterol distribution resembles the surface topography of living cells. Mol Biol Cell 18:211–228

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  44. Maekawa M, Fairn GD (2015) Complementary probes reveal that phosphatidylserine is required for the proper transbilayer distribution of cholesterol. J Cell Sci 128:1422–1433

    Article  CAS  PubMed  Google Scholar 

  45. Iwamoto M, Ohno-Iwashita Y, Ando S (1990) Effect of isolated C-terminal fragment of theta-toxin (perfringolysin O) on toxin assembly and membrane lysis. Eur J Biochem 194:25–31

    Article  CAS  PubMed  Google Scholar 

  46. Sokolov A, Radhakrishnan A (2010) Accessibility of cholesterol in endoplasmic reticulum membranes and activation of SREBP-2 switch abruptly at a common cholesterol threshold. J Biol Chem 285:29480–29490

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  47. McKanna JA, Haigler HT, Cohen S (1979) Hormone receptor topology and dynamics: morphological analysis using ferritin-labeled epidermal growth factor. Proc Natl Acad Sci USA 76:5689–5693

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  48. De Roe C, Courtoy PJ, Baudhuin P (1987) A model of protein-colloidal gold interactions. J Histochem Cytochem 35:1191–1198

    Article  PubMed  Google Scholar 

  49. Cai M, Zhao W, Shang X, Jiang J, Ji H, Tang Z, Wang H (2012) Direct evidence of lipid rafts by in situ atomic force microscopy. Small 8:1243–1250

    Article  CAS  PubMed  Google Scholar 

  50. Bosmann HB, Hagopian A, Eylar EH (1968) Cellular membranes: the isolation and characterization of the plasma and smooth membranes of HeLa cells. Arch Biochem Biophys 128:51–69

    Article  CAS  PubMed  Google Scholar 

  51. Perkins RG, Scott RE (1978) Plasma membrane phospholipid, cholesterol and fatty acyl composition of differentiated and undifferentiated L6 myoblasts. Lipids 13:334–337

    Article  CAS  PubMed  Google Scholar 

  52. Bezrukov L, Blank PS, Polozov IV, Zimmerberg J (2009) An adhesion-based method for plasma membrane isolation: evaluating cholesterol extraction from cells and their membranes. Anal Biochem 394:171–176

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  53. Oglecka K, Rangamani P, Liedberg B, Kraut RS, Parikh AN (2014) Oscillatory phase separation in giant lipid vesicles induced by transmembrane osmotic differentials. Elife 3:e03695

    Article  PubMed Central  PubMed  Google Scholar 

  54. Kusumi A, Suzuki KG, Kasai RS, Ritchie K, Fujiwara TK (2011) Hierarchical mesoscale domain organization of the plasma membrane. Trends Biochem Sci 36:604–615

    Article  CAS  PubMed  Google Scholar 

  55. Fujiwara T, Ritchie K, Murakoshi H, Jacobson K, Kusumi A (2002) Phospholipids undergo hop diffusion in compartmentalized cell membrane. J Cell Biol 157:1071–1081

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  56. Kusumi A, Fujiwara TK, Chadda R, Xie M, Tsunoyama TA, Kalay Z, Kasai RS, Suzuki KG (2012) Dynamic organizing principles of the plasma membrane that regulate signal transduction: commemorating the fortieth anniversary of Singer and Nicolson’s fluid-mosaic model. Annu Rev Cell Dev Biol 28:215–250

    Article  CAS  PubMed  Google Scholar 

  57. Ehrig J, Petrov EP, Schwille P (2011) Near-critical fluctuations and cytoskeleton-assisted phase separation lead to subdiffusion in cell membranes. Biophys J 100:80–89

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  58. Honigmann A, Sadeghi S, Keller J, Hell SW, Eggeling C, Vink R (2014) A lipid bound actin meshwork organizes liquid phase separation in model membranes. Elife 3:e01671

    Article  PubMed Central  PubMed  Google Scholar 

  59. Sinha B, Koster D, Ruez R, Gonnord P, Bastiani M, Abankwa D, Stan RV, Butler-Browne G, Vedie B, Johannes L, Morone N, Parton RG, Raposo G, Sens P, Lamaze C, Nassoy P (2011) Cells respond to mechanical stress by rapid disassembly of caveolae. Cell 144:402–413

