Apoptosis 2004; 9: 363–368 C  2004 Kluwer Academic Publishers Degradation of GFP-labelled POM121, a non-invasive sensor of nuclear apoptosis, precedes clustering of nuclear pores and externalisation of phosphatidylserine M. Beckman, M. Kihlmark, K. Iverfeldt and E. Hallberg Department of Neurochemistry and Neurotoxicology, Stockholm University, SE-106 91 Stockholm, Sweden (M. Beckman, K. Iverfeldt); Section for Natural Sciences, S¨ odert¨ orn University College, SE-141 89 Huddinge, Sweden (M. Kihlmark, E. Hallberg) The nuclear pore membrane protein POM121 is specif- ically degraded during apoptosis by a caspase-3- dependent process enabling early detection of apoptosis in living cells expressing POM121-GFP. Here we fur- ther investigated temporal aspects of apoptotic degra- dation of POM121-GFP. We demonstrate that decreased POM121-GFP fluorescence precedes annexin V-labelling of apoptotic cells. This indicates that degradation of the nuclear pore complex starts prior to redistribution of plasma membrane phosphatidylserine, which serves as a signal for phagocytotic elimination of apoptotic cells. Furthermore, a caspase-resistant GFP-labelled mutant of POM121 resisted degradation even in late apoptosis and was detected in clustered nuclear pores. Thus, it can be concluded that loss of POM121-GFP is a specific sensor of the activation of caspase-3-dependent proteolysis at the nuclear pores. Keywords: annexin V; apoptosis; green fluorescent protein; nuclear envelope; nuclear pore; phosphatidylserine. Introduction Programmed cell death (PCD) is an active signal-induced process, which is essential not only during development but also in the adult for maintenance of tissue homeosta- sis, elimination of damaged or abnormal cells and defence against infections. 1 PCD can be induced by signals from the inside or outside environment of a cell and proceed through multiple intracellular pathways. The most fre- quent phenotype of PCD is termed apoptosis and caspases are one of the key mediators of this process. 2 Apoptotic cells display plasma membrane-associated changes involving loss of the asymmetric distribution of phospholipids which allows for phagocytes to recog- Correspondence to: Dr. Einar Hallberg, Section for Natural Sciences, S¨ odert¨ orn University College, SE-141 89 Huddinge, Sweden; Tel.: +46 (0)8 608 47 33; Fax: +46(0)8 608 45 10; e-mail: einar.hallberg@sh.se nise and engulf these cells. 3 The phospholipid phos- phatidylserine (PtdSer) is normally predominantly located to the inner leaflet of the plasma membrane. However, during apoptosis it is redistributed and exposed on the external cell surface, while the plasma membrane remains intact. This event is considered to be an early manifes- tation of apoptosis that occurs in cells from numerous lineages. 4 The translocation of PtdSer to the outer leaflet of the plasma membrane also occurs during necrosis, but is then accompanied by rupture of the plasma membrane. The nucleus of a cell undergoing apoptosis is charac- terised by chromatin condensation, DNA fragmentation and degradation of nuclear proteins. 5 The distribution of nuclear pores is also altered during apoptosis, where they appear to migrate and cluster in areas distal to con- densed chromatin. 6 POM121 (pore membrane protein of 121 kDa) is an integral membrane protein of the verte- brate nuclear pore complex (NPC). 7 We have previously shown that cleavage of endogenous POM121 occurs at aspartate 531 (DKTD 531 ) by a caspase-3-dependent pro- cess and proceeds in parallel with progressive chromatin condensation and prior to extensive nucleosomal DNA fragmentation. 8–10 POM121 was also shown to be elim- inated significantly more rapid compared to NUP153 (a peripheral protein located on the nucleoplasmic side of the nuclear pore complex) and lamin B (a component of the nuclear lamina). Disappearance of NUP153 and lamin B, in turn, was shown to coincide with both the onset of DNA fragmentation and clustering of nuclear pores. This implies that degradation of POM121 occurs before pore clustering. We have also shown that the chimeric protein POM121-GFP, consisting of the green fluorescent protein (GFP) fused to the C-terminus of rat POM121, is correctly targeted to the nuclear pores in cells with transient or con- stitutive expression of this protein. 11 Since POM121-GFP is correctly targeted and degraded synchronously with the endogenous protein during apoptosis, 9 POM121-GFP Apoptosis · Vol 9 · No 3 · 2004 363