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Monitoring of membrane phospholipid scrambling in human erythrocytes and K562 cells with FM1-43 — a comparison with annexin V-FITC

Cellular & Molecular Biology Letters volume 19, pages 262–276 (2014)Cite this article

Abstract

The styryl dye FM1-43 becomes highly fluorescent upon binding to cell membranes. The breakdown of membrane phospholipid asymmetry in ionophore-stimulated T-lymphocytes further increases this fluorescence [Zweifach, 2000]. In this study, the capacity of FM1-43 to monitor membrane phospholipid scrambling was explored using flow cytometry in human erythrocytes and human erythrocyte progenitor K562 cells. The Ca2+-dependent phosphatidylserine-specific probe annexin V-FITC was used for comparison. The presented data show that the loss of phospholipid asymmetry that could be induced in human erythrocytes by elevated intracellular Ca2+ or by structurally different membrane intercalated amphiphilic compounds increases the FM1-43 fluorescence two- to fivefold. The profile of FM1-43 fluorescence for various treatments resembles that of phosphatidylserine exposure reported by annexin V-FITC. FM1-43 detected the onset of scrambling more efficiently than annexin V-FITC. The amphiphile-induced scrambling was shown to be a Ca2+-independent process. Monitoring of scrambling in K562 cells caused by NEM-induced Ca2+-release from intracellular stores and by Ca2+ and ionophore A23187 treatment showed that the increase in FM1-43 fluorescence correlated well with the number of annexin V-FITC-detected phosphatidylserine-positive cells. The results presented here show the usefulness of FM1-43 as a Ca2+-independent marker of dissipation in asymmetric membrane phospholipid distribution induced by various stimuli in both nucleated and non-nucleated cells.

Abbreviations

A23187:

calcium ionophore

BSA:

bovine serum albumin

C10E8:

octaethylene glycol mono n-decyl ether

C12E8:

octaethylene glycol mono n-dodecyl ether

C12T:

dodecyltrimethylammonium bromide

C12Z:

3-(dodecyldimethylammonio)-1-propanesulphonate

CPZ:

chlorpromazine hydrochloride

DMSO:

dimethyl sulfoxide

FITC:

fluorescein isothiocyanate

Fluo3-AM:

4-(6-acetoxymethoxy-2, 7-dichloro-3-oxo-9-xanthenyl)-4′-methyl-2,2′(ethylenedioxy)dianiline-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl) ester

FM1-43:

(n-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide)

IONO:

ionomycin

NEM:

n-ethylmaleimide

PMA:

phorbol 12-myristate 13-acetate

PS:

phosphatidylserine

S12S:

sodium dodecyl sulphate

S14S:

sodium tetradecyl sulphate

References

  1. Bevers, E.M., Comfurius, P., Dekkers, D.W. and Zwaal, R.F. Lipid translocation across the plasma membrane of mammalian cells. Biochim. Biophys. Acta 1439 (1999) 317–330.

    Article  CAS  PubMed  Google Scholar 

  2. Daleke, D.L. Regulation of phospholipid asymmetry in the erythrocyte membrane. Curr. Opin. Hematol. 15 (2008) 191–195.

    Article  CAS  PubMed  Google Scholar 

  3. Zwaal, R.F., Comfurius, P. and Bevers, E.M. Surface exposure of phosphatidyl-serine in pathological cells. Cell. Mol. Life Sci. 62 (2005) 971–988.

    Article  CAS  PubMed  Google Scholar 

  4. Zhao, J., Zhou, Q., Wiedmer, T. and Sims, P.J. Level of expression of phospholipid scramblase regulates induced movement of phosphatidylserine to the cell surface. J. Biol. Chem. 273 (1998) 6603–6606.

    Article  CAS  PubMed  Google Scholar 

  5. Bassé, F., Stout, J.G., Sims, P.J. and Wiedmer, T. Isolation of an erythrocyte membrane protein that mediates Ca2+-dependent transbilayer movement of phospholipid. J. Biol. Chem. 271 (1996) 17205–17210.

    Article  PubMed  Google Scholar 

  6. Zhou, Q., Zhao, J., Stout, J.G., Luhm, R.A., Wiedmer, T. and Sims, P.J. Molecular cloning of human plasma membrane phospholipid scramblase. A protein mediating transbilayer movement of plasma membrane phospholipids. J. Biol. Chem. 272 (1997) 18240–18244.

    Article  CAS  PubMed  Google Scholar 

  7. Sahu, S.K., Gummadi, S.N., Manoj, N. and Aradhyam, G.K. Phospholipid scramblases: an overview. Arch. Biochem. Biophys. 462 (2007) 103–114.

    Article  CAS  PubMed  Google Scholar 

  8. Bevers, E.M. and Williamson, P.L. Phospholipid scramblase: An update. FEBS Lett. 584 (2010) 2724–2730.

    Article  CAS  PubMed  Google Scholar 

  9. Contreras, F.X., Sánchez-Magraner, L., Alonso, A. and Goñi, F.M. Transbilayer (flip-flop) lipid motion and lipid scrambling in membranes. FEBS Lett. 584 (2010) 1779–1786.

