Insights into UV-induced apoptosis: ultrastructure, trichrome stain and spectral imaging

Nuclear substructures associated with apoptosis in HeLa cells have been examined using light-microscopic morphometry, trichrome staining, spectral imaging and transmission electron microscopy. This detailed analysis reveals several sites where alterations in the normal cellular ultrastructure occur during apoptotic progression. To correlate these ultrastructural changes with the underlying molecular processes, we have characterized and quantified apoptotic cell morphology with and without inhibition of two caspases, which are key effectors of the apoptotic program. Using this analysis, early apoptotic events included: (a) the segregation of nucleolar components, a diminished granular component, and a reduction in number but increase in size of fibrillar centers, (b) an increase in the number of cytoplasmic ribosomes and (c) a very minimal increase in the amount of peripherally condensed DNA. Apoptosis progressed with: (a) an increase in the number of perichromatin granules and perichromatin fibrils, (b) a reduction in number of interchromatin granule centers concomitant with an increase in their size, (c) partial digestion and circumferential condensation of the DNA at the nuclear membrane and (d) rounding of the cytoplasm with an increase in organellar density and shrinkage in cell size. Endstage apoptotic cells showed: (a) one (or two) very large pools of incompletely digested DNA, (b) one (or two) very large interchromatin granule centers, (c) an increased number of perichromatin granules which were distanced from DNA and often closely apposed to the nucleolus, (d) formation of unusually condensed, highly coiled perinucleolar bodies and (e) blebbing of highly dense cytoplasm. In HeLa cells treated with UV and inhibitors of caspase 1 and 3, the length of time from early apoptosis to the formation of apoptotic bodies was greatly extended. Inhibiting caspase activity: (a) prevented the pooling of DNA, (b) retarded the formation of large interchromatin granule centers, (c) increased the number of perichromatin granules, (d) produced disassembly of the nucleolus, (e) prevented the formation of highly coiled perinucleolar bodies, and (f) caused vacuolization in the cell center and a unipolar blebbing of the cytoplasm. Spectral imaging in conjunction with serial section electron microscopy confirmed the staining specificities of the condensed DNA, of the large condensed interchromatin granule centers, and of the nucleoli. The results indicate that the interface between the components of the chromatin domain and the interchromatin space is a critical site of caspase activity in apoptosis, and precedes other events such as internucleosomal DNA degradation.