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.
[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. http://dx.doi.org/10.1016/S1388-1981(99)00110-910.1016/S1388-1981(99)00110-9Search in Google Scholar
[2] Daleke, D.L. Regulation of phospholipid asymmetry in the erythrocyte membrane. Curr. Opin. Hematol. 15 (2008) 191–195. http://dx.doi.org/10.1097/MOH.0b013e3282f97af710.1097/MOH.0b013e3282f97af7Search in 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. http://dx.doi.org/10.1007/s00018-005-4527-310.1007/s00018-005-4527-3Search in 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. http://dx.doi.org/10.1074/jbc.273.12.660310.1074/jbc.273.12.6603Search in 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. http://dx.doi.org/10.1074/jbc.271.29.1720510.1074/jbc.271.29.17205Search in 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. http://dx.doi.org/10.1074/jbc.272.29.1824010.1074/jbc.272.29.18240Search in 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. http://dx.doi.org/10.1016/j.abb.2007.04.00210.1016/j.abb.2007.04.002Search in Google Scholar
[8] Bevers, E.M. and Williamson, P.L. Phospholipid scramblase: An update. FEBS Lett. 584 (2010) 2724–2730. http://dx.doi.org/10.1016/j.febslet.2010.03.02010.1016/j.febslet.2010.03.020Search in 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. http://dx.doi.org/10.1016/j.febslet.2009.12.04910.1016/j.febslet.2009.12.049Search in Google Scholar
[10] Zwaal, R.F. and Schroit, A.J. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 89 (1997) 1121–1132. Search in 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. http://dx.doi.org/10.1016/S1388-1981(02)00324-410.1016/S1388-1981(02)00324-4Search in 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. http://dx.doi.org/10.1042/0264-6021:362074110.1042/bj3620741Search in 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. http://dx.doi.org/10.1021/bi001929z10.1021/bi001929zSearch in 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. http://dx.doi.org/10.1074/jbc.M70020220010.1074/jbc.M700202200Search in 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. http://dx.doi.org/10.1054/ceca.1999.002910.1054/ceca.1999.0029Search in 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. http://dx.doi.org/10.1042/0264-6021:362070110.1042/bj3620701Search in 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. http://dx.doi.org/10.1006/excr.1997.373810.1006/excr.1997.3738Search in 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. http://dx.doi.org/10.1006/bbrc.1999.182010.1006/bbrc.1999.1820Search in 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. http://dx.doi.org/10.1111/j.1432-1033.1982.tb05898.x10.1111/j.1432-1033.1982.tb05898.xSearch in 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. http://dx.doi.org/10.1016/0005-2736(83)90169-410.1016/0005-2736(83)90169-4Search in 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. Search in 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. Search in 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. Search in 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. Search in 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. Search in 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. http://dx.doi.org/10.1084/jem.182.5.154510.1084/jem.182.5.1545Search in Google Scholar PubMed PubMed Central
[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. Search in 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. Search in 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. Search in 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. Search in 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. http://dx.doi.org/10.1021/bi010749210.1021/bi0107492Search in 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. http://dx.doi.org/10.1016/j.bmc.2005.04.07110.1016/j.bmc.2005.04.071Search in Google Scholar
[33] Zweifach, A. FM1-43 reports plasma membrane phospholipid scrambling in T-lymphocytes. Biochem. J. 349 (2000) 255–260. http://dx.doi.org/10.1042/0264-6021:349025510.1042/0264-6021:3490255Search in 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. http://dx.doi.org/10.1146/annurev.neuro.22.1.110.1146/annurev.neuro.22.1.1Search in Google Scholar
[35] Betz, W.J., Mao, F. and Smith, C.B. Imaging exocytosis and endocytosis. Curr. Opin. Neurobiol. 6 (1996) 365–371. http://dx.doi.org/10.1016/S0959-4388(96)80121-810.1016/S0959-4388(96)80121-8Search in 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. http://dx.doi.org/10.1016/S0005-2736(98)00188-610.1016/S0005-2736(98)00188-6Search in 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. http://dx.doi.org/10.3109/0968768980902752310.3109/09687689809027523Search in Google Scholar
[38] Lozzio, C.B. and Lozzio, B.B. Human chronic myelogenous leukemia cellline with positive Philadelphia chromosome. Blood 45 (1975) 321–334. Search in Google Scholar
[39] Isomaa, B., Hägerstrand, H. and Paatero, G. Shape transformations induced by amphiphiles in erythrocytes. Biochim. Biophys. Acta 899 (1987) 93–103. http://dx.doi.org/10.1016/0005-2736(87)90243-410.1016/0005-2736(87)90243-4Search in 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. http://dx.doi.org/10.1016/0005-2736(83)90055-X10.1016/0005-2736(83)90055-XSearch in 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. http://dx.doi.org/10.1016/j.bpj.2009.04.02810.1016/j.bpj.2009.04.028Search in Google Scholar PubMed PubMed Central
© 2013 University of Wrocław, Poland
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.