Phosphatidylserine Externalization during CD95-induced Apoptosis of Cells and Cytoplasts Requires ICE/CED-3 Protease Activity

Abstract

Phosphatidylserine (PS), a lipid normally confined to the inner leaflet of the plasma membrane, is exported to the outer plasma membrane leaflet during apoptosis to serve as a trigger for recognition of apoptotic cells by phagocytes. The mechanism of PS export during apoptosis is not known nor is it clear whether the nuclear changes that typify apoptosis contribute in any way to this event. Here, we demonstrate that ligation of the CD95 (Fas/APO-1) molecule on Jurkat cytoplasts induces dramatic PS externalization similar to that observed during apoptosis of intact cells. Apoptosis of both cells and cytoplasts was associated with proteolytic processing of CPP32, a member of the interleukin-1␤ converting enzyme (ICE)/CED-3 protease family, to its active form. Fodrin, a component of the cortical cytoskeleton, also underwent proteolytic cleavage during apoptosis of both cytoplasts and intact cells. Strikingly, CPP32 activation, fodrin proteolysis, and PS externalization were all inhibited in the presence of peptide inhibitors of ICE/CED-3 family proteases. These data provide strong support for the notion that the cell death machinery is extranuclear and is likely to be comprised of one or more members of the ICE/CED-3 family and that activation of this machinery does not require nuclear participation.Apoptosis is a mode of cell death known to be under molecular control and is central to numerous physiological and pathological processes where cells are eliminated (1-3). Although the most prominant morphological changes that occur during apoptosis typically involve the cell nucleus, studies using cytoplasts have demonstrated that the nucleus is not required for the dramatic membrane blebbing events and the subsequent loss of viability that occurs during this process (4 -6). However, it is unclear whether enucleated cells are truly apoptotic, that is, whether they can be recognized as such by macrophages and other phagocytes, as apoptotic cells are known to be (7,8). It is formally possible that although cytoplasts undergo plasma membrane blebbing and other gross features of apoptosis, they may not acquire the membrane changes that would stimulate uptake of these cells by phagocytes.Two major mechanisms appear to exist for the recognition of apoptotic cells by phagocytes; one mediated by a receptor that engages PS 1 exported to the plasma membrane during apoptosis and the other mediated by a vitronectin receptor/CD36/ thrombospondin interaction that sees an as yet unidentified ligand on the apoptotic cell (9, 10). These two mechanisms appear to be mutually exclusive because macrophages that express the PS receptor do not appear to be capable of utilizing the vitronectin receptor-associated pathway (11). Thus far, the molecular events underlying these plasma membrane changes remain unknown.Recent evidence indicates that members of the emerging ICE/CED-3 family of proteases may occupy a critical position in the cellular apparatus that effects the destructive changes within the cell during apoptosis (see Refs. 12-14 for r...

Bcl-2 family proteins are essential for platelet survival

“…Previous studies have shown that apoptotic agents promote morphological changes in anucleated cytoplast that resemble characteristics of programmed cell death. 23,24 In platelets, the morphological changes of apoptosis closely resemble those of activation, including PS externalization, microparticle release (i.e., apoptotic bodies), and cell shrinkage. Platelet aging is also characterized by PS externalization; however, it is unclear whether this is reflective of an apoptotic process or an incomplete platelet activation event.…”

The CASBAH: a searchable database of caspase substrates

“…A number of alterations to the composition of the membranes of apoptotic cells have been documented, 54 perhaps the most well known of which is the externalisation of phosphatidylserine on the outer leaflet of the plasma membrane. 55 Although this event is also blocked by inhibition of caspase activity, 56 it remains unclear how phosphatidylserine externalisation or other apoptosis-associated membrane events, is regulated by caspases. In addition to becoming licensed for removal by phagocytes, apoptotic cells may also actively attract the attentions of such cells by secreting molecules with chemotactic properties.…”

In vitro measurement of cell death with the annexin A5 affinity assay

“…Expression of PS relies on activated caspase 3 in CD95-induced apoptotic cell death, whereas cathepsin B activity is necessary for PS expression in tumor necrosis factor-α-induced caspase-independent cell death 8,9 . Several studies have shown that pathways exist that cause cell surface expression of PS by living cells in specific conditions.…”

“…Activated caspases can be detected by western blot with antibodies to cleaved caspase fragments and by flow cytometry using fluorescent probes 17,18 . Caspases are activated early in the apoptotic process before PS expression, so with this method it is possible to detect cells at the onset of apoptosis 8 . Assessment of caspase activity consumes more time than the annexin A5 affinity assay does.…”