Sequential degradation of proteins from the nuclear envelope during apoptosis.
We have produced new antibodies specific for the integral pore membrane protein POM121. Using these antibodies we show that during apoptosis POM121 becomes proteolytically degraded in a caspase-dependent manner. The POM121 antibodies and antibodies specific for other proteins of the nuclear envelope were used in a comparative study of nuclear apoptosis in staurosporine-treated buffalo rat liver cells. Nuclei from these cells were classified in three different stages of apoptotic progression: stage I, moderately condensed chromatin surrounded by a smooth nuclear periphery; stage II, compact patches of condensed chromatin collapsing against a smooth nuclear periphery; stage III, round compact chromatin bodies surrounded by grape-shaped nuclear periphery. We have performed double labeling immunofluorescence microscopy of individual apoptotic cells and quantitative immunoblotting analysis of total proteins from apoptotic cell cultures. The results showed that degradation of nuclear envelope marker proteins occurred in a specific order. POM121 degradation occurred surprisingly early and was initiated before nucleosomal DNA degradation could be detected using TUNEL assay and completed before clustering of the nuclear pores. POM121 was eliminated significantly more rapid compared with NUP153 (a peripheral protein located in the nucleoplasmic basket of the nuclear pore complex) and lamin B (a component of the nuclear lamina). Disappearance of NUP153 and lamin B was coincident with onset of DNA fragmentation and clustering of nuclear pores. By contrast, the peripheral NPC protein p62 was degraded much later. The results suggest that degradation of POM121 may be an important early step in propagation of nuclear apoptosis.
Kihlmark
M
M
Södertörns Högskola (University College), Box 4101, 141 04 Huddinge, Sweden.
Imreh
G
G
Hallberg
E
E
D000818:Animals; D017209:Apoptosis / Q000502:physiology* / Q000528:radiation effects; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D034921:Lamin Type B; D034882:Lamins; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D009685:Nuclear Envelope / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D051381:Rats; D011915:Rats, Inbred BUF; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D034921:Lamin Type B; D034882:Lamins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; C513083:Nup153 protein, rat; C082264:Pom121 protein, rat; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019311:Staurosporine