Relationship between Membrane Fluidity Changes, Phospholipid Protrusion Probability and Phospholipase A2 Activity during Thapsigargin-Induced Apoptosis

Abstract

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 lymphoma cells using probes specific for the plasma membrane: Patman and trimethylammonium-diphenylhexatriene. Alterations in emission spectra or anisotropy of these probes corresponded with enhanced susceptibility of the cells to hydrolysis by secretory phospholipase A2. Furthermore, these alterations appeared to correlate temporally with fragmentation of actin filaments detected by confocal microscopy of phalloidin fluorescence. 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.