PLASMA-MEMBRANE ALTERATIONS AND CYTOSKELETAL CHANGES IN APOPTOSIS

During apoptosis, one of the first membrane changes that can be detect ed is exposure of phosphatidylserine residues at the outer plasma memb rane leaflet, while early apoptosis is also accompanied by changes in the cytoskeletal organization. In this study we investigated the relat ionship between these two phenomena during olomoucine- and rescovitin- induced apoptosis in human lung cancer and neuroblastoma cell lines. L oss of membrane asymmetry was detected by biotin-labeled or FITC-label ed annexin V binding to negatively charged phosphatidylserine, while c ytoskeletal components were visualized by immunocytochemistry. The apo ptotic, annexin V-positive, cells were analyzed by flow cytometry, con focal scanning laser microscopy, and Western blotting. We report that cytokeratin and vimentin aggregation in early apoptosis occurs simulta neously with phosphatidylserine exposure and chromatin condensation. I n contrast to these intermediate filament proteins, which were disasse mbled and proteolytically cleaved in early apoptosis, microfilaments a nd microtubuli were not proteolytically degraded but were found to be present as aggregated filaments in the apoptotic bodies. We also show that loss of membrane asymmetry and cytokeratin aggregation are indepe ndent processes, since N-ethylmaleimide-induced phosphatidylserine exp osure does not cause cytokeratin disassembly. Vice versa, phorbol 12-m yristate 13-acetate-induced cytokeratin filament aggregation does not result in phosphatidylserine exposure. (C) 1997 Academic Press.