During apoptosis, physical changes in the plasma membrane prepare the cell for clearance by phagocytes and hydrolysis by secretory phospholipase A(2) (sPLA(2)). The relationships among these changes have not been adequately established, especially for hormone-stimulated apoptosis. This study addresses these issues for glucocorticoid-induced apoptosis in S49 lymphoma cells. Flow cytometry, microscopy, and fluorescence spectroscopy were used to assess merocyanine 540 emission, laurdan generalized polarization, phosphatidylserine exposure, caspase activation, and membrane permeability to propidium iodide in the absence and presence of sPLA(2). The earliest event observed was activation of cellular caspases. Results with membrane probes suggest that interlipid spacing also increases early during apoptosis and precedes transbilayer migration of phosphatidylserine, DNA fragmentation, and a general increase in lipid order associated with blebbing and dissolution of the cells. The activity of sPLA(2) appeared to be linked more to lipid spacing than to loss of membrane asymmetry. The early nature of some of these events and their ability to promote activity of a proinflammatory enzyme suggests the possibility of an inflammatory response during T-lymphocyte apoptosis.

Some isoforms of secretory phospholipase A 2 (sPLA 2 ) distinguish between healthy and damaged or apoptotic cells. This distinction reflects differences in membrane physical properties. Because various sPLA 2 isoforms respond differently to properties of artificial membranes such as surface charge, they should also behave differently as these properties evolve during a dynamic physiological process such as apoptosis. To test this idea, S49 lymphoma cell death was induced by glucocorticoid (6 -48 h) or calcium ionophore. Rates of membrane hydrolysis catalyzed by various concentrations of snake venom and human groups IIa, V, and X sPLA 2 were compared after each treatment condition. The data were analyzed using a model that evaluates the adsorption of enzyme to the membrane surface and subsequent binding of substrate to the active site. Results were compared temporally to changes in membrane biophysics and composition. Under control conditions, membrane hydrolysis was confined to the few unhealthy cells present in each sample. Increased hydrolysis during apoptosis and necrosis appeared to reflect substrate access to adsorbed enzyme for the snake venom and group X isoforms corresponding to weakened lipid-lipid interactions in the membrane. In contrast, apoptosis promoted initial adsorption of human groups V and IIa concurrent with phosphatidylserine exposure on the membrane surface. However, this observation was inadequate to explain the behavior of the groups V and IIa enzymes toward necrotic cells where hydrolysis was reduced or absent. Thus, a combination of changes in cell membrane properties during apoptosis and necrosis capacitates the cell for hydrolysis differently by each isoform.During programmed cell death, or apoptosis, a variety of changes occur in the plasma membrane of the cell. These include morphological alterations that emerge late in the process such as blebbing and increased permeability of the membrane. Earlier in the process, several more subtle membrane changes occur. The best studied is a loss of the normal asymmetrical transmembrane distribution of phospholipid species. Consequently, anionic lipids like phosphatidylserine, which are typically confined to the inner leaflet of the membrane, become exposed on the outer surface (1). In addition, studies with fluorescent membrane probes have revealed possible increases in fluidity and/or the spacing between lipid molecules that may precede or coincide with the loss of membrane asymmetry, depending on the cell type and mode of apoptosis (2-9). Recently, a latent increase in the order of membrane lipids has also been reported (9).A potential consequence of these events during apoptosis is enzymatic attack of the cell membrane by secretory phospholipase A 2 (sPLA 2 ).2 Ordinarily, healthy cells resist hydrolysis, but during apoptosis they become vulnerable to destruction by the enzyme (9 -11). Studies with snake venom phospholipase A 2 have identified possible ways by which this phenomenon relates to membrane physical properties (8,9,12). Prelimina...

Exposure of human erythrocytes to elevated intracellular calcium causes fragments of the cell membrane to be shed as microvesicles. This study tested the hypothesis that microvesicle release depends on microscopic membrane physical properties such as lipid order, fluidity, and composition. Membrane properties were manipulated by varying the experimental temperature, membrane cholesterol content, and the activity of the trans-membrane phospholipid transporter, scramblase. Microvesicle release was enhanced by increasing the experimental temperature. Reduction in membrane cholesterol content by treatment with methyl-beta-cyclodextrin also facilitated vesicle shedding. Inhibition of scramblase with R5421 impaired vesicle release. These data were interpreted in the context of membrane characteristics assessed previously by fluorescence spectroscopy with environment-sensitive probes such as laurdan, diphenylhexatriene, and merocyanine 540. The observations supported the following conclusions: 1) calcium-induced microvesicle shedding in erythrocytes relates more to membrane properties detected by diphenylhexatriene than by the other probes; 2) loss of trans-membrane phospholipid asymmetry is required for microvesicle release.PACS Codes: 87.16.dj, 87.16.dt.

Accumulating evidence implicates the voltage-dependent anion channel (VDAC) as functioning in mitochondria-mediated apoptosis involving cytochrome c release, leading to caspases activation and apoptosis. The mechanisms regulating cytochrome c release and the molecular architecture of the cytochrome c conducting channel remain unknown. Previously, we demonstrated that apoptosis induction was accompanied by VDAC oligomerization, as revealed by cross-linking and directly monitored in living cells using Bioluminescence Resonance Energy Transfer technology. Moreover, apoptosis inhibitors inhibited VDAC oligomerization and a correlation between the levels of VDAC oligomerization and apoptosis was observed. Here, we combined sitedirected mutagenesis with chemical cross-linking to reveal the contact sites between VDAC1 molecules in dimers and higher oligomers. Replacing hydrophobic amino acids with charged amino acids in b-strands 1,2 and 19, but not 14, interfered with VDAC1 oligomerization and apoptosis induction. Cysteine cross-linking results, from introducing cysteine at a defined position in cysteineless VDAC1 and applying the cysteine-specific cross-linker, BMOE, supported the close vicinity of b-strands 1,2 and 19 in VDAC1 dimer. Moreover, the results suggest that VDAC1 exists as a dimer that undergoes conformational changes upon apoptosis induction to assemble into a higher oligomeric state. Additionally we demonstrated that the N-terminal region of VDAC1 lies inside the pore, but could also move and interact with the N-terminus from a second molecule to form a dimer. Our results suggest that the glycine rich sequence 21-GYGFG-25 is involved in the N-terminus translocation from the internal pore to the channel face. These results provide structural insight into cellular VDAC1's oligomeric state and its N-terminal region location and translocation.

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