Fig. 1.

Redistribution of histone 2B-green fluorescent protein (H2B^GFP) during ultraviolet (UV)-B-induced apoptosis in HeLa cells. Adherent HeLa cells expressing fluorescent H2B^GFP histone were cultured to a density of 3 × 10⁴ cells/cm². After UV-B irradiation with 900 mJ/cm² the distribution of H2B^GFP was monitored by video-microscopy. (a–d) The characteristic nuclear changes that had been described for adherent cells undergoing apoptosis. After 270 min the cell marked with ‘1’ had undergone waves of formation of apoptotic cell-derived membranous vesicles (ACMV). None of these contained detectable amounts of H2B^GFP; 30 min later two of the vesicles were filled with substantial amounts of H2B^GFP (arrows). Note that (1) not all ACMV contain degraded chromatin (H2B^GFP); (2) ACMV are formed initially without nuclear material, and are loaded in a second step (arrows). (e,f) Some large ACMV^L remain unloaded even 10 h after their formation. (g,h) In very late phases of apoptosis some ACMV^L which had been loaded with H2B^GFP disintegrate and release their load into the culture supernatant, most probably during secondary necrosis. (i) mean fluorescence intensity (MFI) of the nuclei during the 10 h after irradiation. Note a significant increase of the MFI starting 6 h after irradiation (Student's t-test of cellular MFI at t = 0 versus t = × hours after irradiation). (j) nuclear area during the 10 h after irradiation. Note a significant shrinkage of the nuclei 9 h after irradiation (Student's t-test of cellular nuclear area at t = 0 versus t = × hours after irradiation).