Partial cleavage of A-type lamins concurs with their total disintegration from the nuclear lamina during apoptosis.
Although activated caspase 6 is capable of cleaving both A- and B-type lamins during apoptosis, the higher-order structure of the nuclear lamina may cause a differential breakdown of these two types of lamins. In order to obtain a better understanding of the dynamics and the consequences of the rapid, coordinated breakdown of the lamina complex, we applied the green fluorescent protein (GFP) technology in living cells, in which the fate of individual caspase cleavage fragments of A- and B-type lamins was examined. CHO-K1 cells were stably transfected with cDNA constructs encoding N-terminally GFP-labelled hybrids of lamin A, lamin Adelta10, lamin C or lamin B1. The course of the apoptotic process, induced by the kinase inhibitor staurosporine or by the proteasome inhibitor MG132, was monitored by digital imaging microscopy or confocal microscopy. Time-lapse recordings showed that parallel to DNA condensation N-terminally GFP-tagged A-type lamins became diffusely dispersed throughout the nucleoplasm and rapidly translocated to the cytoplasm. In contrast, the majority of GFP-lamin B1 signal remained localised at the nuclear periphery, even after extensive DNA condensation. Comparison of lamin B1-GFP signal with A-type lamin antibody staining in the same apoptotic cells confirmed the temporal differences between A- and B-type lamina dispersal. Immunoblotting revealed only a partial cleavage of A-type lamins and an almost complete cleavage of lamin B1 during apoptosis. In contrast to lamin B1 in normal cells, this cleaved lamin B1, which is apparently still associated with the nuclear membrane, can be completely extracted by methanol or ethanol. Fluorescence loss of intensity after photobleaching experiments showed that in apoptotic cells A-type lamin-GFP molecules diffuse almost freely in both nucleoplasm and cytoplasm, while the lamin B1-GFP fragments remain more stably associated with the nuclear membrane, which is confirmed by co-localisation immunofluorescence studies with a nucleoporin p62 antibody. Our results therefore clearly show a differential behaviour of A- and B-type lamins during apoptosis, suggesting not only distinct differences in the organisation of the lamina filaments, but also that caspase cleavage of only a small fraction of A-type lamins is needed for its complete disintegration.
Broers
Jos L V
JL
Department of Molecular Cell Biology, Research Institute Growth & Development (GROW), University of Maastricht, Maastricht, The Netherlands. jos.broers@molcelb.unimaas.nl
Bronnenberg
Nancy M H J
NM
Kuijpers
Helma J H
HJ
Schutte
Bert
B
Hutchison
Christopher J
CJ
Ramaekers
Frans C S
FC
D021581:Active Transport, Cell Nucleus / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D016466:CHO Cells; D053178:Caspase 6; D020169:Caspases / Q000378:metabolism; D006224:Cricetinae; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D004058:Diffusion / Q000187:drug effects; D005057:Eukaryotic Cells; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins; D034904:Lamin Type A / Q000235:genetics / Q000378:metabolism*; D034921:Lamin Type B / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins; D009685:Nuclear Envelope / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins
D034904:Lamin Type A; D034921:Lamin Type B; D008164:Luminescent Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D053178:Caspase 6; D020169:Caspases