    Article  CAS  PubMed  Google Scholar 

  10. Zwaal, R.F. and Schroit, A.J. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 89 (1997) 1121–1132.

    CAS  PubMed  Google Scholar 

  11. Williamson, P. and Schlegel, R.A. Transbilayer phospholipid movement and the clearance of apoptotic cells. Biochim. Biophys. Acta 1585 (2002) 53–63.

    Article  CAS  PubMed  Google Scholar 

  12. Dekkers, D.W., Comfurius, P., Bevers, E.M. and Zwaal, R.F. Comparison between Ca2+-induced scrambling of various fluorescently labeled lipid analogues in red blood cells. Biochem. J. 362 (2002) 741–747.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. Williamson, P., Christie, A., Kohlin, T., Schlegel, R.A., Comfurius, P., Harmsma, M., Zwaal, R.F. and Bevers, E.M. Phospholipid scramblase activation pathways in lymphocytes. Biochemistry 40 (2001) 8065–8072.

    Article  CAS  PubMed  Google Scholar 

  14. Balasubramanian, K., Mirnikjoo, B. and Schroit, A.J. Regulated externalization of phosphatidylserine at the cell surface: implications for apoptosis. J. Biol. Chem. 282 (2007) 18357–18364.

    Article  CAS  PubMed  Google Scholar 

  15. Woon, L.A., Holland, J.W., Kable, E.P. and Roufogalis, B.D. Ca2+ sensitivity of phospholipid scrambling in human red cell ghosts. Cell Calcium 25 (1999) 313–320.

    Article  CAS  PubMed  Google Scholar 

  16. Wurth, G.A. and Zweifach, A. Evidence that cytosolic calcium increases are not sufficient to stimulate phospholipid scrambling in human T-lymphocytes. Biochem. J. 362 (2002) 701–708.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  17. van Engeland, M., Kuijpers, H.J., Ramaekers, F.C., Reutelingsperger, C.P. and Schutte, B. Plasma membrane alterations and cytoskeletal changes in apoptosis. Exp. Cell. Res. 235 (1997) 421–430.

    Article  PubMed  Google Scholar 

  18. Fadeel, B., Gleiss, B., Högstrand, K., Chandra, J., Wiedmer, T., Sims, P.J., Henter, J.I., Orrenius, S. and Samali, A. Phosphatidylserine exposure during apoptosis is a cell-type-specific event and does not correlate with plasma membrane phospholipid scramblase expression. Biochem. Biophys. Res. Commun. 266 (1999) 504–511.

    Article  CAS  PubMed  Google Scholar 

  19. Bevers, E.M., Comfurius, P., van Rijn, J.L., Hemker, H.C. and Zwaal, R.F. Generation of prothrombin-converting activity and the exposure of phosphatidylserine at the outer surface of platelets. Eur. J. Biochem. 122 (1982) 429–436.

    Article  CAS  PubMed  Google Scholar 

  20. Bevers, E.M., Comfurius, P. and Zwaal, R.F. Changes in membrane phospholipid distribution during platelet activation. Biochim. Biophys. Acta 736 (1983) 57–66.

    Article  CAS  PubMed  Google Scholar 

  21. Blumenfeld, N., Zachowski, A., Galacteros, F., Beuzard, Y. and Devaux, P.F. Transmembrane mobility of phospholipids in sickle erythrocytes: effect of deoxygenation on diffusion and asymmetry. Blood 77 (1991) 849–854.

    CAS  PubMed  Google Scholar 

  22. Utsugi, T., Schroit, A.J., Connor, J., Bucana, C.D. and Fidler, I.J. Elevated expression of phosphatidylserine in the outer membrane leaflet of human tumor cells and recognition by activated human blood monocytes. Cancer Res. 51 (1991) 3062–3066.

    CAS  PubMed  Google Scholar 

  23. Schroit, A.J., Madsen, J.W. and Tanaka, Y. In vivo recognition and clearance of red blood cells containing phosphatidylserine in their plasma membranes. J. Biol. Chem. 260 (1985) 5131–5138.

    CAS  PubMed  Google Scholar 

  24. Connor, J., Pak, C.C. and Schroit, A.J. Exposure of phosphatidylserine in the outer leaflet of human red blood cells. Relationship to cell density, cell age, and clearance by mononuclear cells. J. Biol. Chem. 269 (1994) 2399–2404.

    CAS  PubMed  Google Scholar 

  25. Fadok, V.A., Voelker, D.R., Campbell, P.A., Cohen, J.J., Bratton, D.L. and Henson, P.M. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148 (1992) 2207–2216.

    CAS  PubMed  Google Scholar 

  26. Martin, S.J., Reutelingsperger, C.P., McGahon, A.J., Rader, J.A., van Schie, R.C., LaFace, D.M. and Green, D.R. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J. Exp. Med. 182 (1995) 1545–1556.

