Correlation of mammalian cell killing by heat shock to intramembranous particle aggregation and lateral phase...

Heat shock induces a dose-dependent increase in the fraction of Chinese hamster ovary cells that stain the fluorescent membrane probe N-epsilon-dansyl-L-lysine (DL). Dansyl lysine has previously been shown to select for cholesterol-free membrane domains in phospholipid liposomes. We found that the fraction of cells excluding DL could be closely correlated to cell survival as assayed by 37 degrees C incubation following heat treatment. Fluorescence-activated cell sorting indicated that essentially all of the DL-staining cells were nonviable. Freeze fracture electron microscopy of sorted cells showed that all the cells that stained with DL also had highly suggested intramembranous particle (IMP) aggregation while DL-excluding cells did not. Furthermore, IMP aggregation was shown to occur immediately after heat shock and to precede DL staining. Treatment with other membrane-active agents such as ethanol, amphotericin B, filipin, procaine, and lidocaine (i) induced DL staining that was closely correlated to survival, (ii) induced dramatic cytotoxic sensitization when combined with heat, and (iii) induced aggregated IMPs at relevant cytotoxic concentrations. Several nonmembrane-active agents were examined; none induced DL staining, dramatic cytotoxic sensitization, or IMP aggregation. These results raise the possibility that heat shock inactivates mammalian cells primarily via nonspecific aggregation and denaturation of membrane proteins resulting in a lateral phase separation of membrane components, including the generation of phospholipid domains.