Sequential degradation of proteins from the nuclear envelope during apoptosis

We have produced new antibodies specific for the integral pore membrane pro tein POM121. Using these antibodies we show that during apoptosis POM121 be comes proteolytically degraded in a caspase-dependent manner. The POM121 an tibodies and antibodies specific for other proteins of the nuclear envelope were used in a comparative study of nuclear apoptosis in staurosporine-tre ated buffalo rat liver cells. Nuclei from these cells were classified in th ree different stages of apoptotic progression: stage I, moderately condense d chromatin surrounded by a smooth nuclear periphery; stage II, compact pat ches of condensed chromatin collapsing against a smooth nuclear periphery; stage III, round compact chromatin bodies surrounded by grape-shaped nuclea r periphery. We have performed double labeling immunofluorescence microscop y of individual apoptotic cells and quantitative immunoblotting analysis of total proteins from apoptotic cell cultures. The results showed that degra dation of nuclear envelope marker proteins occurred in a specific order. PO M121 degradation occurred surprisingly early and was initiated before nucle osomal DNA degradation could be detected using TUNEL assay and completed be fore clustering of the nuclear pores. POM121 was eliminated significantly m ore rapid compared with NUP153 (a peripheral protein located in the nucleop lasmic basket of the nuclear pore complex) and lamin B (a component of the nuclear lamina). Disappearance of NUP153 and lamin B was coincident with on set of DNA fragmentation and clustering of nuclear pores. By contrast, the peripheral NPC protein p62 was degraded much later. The results suggest tha t degradation of POM121 may be an important early step in propagation of nu clear apoptosis.