Membrane Changes during Apoptosis: Part of the Process or Characteristics of the Corpse?

Abstract

Thapsigargin-induced apoptosis in S49 lymphoma cells causes biophysical changes in the plasma membrane. Thapsigargin (TG) is a sarco/endoplasmic reticulum Ca2+ ATPase inhibitor that causes depletion of intracellular calcium stores and increased cytosolic calcium levels. It is used to model endoplasmic reticulum stress-induced apoptosis. This study focuses on membrane physical changes involved in this specific apoptotic pathway and possible cellular mechanisms that could account for these alterations. The fluorescent probes merocyanine 540 (MC540), laurdan, patman, and diphenylhexatriene (DPH) were used to assess lipid spacing, order, and fluidity using fluorescence spectroscopy, two-photon excitation microscopy, and confocal microscopy. MC540 fluorescence intensity increased throughout the apoptotic process, suggesting an increase in interlipid spacing. Two-photon microscopy images with laurdan showed a transient reduction in membrane order. Alexa Fluor-labeled annexin V was used to assess phosphatidylserine exposure in the outer leaflet of the plasma membrane. Flow cytometry experiments showed a sharp increase in the population showing this flip-flop after 2.5 h incubation with TG. Susceptibility of the cells to secretory phospholipase A2 (sPLA2), a hydrolytic enzyme that can distinguish apoptotic membranes from healthy ones, was also evaluated by measuring membrane permeability to propidium iodide. Two hours after addition of TG, a small population of cells became susceptible to sPLA2, and that population increased steadily with longer incubations. All of these alterations in the plasma membrane were compared temporally with caspase activation using a fluorescently labeled caspase inhibitor, FAM-VAD-fmk. This comparison suggested that caspase activation, susceptibility to sPLA2, and decreased lipid order detected by laurdan precede PS exposure, elevated MC540 fluorescence, and eventual cellular demise. Experiments are in progress with multiple pharmacological agents to assess cause and effect relationships among these events and a possible role of ceramide.