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Sequential degradation of proteins from the nuclear envelope during apoptosis

¹ Södertörns Högskola (University College), Box 4101, 141 04 Huddinge, Sweden
² Department of Biochemistry and Biophysics, Stockholm University, 106 91 Stockholm, Sweden

View larger version (16K): [in a new window]   Fig. 1. Characterization of new antibodies against POM121. (A) Immunostaining of monolayer BRL cells with anti-POM121 antibodies. A grazing section (a), an equatorial section (b) and the corresponding DNA staining (c) of nuclei of BRL cells are shown. Bar, 20 µm. (B) Western blotting of SDS-PAGE separated proteins of rat nuclear envelope membranes extracted with 7 M urea (7M up) or of whole cell lysates of monolayer cultures of BRL cells (BRL lyste), using anti-POM121 antibodies.

View larger version (59K): [in a new window]   Fig. 2. Proteolysis of overexpressed POM121-GFP and endogenous POM121 during apoptosis. Monolayer cultures of stably transfected neuroblastoma SH-SY5Y cells (GI97-4 cells), stably transfected PC-12 cells or BRL cells, were treated with staurosporine for different times. (A) Fluorescence micrographs showing equatorial sections of GI97-4 cells (a,b,e,f) and BRL cells (c,d,g,h), treated for 15 hours. Pairs of images of control (-STSP) and treated (+STSP) cells were acquired and processed identically. The GFP fluorescence (a,e) and the immunostaining using anti-POM121 antibodies (c,g) are shown to the left of the corresponding images of DNA staining (b,d,f,h). Bar, 20 µm. (B) Western blot analysis of POM121 and POM121-GFP. Treated (+STSP) and control (-STSP) cells were solubilized in sample buffer after 8 hours of treatment and subjected to SDS-PAGE and western blotting using anti-POM121 or anti-GFP antibodies. In apoptotic cells, overexpressed POM121-GFP () as well as endogenous POM121 () decreased. (C) Western blot analysis of overexpressed POM121-GFP () and endogenous POM121 () in stably transfected PC12 cells undergoing apoptosis (+STSP). After 8 hours of treatment 56% of the overexpressed and 47% of the endogenous protein remained. After 16 hours the corresponding figures were 14% and 17%, respectively. Data are from one representative experiment.

View larger version (56K): [in a new window]   Fig. 3. Proteolytic breakdown of POM121 during apoptosis induced by UV-irradiation. Monolayer cultures of BRL cells were exposed to UV-irradiation and incubated for 24 hours. (A) Fluorescence micrographs of UV-irradiated BRL cells showing three cells in different stages of apoptotic progression. Control cells were similar to those in Fig. 2A panels c,d. Note the almost complete lack of POM121 immunostaining in the cell with condensed chromatin in the nuclear periphery (upper right). Bar, 20 µm. (B) Western blot of SDS-PAGE separated proteins of whole cell lysates of BRL cells, exposed to different doses of UV-irradiation (as indicated).

View larger version (32K): [in a new window]   Fig. 4. Classification of apoptotic nuclei. BRL cells treated with 1 µM staurosporine for 15 hours were fixed, permeabilized and stained for DNA using BoBo-3-iodide or for lamin B before analysis by CLSM. The panels show chromatin in 0.3 µm equatorial sections of apoptotic nuclei classified as stage I-III as indicated. The extremely weak lamin B immunofluorescence signal remaining in a stage III nucleus delineating the grape-shaped nuclear periphery is also shown. Bars, 10 µm (stage I,II); 5 µm (stage III).

View larger version (84K): [in a new window]   Fig. 5. Subcellular distribution of proteins form the nuclear envelope during apoptosis. Monolayer cell cultures were incubated in the absence, -STSP (a-r) or presence, +STSP (a’-r’) of 1 µM staurosporine. After 15 hours of treatment the cells were fixed and permeabilized and subjected to double immunolabeling using the antibodies indicated. The chromatin was stained using Hoechst 33258. In stably transfected BHK cells, POM121-GFP was visualized by its own fluorescence (g,g’). The smaller panels show fields of cells. The larger panels show enlargements of control or stage II apoptotic nuclei inside the frames. Pairs of images of control and treated cells were acquired and processed identically. Control cells displayed an intense staining around the nuclear rim with all antibodies tested and nuclei with dispersed chromatin and large diameters (a-r). Apoptotic cells (a’-r’) showed nuclei with condensed chromatin and a smaller nuclear diameter. The immunolabeling of the periphery of apoptotic nuclei was weaker or absent. However, the immunofluorescence using mAb414 antibodies was retained (b’,j’,m’) in most apoptotic cells, although it frequently displayed a clustered distribution (j’). Bar, 20 µm.

View larger version (70K): [in a new window]   Fig. 6. High resolution confocal images of NPC clustering. BRL cells were incubated with 1 µM staurosporine for 15 hours and subjected to immunostaining using mAb414 and/or anti-lamin B antibodies. DNA was visualized using BoBo-3-iodide. (A) Distribution of nuclear pore proteins (green) and lamin B (red) in 0.3 µm equatorial sections of nuclei in different stages of apoptotic progression. The right panels show enhanced versions of the merged mAb414 and anti-lamin B staining in order to better visualize the weak lamin B staining. Note the interrupted and accumulated pattern of mAb414 immunofluorescence in stage II and III. Bar, 5 µm. (B) Distribution of nuclear pore proteins (green) and chromatin (red) in (0.3 µm) sections of stage II and III nuclei, respectively. Bar, 10 µm. (C) A projection of a stack of optical sections of a nucleus between stage II and III that had been stained with mAb414 antibodies (green) and Bobo-3-iodide (red).

View larger version (22K): [in a new window]   Fig. 7. Sequential proteolysis of proteins from the nuclear envelope in cell cultures undergoing apoptosis. (A) BRL cells were treated with 1 µM staurosporine for different times. Equivalent amounts of total proteins of control or treated cell cultures were separated by SDS-PAGE and analyzed by western blotting using anti-POM121, mAb414 (NUP153 and p62) or anti-lamin B antibodies. The panel of western blots is representative of one experiment. (B) Mean t50 values of quantified western blots from four separate experiments. Bars denoted with different letters are significantly different (a-c). (C) BRL cells were treated with 1 µM staurosporine and the caspase inhibitor was added either 1 hour before (1h inhibitor +STSP), or 1 hour after addition of staurosporine (1 h STSP +inhibitor) or cells were incubated with staurosporine alone (STSP). The panel of western blots is representative of one experiment.

View larger version (75K): [in a new window]   Fig. 8. Distribution of gp210 in apoptotic cells lacking POM121. BRL cells were treated with 1 µM staurosporine for 15 hours and subjected to double immunolabeling using antibodies specific for POM121 (a,d) and gp210 (b,e), respectively. DNA was visualized using Hoechst 33258 (c,f). Control cells (a-c) displayed a punctate fluorescence pattern along the nuclear rim. The gp210 antibodies also gave rise to a background staining seen as small dots in the nucleoplasm. In staurosporine-treated cells (d-f) both antibodies gave rise to a bright peripheral immunostaining of a type I cell (left), reminiscent of control cells. By contrast, the nuclear periphery of an early type II cell (right) only stained positive for gp210 (e), whereas POM121 was absent (d). Bar, 20 µm.

View larger version (19K): [in a new window]   Fig. 9. Sequential steps in nuclear envelope breakdown during apoptosis.