    Article  CAS  PubMed  Google Scholar 

  27. Kuypers, F.A., Lewis, R.A., Hua, M., Schott, M.A., Discher, D., Ernst, J.D. and Lubin, B.H. Detection of altered membrane phospholipid asymmetry in subpopulations of human red blood cells using fluorescently labeled annexin V. Blood 87 (1996) 1179–1187.

    CAS  PubMed  Google Scholar 

  28. Wood, B.L., Gibson, D.F. and Tait, J.F. Increased erythrocyte phosphatidylserine exposure in sickle cell disease: flow-cytometric measurement and clinical associations. Blood 88 (1996) 1873–1880.

    CAS  PubMed  Google Scholar 

  29. Trotter, P.J., Orchard, M.A. and Walker, J.H. Ca2+ concentration during binding determines the manner in which annexin V binds to membranes. Biochem. J. 308 (1995) 591–598.

    CAS  PubMed Central  PubMed  Google Scholar 

  30. Koopman, G., Reutelingsperger, C.P., Kuijten, G.A., Keehnen, R.M., Pals, S.T. and van Oers, M.H. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84 (1994) 1415–1420.

    CAS  PubMed  Google Scholar 

  31. Kamp, D., Sieberg, T. and Haest, C.W. Inhibition and stimulation of phospholipid scrambling activity. Consequences for lipid asymmetry, echinocytosis, and microvesiculation of erythrocytes. Biochemistry 40 (2001) 9438–9446.

    Article  CAS  PubMed  Google Scholar 

  32. Hanshaw, R.G. and Smith, B.D. New reagents for phosphatidylserine recognition and detection of apoptosis. Bioorg. Med. Chem. 13 (2005) 5035–5042.

    Article  CAS  PubMed  Google Scholar 

  33. Zweifach, A. FM1-43 reports plasma membrane phospholipid scrambling in T-lymphocytes. Biochem. J. 349 (2000) 255–260.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Cochilla, A.J., Angleson, J.K. and Betz, W.J. Monitoring secretory membrane with FM1-43 fluorescence. Annu. Rev. Neurosci. 22 (1999) 1–10.

    Article  CAS  PubMed  Google Scholar 

  35. Betz, W.J., Mao, F. and Smith, C.B. Imaging exocytosis and endocytosis. Curr. Opin. Neurobiol. 6 (1996) 365–371.

    Article  CAS  PubMed  Google Scholar 

  36. Schote, U. and Seelig, J. Interaction of the neuronal marker dye FM1-43 with lipid membranes. Thermodynamics and lipid ordering. Biochim. Biophys. Acta 1415 (1998) 135–146.

    Article  CAS  PubMed  Google Scholar 

  37. Hägerstrand, H., Holmström, T.H., Bobrowska-Hägerstrand, M., Eriksson, J.E. and Isomaa, B. Amphiphile-induced phosphatidylserine exposure in human erythrocytes. Mol. Membr. Biol. 15 (1998) 89–95.

    Article  PubMed  Google Scholar 

  38. Lozzio, C.B. and Lozzio, B.B. Human chronic myelogenous leukemia cellline with positive Philadelphia chromosome. Blood 45 (1975) 321–334.

    CAS  PubMed  Google Scholar 

  39. Isomaa, B., Hägerstrand, H. and Paatero, G. Shape transformations induced by amphiphiles in erythrocytes. Biochim. Biophys. Acta 899 (1987) 93–103.

    Article  CAS  PubMed  Google Scholar 

  40. Williamson, P., Mattocks, K. and Schlegel, R.A. Merocyanine 540, a fluorescent probe sensitive to lipid packing. Biochim. Biophys. Acta 732 (1983) 387–393.

    Article  CAS  PubMed  Google Scholar 

  41. Wu, Y., Yeh, F.L., Mao, F. and Chapman, E.R. Biophysical characterization of styryl dye-membrane interactions. Biophys. J. 97 (2009) 101–109.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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Authors and Affiliations

  1. Institute of Physics, Wrocław University of Technology, 50-370, Wrocław, Poland

    Anna Wróbel

  2. Department of Biosciences, Åbo Akademi University, 20520, Åbo/Turku, Finland

    Małgorzata Bobrowska-Hägerstrand, Christer Lindqvist & Henry Hägerstrand

  3. Department of Biosciences, Cell Biology, Åbo Akademi University, Artillerigatan 6, FIN-20520, Åbo/Turku, Finland

    Henry Hägerstrand

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  1. Anna Wróbel
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  2. Małgorzata Bobrowska-Hägerstrand
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  4. Henry Hägerstrand
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Correspondence to Henry Hägerstrand.

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Wróbel, A., Bobrowska-Hägerstrand, M., Lindqvist, C. et al. Monitoring of membrane phospholipid scrambling in human erythrocytes and K562 cells with FM1-43 — a comparison with annexin V-FITC. Cell Mol Biol Lett 19, 262–276 (2014). https://doi.org/10.2478/s11658-014-0195-3

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Keywords

Cellular & Molecular Biology Letters

ISSN: 1689-1392

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