Phosphatidylserine exposure during apoptosis reflects bidirectional trafficking between plasma membrane and cytoplasm S-H Lee 1,2 , XW Meng 1,2 , KS Flatten 2 , DA Loegering 2 and SH Kaufmann* ,1,2 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. Cell Death and Differentiation advance online publication, 3 August 2012; doi:10.1038/cdd.2012.93 A common feature of apoptosis from Caenorhabditis elegans to man is the transfer of phosphatidylserine (PS) and phosphatidylethanolamine, which ordinarily reside on the cytoplasmic surface of the membranes, to the cell surface. 1–4 The PS that is externalized not only contributes to the recognition and subsequent removal of apoptotic bodies by phagocytes, 1,5,6 but also provides a binding site for the anionic lipid binding protein annexin V, 7 which is widely used to detect apoptotic cells. 8 In addition to its use in laboratory studies in vitro, annexin V binding is being explored as a potential early marker of treatment efficacy in cancer patients. 9 The biochemical basis for apoptotic PS externalization remains incompletely understood. The most widely accepted model suggests that an enzyme capable of randomizing PS across the plasma membrane – a so-called scramblase – is activated during apoptosis. Consistent with this model, a polypeptide capable of scrambling PS in liposomes in vitro was purified; and the cDNA encoding this phospholipid scramblase (PLSCR1) was cloned. 10 Plscr1 deletion in mice, however, had no effect on apoptosis-induced PS externaliza- tion. 11 Additional studies suggested a role for the ATP-binding cassette (ABC) transporters ABC1 12 and Tat-1 4 in PS externalization, although the latter claim has also been questioned. 13 More recently, TMEM16F was implicated in Ca 2 þ -dependent phospholipid scrambling in blood cells, 14 although its role in apoptotic PS exposure has not been confirmed. An alternative model suggests that apoptotic PS externa- lization reflects trafficking of lysosomes to the cell surface rather than a simple enzymological process. 15 The demon- stration that annexin V binding to staurosporine-treated HeLa cells could be inhibited by the lysosomotropic agent chloro- quine, coupled with studies of NBD-PS dynamics, suggested that PS externalization reflects Ca 2 þ -induced trafficking of lysosomes to the plasma membrane. 15 Whether this occurs in response to other stimuli and in different cells remains uncertain. Despite the widespread use of PS externalization as a marker for apoptosis, certain cell lines undergo apoptosis with very little increase in annexin V binding. 16 Although special assay conditions have been developed to detect PS on the surface of these apoptotic cells, 8 the biological basis for the diminished PS externalization is unclear. Based on the results of the present study, we propose a different mechanism for PS externalization during apoptosis. We also report that the second step in this process is impaired in a number of human cancer cell lines, providing an explanation for the low PS externalization observed during apoptosis in those lines. 1 Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, Rochester, MN 55905, USA and 2 Department of Oncology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA *Corresponding author: SH Kaufmann, Division of Oncology Research, Mayo Clinic, Gonda 19-212, 200 First Street, SW, Rochester, MN 55905, USA. Tel: þ 507 284 8950; Fax: þ 507 293 0107; E-mail: Kaufmann.scott@mayo.edu Received 16.3.12; revised 25.6.12; accepted 26.6.12; Edited by G Melino Keywords: Annexin V; phosphatidylserine; membrane vesicles 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-a-related apoptosis inducing ligand Cell Death and Differentiation (2012), 1–13 & 2012 Macmillan Publishers Limited All rights reserved 1350-9047/12 www.nature.com/cdd