Abstract
Phosphatidylserine (PS) exposure on the external leaflet of the plasma membrane is widely observed during apoptosis and forms the basis for the annexin V binding assay to detect apoptotic cell death. Current efforts to explain PS exposure focus on two potential mechanisms, activation of a phospholipid scramblase or calcium-mediated trafficking of lysosomes to the cell surface. Here, we provide evidence that apoptotic PS exposure instead reflects bidirectional trafficking of membrane between the cell surface and cytoplasm. Using a series of cell lines, some of which expose large amounts of PS during apoptosis and some of which do not, we demonstrate that accumulation of plasma membrane-derived cytoplasmic vesicles in a dynamin-, clathrin- and Cdc42-independent manner is a previously undescribed but widely occurring feature of apoptosis. The apoptotic exposure of PS occurs when these vesicles traffic back to cell surface in a calcium-dependent process that is deficient in a substantial fraction of human cancer cell lines. These observations provide a new model for PS externalization during apoptosis and simultaneously identify an altered step that accounts for the paucity of apoptotic PS exposure in many cell lines.
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Abbreviations
- PS:
-
phosphatidylserine
- ABC:
-
ATP-binding cassette
- APC:
-
allophycocyanin
- CHX:
-
cycloheximide
- EBFP:
-
enhanced blue fluorescent protein
- EGFP:
-
enhanced green fluorescent protein
- FBS:
-
heat-inactivated fetal bovine serum
- FITC:
-
fluorescein isothiocyanate
- PLSCR1:
-
phospholipid scramblase 1
- PBS:
-
calcium- and magnesium-free Dulbecco’s phosphate-buffered saline
- PCR:
-
polymerase chain reaction
- Q-VD-OPh:
-
N-(2-quinolyl)valylaspartyl-(2,6-difluorophenoxy)methyl ketone
- TRAIL:
-
tumor necrosis factor-α-related apoptosis inducing ligand
References
Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM . Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 1992; 148: 2207–2216.
Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH . Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 1994; 84: 1415–1420.
Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie RC, LaFace DM et al. 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 1995; 182: 1545–1556.
Zullig S, Neukomm LJ, Jovanovic M, Charette SJ, Lyssenko NN, Halleck MS et al. Aminophospholipid translocase TAT-1 promotes phosphatidylserine exposure during C. elegans apoptosis. Curr Biol 2007; 17: 994–999.
Schlegel RA, Williamson P . P.S. to PS (phosphatidylserine)--pertinent proteins in apoptotic cell clearance. Sci STKE 2007; 2007: pe57.
Zhou Z . New phosphatidylserine receptors: clearance of apoptotic cells and more. Developmental Cell 2007; 13: 759–760.
Zwaal RF, Comfurius P, Bevers EM . Surface exposure of phosphatidylserine in pathological cells. Cell Mol Life Sci 2005; 62: 971–988.
van Genderen H, Kenis H, Lux P, Ungeth L, Maassen C, Deckers N et al. In vitro measurement of cell death with the annexin A5 affinity assay. Nature Protocols 2006; 1: 363–367.
Neves AA, Brindle KM . Assessing responses to cancer therapy using molecular imaging. Biochim Biophys Acta 2006; 1766: 242–261.
Zhou Q, Zhao J, Stout JG, Luhm RA, Wiedmer T, Sims PJ . Molecular cloning of human plasma membrane phospholipid scramblase. A protein mediating transbilayer movement of plasma membrane phospholipids. J Biol Chem 1997; 272: 18240–18244.
Zhou Q, Zhao J, Wiedmer T, Sims PJ . Normal hemostasis but defective hematopoietic response to growth factors in mice deficient in phospholipid scramblase 1. Blood 2002; 99: 4030–4038.
Hamon Y, Broccardo C, Chambenoit O, Luciani MF, Toti F, Chaslin S et al. ABC1 promotes engulfment of apoptotic cells and transbilayer redistribution of phosphatidylserine. Nat Cell Biol 2000; 2: 399–406.
Darland-Ransom M, Wang X, Sun CL, Mapes J, Gengyo-Ando K, Mitani S et al. Role of C. elegans TAT-1 protein in maintaining plasma membrane phosphatidylserine asymmetry. Science 2008; 320: 528–531.
Suzuki J, Umeda M, Sims PJ, Nagata S . Calcium-dependent phospholipid scrambling by TMEM16F. Nature 2010; 468: 834–838.
Mirnikjoo B, Balasubramanian K, Schroit AJ . Suicidal membrane repair regulates phosphatidylserine externalization during apoptosis. J Biol Chem 2009; 284: 22512–22516.
Fadeel B, Gleiss B, Hogstrand K, Chandra J, Wiedmer T, Sims PJ et al. Phosphatidylserine exposure during apoptosis is a cell-type-specific event and does not correlate with plasma membrane phospholipid scramblase expression. Biochem Biophys Res Commun 1999; 266: 504–511.
Meng XW, Heldebrant MP, Flatten KS, Loegering DA, Dai H, Schneider PA et al. Protein kinase Cbeta modulates ligand-induced cell surface death receptor accumulation: a mechanistic basis for enzastaurin-death ligand synergy. J Biol Chem 2010; 285: 888–902.
Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C . A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 1995; 184: 39–51.
