Molecular details of membrane fluidity changes during apoptosis and relationship to phospholipase A2 activity

Abstract

Summary Secretory phospholipase A2 exhibits much greater activity toward apoptotic versus healthy cells. Various plasma membrane changes responsible for this phenomenon have been proposed, including biophysical alterations described as “membrane fluidity” and “order.” Understanding of these membrane perturbations was refined by applying studies with model membranes to fluorescence measurements during thapsigargin-induced apoptosis of S49 cells using probes specific for the plasma membrane: Patman and trimethylammonium-diphenylhexatriene. Alterations in emission properties of these probes corresponded with enhanced susceptibility of the cells to hydrolysis by secretory phospholipase A2. By applying a quantitative model, additional information was extracted from the kinetics of Patman equilibration with the membrane. Taken together, these data suggested that the phospholipids of apoptotic membranes display greater spacing between adjacent headgroups, reduced interactions between neighboring lipid tails, and increased penetration of water among the heads. The phase transition of artificial bilayers was used to calibrate quantitatively the relationship between probe fluorescence and the energy of interlipid interactions. This analysis was applied to results from apoptotic cells to estimate the frequency with which phospholipids protrude sufficiently at the membrane surface to enter the enzyme’s active site. The data suggested that this frequency increases 50–100-fold as membranes become susceptible to hydrolysis during apoptosis.

Synergistic Effects of Secretory Phospholipase A2from the Venom ofAgkistrodon piscivorus piscivoruswith Cancer Chemotherapeutic Agents

“…Prior investigations by our lab using calcium ionophore, glucocorticoid, and thapsigargin have successfully identified several biophysical changes that occur in the cellular membrane during apoptosis [3, 4, 7–9]. The results of this current study confirm that these changes are a generalizable feature of programmed cell death that occur regardless of the type of apoptotic inducer.…”

“…Methotrexate, paclitaxel, actinomycin D, and daunorubicin induce cell death by disparate means; however, in each case, the interlipid spacing and fluidity of the membrane increased compared to the control. The idea is that these physical changes improve the likelihood of vertical movements of phospholipids in the membrane sufficient for individual phospholipids to enter the active site of adsorbed enzyme [3, 4, 7, 10–12]. For example, a recent study on thapsigargin-treated cells estimated that the chance of such lipid protrusions from the membrane increases approximately 100 fold when TMA-DPH anisotropy decreases to levels comparable to those displayed in Figure 4 [7].…”

“…The idea is that these physical changes improve the likelihood of vertical movements of phospholipids in the membrane sufficient for individual phospholipids to enter the active site of adsorbed enzyme [3, 4, 7, 10–12]. For example, a recent study on thapsigargin-treated cells estimated that the chance of such lipid protrusions from the membrane increases approximately 100 fold when TMA-DPH anisotropy decreases to levels comparable to those displayed in Figure 4 [7]. In each case, the changed biophysical state of the membrane appeared to render it vulnerable to attack by snake venom sPLA 2 as evidenced by the emergence of a significant population of cells that were alive and susceptible to hydrolysis.…”

Optimization of microelectrophoresis to select highly negatively charged sperm

“…During apoptosis in sperm, the integrity of the membrane is lost due to the translocation of phosphatidylserine [53], during which the phosphatidylserine is shifted from the interior to the exterior of the plasma membrane [54,55]. In addition, apoptosis results in DNA fragmentation and degradation of cytoskeletal and nuclear proteins (reviewed by Elmore [56],), and affects fluidity of the plasma membrane [57]. The externalization of phosphatidylserine and loss of membrane fluidity may also facilitate the loss of epididymal glycoproteins anchored to the membrane, thereby causing a loss in negative charge.…”

Relationships between membrane water molecules and Patman equilibration kinetics at temperatures far above the phosphatidylcholine melting point

“…We have considered the hypothesis proposed above (faster water rotation) as well as an alternative that these effects result instead from increases in the number and/or penetration depth of bilayer water molecules. We take advantage of recent observations from kinetic analyses of probe equilibration suggesting that these naphthalene derivatives may reside in at least two configurations in the membrane and that one can learn additional detail about membrane dynamics by considering the properties of those configurations [20,21]. For such studies, Patman appears ideally suited [20], and we have therefore focused our attention on the behavior of that probe with unilamellar vesicles of various saturated and unsaturated phosphatidylcholines.…”