Appearance of Phosphatidylserine on Apoptotic Cells Requires Calcium-mediated Nonspecific Flip-Flop and Is Enhanced by Loss of the Aminophospholipid Translocase

Abstract

Phosphatidylserine (PS), ordinarily sequestered in the plasma membrane inner leaflet, appears in the outer leaflet during apoptosis, where it triggers non-inflammatory phagocytic recognition of the apoptotic cell. The mechanism of PS appearance during apoptosis is not well understood but has been associated with loss of aminophospholipid translocase activity and nonspecific flip-flop of phospholipids of various classes. The human leukemic cell line HL-60, the T cell line Jurkat, and peripheral blood neutrophils, undergoing apoptosis induced either with UV irradiation or anti-Fas antibody, were probed in the cytofluorograph for (i) surface PS using fluorescein isothiocyanate-labeled annexin V, (ii) PS uptake by the aminophospholipid translocase using [6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl] (NBD)-labeled PS, (iii) nonspecific uptake of phospholipids (as a measure of transbilayer flip-flop) using NBDlabeled phosphatidylcholine, and (iv) the appearance of hypodiploid DNA. In all three types of cells undergoing apoptosis, the appearance of PS followed loss of aminophospholipid translocase and was accompanied by nonspecific phospholipid flip-flop. Importantly, however, in the absence of extracellular calcium, the appearance of PS was completely inhibited despite DNA fragmentation and loss of aminophospholipid translocase activity, the latter demonstrating that loss of the translocase is insufficient for PS appearance during apoptosis. Furthermore, while both the appearance of PS and nonspecific phospholipid uptake demonstrated identical extracellular calcium requirements with an ED 50 of nearly 100 M, the magnitude of PS appearance depended on the level of aminophospholipid translocase activity. Taken together, the data strongly suggest that while nonspecific flip-flop is the driving event for PS appearance in the plasma membrane outer leaflet, aminophospholipid translocase activity ultimately modulates its appearance.The appearance of phosphatidylserine in the outer leaflet of the plasma membrane appears to be a universal phenomenon in cells undergoing apoptosis, or programmed cell death (1). Importantly, outer leaflet PS likely serves as a signal in tissues for the noninflammatory engulfment of apoptotic cells (Ref. 2 and see "Discussion"), a distinctly different response than the pro-inflammatory events following tissue necrosis. While phosphatidylserine (PS) 1 is actively transported from the outer to the inner leaflet by the aminophospholipid translocase (3-5), the mechanism(s) by which PS appears in apoptosis is not well understood. Verhoven et al. (3) reported that PS appearance was accompanied by both loss of aminophospholipid translocase activity and enhanced nonspecific transbilayer movement of phospholipids, perhaps due to activation of a "scramblase." Hence, they proposed that PS may become detectable in the outer leaflet due to the combination of these two events. However, to date, the relative contribution of these two events has not been clarified. Additionally, it is not known what role...

Chondrocytes Utilize a Cholesterol-Dependent Lipid Translocator To Externalize Phosphatidylserine

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“…PLSCR1 appeared to localize to the plasma membrane in prehypertrophic (day 3) chondrocytes and a portion redistributed to the nucleus as the cells underwent late hypertrophy ( Figure 3B). As shown in Figure 3C, PLSCR1 protein levels in maturing chondrocytes remained constant.Lipid translocator activity was measured by following NBD fluorescent lipid uptake using flow cytometry (36,41,55). This well-characterized assay measures APLT activity as uptake of NBD-PS and scramblase activity as uptake of PC.…”

Apoptosis in the pathogenesis of systemic lupus erythematosus

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Cinétique et caractérisation du phénomène “apoptosis-like” induit au cours de la congélation des spermatozoïdes bovins

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