Meng XW, Lee SH, Dai H, Loegering D, Yu C, Flatten K et al. Mcl-1 as a buffer for proapoptotic Bcl-2 family members during TRAIL-induced apoptosis: a mechanistic basis for sorafenib (Bay 43-9006)-induced TRAIL sensitization. J Biol Chem 2007; 282: 29831–29846.
Ding H, Hackbarth J, Schneider PA, Peterson KL, Meng XW, Dai H et al. Cytotoxicity of farnesyltransferase inhibitors in lymphoid cells mediated by MAPK pathway inhibition and Bim up-regulation. Blood 2011; 118: 4872–4881.
Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE, Poirier GG . Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res 1993; 53: 3976–3985.
Ford MG, Pearse BM, Higgins MK, Vallis Y, Owen DJ, Gibson A et al. Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes. Science 2001; 291: 1051–1055.
Wyllie AH, Kerr JF, Currie AR . Cell death: the significance of apoptosis. International Review of Cytology 1980; 68: 251–306.
Bortner CD, Cidlowski JA . Life and death of lymphocytes: a volume regulation affair. Cell Physiol Biochem 2011; 28: 1079–1088.
Maeno E, Ishizaki Y, Kanaseki T, Hazama A, Okada Y . Normotonic cell shrinkage because of disordered volume regulation is an early prerequisite to apoptosis. Proc Natl Acad Sci USA 2000; 97: 9487–9492.
Frasch SC, Henson PM, Kailey JM, Richter DA, Janes MS, Fadok VA et al. Regulation of phospholipid scramblase activity during apoptosis and cell activation by protein kinase Cdelta. J Biol Chem 2000; 275: 23065–23073.
Williamson P, Bevers EM, Smeets EF, Comfurius P, Schlegel RA, Zwaal RF . Continuous analysis of the mechanism of activated transbilayer lipid movement in platelets. Biochemistry 1995; 34: 10448–10455.
McNeil PL, Kirchhausen T . An emergency response team for membrane repair. Nat Rev Mol Cell Biol 2005; 6: 499–505.
Rodriguez A, Webster P, Ortego J, Andrews NW . Lysosomes behave as Ca2+-regulated exocytic vesicles in fibroblasts and epithelial cells. J Cell Biol 1997; 137: 93–104.
Yoshida H, Kawane K, Koike M, Mori Y, Uchiyama Y, Nagata S . Phosphatidylserine-dependent engulfment by macrophages of nuclei from erythroid precursor cells. Nature 2005; 437: 754–758.
Keerthivasan G, Small S, Liu H, Wickrema A, Crispino JD . Vesicle trafficking plays a novel role in erythroblast enucleation. Blood 2010; 116: 3331–3340.
Elliott JI, Sardini A, Cooper JC, Alexander DR, Davanture S, Chimini G et al. Phosphatidylserine exposure in B lymphocytes: a role for lipid packing. Blood 2006; 108: 1611–1617.
Fischer K, Voelkl S, Berger J, Andreesen R, Pomorski T, Mackensen A . Antigen recognition induces phosphatidylserine exposure on the cell surface of human CD8+ T cells. Blood 2006; 108: 4094–4101.
Keller S, Konig AK, Marme F, Runz S, Wolterink S, Koensgen D et al. Systemic presence and tumor-growth promoting effect of ovarian carcinoma released exosomes. Cancer Lett 2009; 278: 73–81.
Luzio JP, Pryor PR, Bright NA . Lysosomes: fusion and function. Nat Rev Mol Cell Biol 2007; 8: 622–632.
Mellgren RL . A plasma membrane wound proteome: reversible externalization of intracellular proteins following reparable mechanical damage. J Biol Chem 2010; 285: 36597–36607.
Cai C, Masumiya H, Weisleder N, Matsuda N, Nishi M, Hwang M et al. MG53 nucleates assembly of cell membrane repair machinery. Nat Cell Biol 2009; 11: 56–64.
Leventis PA, Grinstein S . The distribution and function of phosphatidylserine in cellular membranes. Annu Rev Biophys 2010; 39: 407–427.
Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C . A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 1991; 139: 271–279.
Walev I, Bhakdi SC, Hofmann F, Djonder N, Valeva A, Aktories K et al. Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O. Proc Natl Acad Sci USA 2001; 98: 3185–3190.
Dekkers DW, Comfurius P, Bevers EM, Zwaal RF . Comparison between Ca2+-induced scrambling of various fluorescently labelled lipid analogues in red blood cells. Biochem J 2002; 362 (Pt 3): 741–747.
Kaufmann SH . Reutilization of immunoblots after chemiluminescent detection. Anal Biochem 2001; 296: 283–286.
Kaufmann SH . Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: a cautionary note. Cancer Res 1989; 49: 5870–5878.
Acknowledgements
This work was supported in part by R01 CA69008 and a predoctoral fellowship to S-HL from the Mayo Foundation for Education and Research. We gratefully acknowledge use of the Mayo Clinic Flow Cytometry and Electron Microscopy Core Facilities; assistance of Trace Christensen, Scott Gamb and Cindy Uhl with the electron micrographs; assistance of Anand Patel and Kevin Peterson with confocal microscopy and cell size determinations, respectively; and helpful conversations with Greg Gores.
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Lee, SH., Meng, X., Flatten, K. et al. Phosphatidylserine exposure during apoptosis reflects bidirectional trafficking between plasma membrane and cytoplasm. Cell Death Differ 20, 64–76 (2013). https://doi.org/10.1038/cdd.2012.93
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DOI: https://doi.org/10.1038/cdd.2012.93
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