Unique behaviour of NuMA during heat-induced apoptosis of lymphocytes

Nuclear collapse is a key feature of apoptosis, reflecting the DNA and protein fragmentation observed biochemically. We have compared nuclear events during spontaneous and heat-induced (42 degrees C for 30 min) apoptosis at the level of individual cells, monitoring overall chromatin organization by staining with 4,6'-diamidino-2-phenylindole (DAPI), DNA cleavage by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL), and the nuclear antigens NuMA, PI2, and lamin B by immunofluorescence microscopy. The data were correlated with analyses at the population level by flow cytometry and immunoblotting. We show that heat treatment doubles the level of apoptotic splenocytes with respect to the spontaneous apoptosis in controls within 6 honThe organization and disassembly of nuclear envelope antigens is identical during spontaneous and heat-induced apoptosis and proceeds in a temporally and spatially ordered manner relative to DNA fragmentation and chromatin collapse. In contrast, NuMA organization is affected by heat treatment, with about half of the cells containing many bright spots in addition to the usual diffuse labelling. The spots were still visible in many fully collapsed apoptotic nuclei. Further, this novel reorganization of NuMA and the hyperinduction of apoptosis by heat are correlated, suggesting that lymphocytes with reorganized NuMA are destined to undergo apoptosis. The data also indicate that DNA fragmentation precedes extensive remodelling of the nucleoplasm in these cells, and that chromatin begins to collapse before disassembly of nuclear envelope components.