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  60. Guo Q, Park S, Ma H (2012) Microfluidic micropipette aspiration for measuring the deformability of single cells. Lab Chip 12:2687–2695

    Article  CAS  PubMed  Google Scholar 

  61. Bassereau P, Sorre B, Levy A (2014) Bending lipid membranes: experiments after W. Helfrich’s model. Adv Colloid Interface Sci 208:47–57

    Article  CAS  PubMed  Google Scholar 

  62. Willekens FL, Werre JM, Groenen-Dopp YA, Roerdinkholder-Stoelwinder B, de Pauw B, Bosman GJ (2008) Erythrocyte vesiculation: a self-protective mechanism? Br J Haematol 141:549–556

    Article  CAS  PubMed  Google Scholar 

  63. Salzer U, Zhu R, Luten M, Isobe H, Pastushenko V, Perkmann T, Hinterdorfer P, Bosman GJ (2008) Vesicles generated during storage of red cells are rich in the lipid raft marker stomatin. Transfusion 48:451–462

    Article  CAS  PubMed  Google Scholar 

  64. Bosman GJ, Lasonder E, Groenen-Dopp YA, Willekens FL, Werre JM (2012) The proteome of erythrocyte-derived microparticles from plasma: new clues for erythrocyte aging and vesiculation. J Proteomics 76:203–210

    Article  CAS  PubMed  Google Scholar 

  65. van Galen J, Campelo F, Martinez-Alonso E, Scarpa M, Martinez-Menarguez JA, Malhotra V (2014) Sphingomyelin homeostasis is required to form functional enzymatic domains at the trans-Golgi network. J Cell Biol 206:609–618

    Article  PubMed Central  PubMed  Google Scholar 

  66. van den Bogaart G, Meyenberg K, Risselada HJ, Amin H, Willig KI, Hubrich BE, Dier M, Hell SW, Grubmuller H, Diederichsen U, Jahn R (2011) Membrane protein sequestering by ionic protein-lipid interactions. Nature 479:552–555

    Article  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Drs. A. Miyawaki, M. Abe and T. Kobayashi at Riken Brain Science Institute (Saitama, Japan) as well as H. Mizuno (KU Leuven, Belgium) for generously supplying the Dronpa-theta-D4 plasmid, Dr. A. De Matteis (Naples, Italy) for mCherry-Rab5 plasmid and P. Gailly for C2C12 myoblasts; P. Henriet, H. Emonard and J. Lorent for help in producing toxin* and liposomes and T. Lac and N. Chevalier for technical support (UCL, Belgium). This work was supported by grants from UCL (FSR), the F.R.S.-FNRS, Salus Sanguinis foundation, ARC, IUAP and the Région Wallonne. DT and MVdC are Research associates at Fonds de la Recherche Scientifique (F.R.S.-FNRS).

Author information

Authors and Affiliations

  1. CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium

    Mélanie Carquin, Louise Conrard, Hélène Pollet, Patrick Van Der Smissen, Antoine Cominelli, Pierre J. Courtoy & Donatienne Tyteca

  2. Laboratory of Physiological Chemistry, de Duve Institute and Université Catholique de Louvain, 1200, Brussels, Belgium

    Maria Veiga-da-Cunha

Authors
  1. Mélanie Carquin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Louise Conrard
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hélène Pollet
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Patrick Van Der Smissen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Antoine Cominelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Maria Veiga-da-Cunha
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Pierre J. Courtoy
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Donatienne Tyteca
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Donatienne Tyteca.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (AVI 108863 kb)

Supplementary material 2 (DOCX 773 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Carquin, M., Conrard, L., Pollet, H. et al. Cholesterol segregates into submicrometric domains at the living erythrocyte membrane: evidence and regulation. Cell. Mol. Life Sci. 72, 4633–4651 (2015). https://doi.org/10.1007/s00018-015-1951-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00018-015-1951-x

Keywords

Search

Navigation