Phosphatidylserine exposure during apoptosis reflects bidirectional trafficking between plasma membrane and cytoplasm

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. Cell Death and Differentiation (2013) 20, 64-76; doi:10.1038/cdd.2012; published online 3 August 2012A 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.

Autophagic vesicles on mature human reticulocytes explain phosphatidylserine-positive red cells in sickle cell disease

“…We assume that autophagic vesicles expelled by maturing reticulocytes would be phagocytosed in vivo by splenic macrophages in individuals with a functional spleen. Our data suggests that a bidirectional trafficking process, as proposed by Lee et al, 8 is used to generate erythrocytes from mature reticulocytes. However, in the maturing human reticulocyte, this apoptotic-like process combines with autophagy to achieve the reduction in surface area and volume, and simultaneously eliminate unwanted residual intracellular organelles in order to form the biconcave erythrocyte.…”

MLN4924 induces Noxa upregulation in acute myelogenous leukemia and synergizes with Bcl-2 inhibitors

“…Consistent with these data, apoptosis was also evident from western blots that revealed cleavage of wellestablished caspase substrates 30 such as poly(ADP-ribose) polymerase 1 (PARP1) and procaspase-3 (Figure 1c), as well as flow cytometry showing increased annexin V staining (see below), another hallmark of apoptosis. 31 Although previously noted, 5 formation of reactive oxygen species (ROS) was not observed in any of the three AML lines treated with MLN4924 (Supplementary Figure S1c), whereas increased ROS were readily detected upon exposure to the positive control adaphostin, an agent that inhibits mitochondrial electron transport to induce ROS. 32 Evidence of endoplasmic reticulum (ER) stress also was not present, as levels of ATF4, GRP78, and phospho-EIF2α remained unchanged in response to MLN4924 treatment (Supplementary Figure S1d).…”

“…Cells were exposed to increasing concentrations of MLN4924 diluted in DMSO (final concentration 0.1% v/v) for 4-48 h, then pelleted and stained with PI as previously described. 29,31 After 30 min, samples were subjected to flow cytometry using a FACSCanto II (BD Bioscience) to assess PI fluorescence intensity. Analysis and quantification of the sub-G 0 /G 1 population was completed using FlowJo Software (TreeStar, Ashland, OR, USA).…”

“…Annexin V staining was performed as previously described. 31 Western blotting and qRTPCR. Cells exposed to increasing concentrations of MLN4924 or 0.1% (v/v) DMSO for 24 h were pelleted for protein isolation.…”

Human c1q induces apoptosis in an ovarian cancer cell line via tumor necrosis factor pathway

“…Significance was ascertained using the unpaired one-way ANOVA test (*p < 0.05, *p < 0.01, and ***p < 0.001) (n = 3). During early apoptosis, the integrity of the cell membrane is lost and phosphatidylserine (PS) is exposed for Annexin V binding (21). A significant number of SKOV3 cells were stained positive for Annexin V, when examined under immunofluorescence microscope, suggesting that the cell membrane integrity was no longer intact as Annexin V labeled with FITC was able to bind to PS, which is usually present on the intracellular leaflet of the healthy cells.…”