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First published online 11 December 2002
doi: 10.1242/jcs.00227

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NuMA and nuclear lamins behave differently in Fas-mediated apoptosis

¹ Department of Pathology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland
² Turku Graduate School of Biomedical Sciences, Turku, Finland

View larger version (44K): [in a new window]   Fig. 1. Immunofluorescence microscopy of cultured Jurkat T cells during Fas-mediated apoptosis. Cells grown in suspension were untreated (A-E) or treated (F-J) with Fas antibody for 120 minutes, fixed and double-stained with NuMA (A and F) and lamin B (B and G) primary antibodies and FITC-conjugated rabbit-anti-mouse (A and F) and TRITC-conjugated rabbit-anti-goat (B and G) secondary antibodies. Samples were counterstained for DNA with Hoechst (D and I). Note the uneven condensed distribution of NuMA and wrinkled distribution of lamin B in early apoptotic cell (arrow) and the presence of NuMA and lamin B around fragmented nuclei in apoptotic bodies (arrowheads). Bar, 5 µm.

View larger version (47K): [in a new window]   Fig. 2. The cleavage of NuMA, lamin B and poly(ADP-ribose) polymerase-1 (PARP-1) during Fas-mediated apoptosis in Jurkat T cells. Jurkat T cells were treated with Fas antibody and prepared for SDS-PAGE at different time points as indicated. Identically loaded gels were run and immunoblotted with NuMA, lamin A/C, lamin B or PARP-1 antibody. Arrowheads indicate the cleavage products of NuMA, lamin B and PARP-1. Lamin A/C antibody does not detect lamin A or C in Jurkat T cells. Fas-antibody- and PD 98059-treated and untreated HeLa cells were used as controls (both 36 hours, see later).

View larger version (56K): [in a new window]   Fig. 3. Localization of NuMA, p85 fragment of PARP-1 and cleaved caspase-3 in cultured HeLa cells during Fas-mediated apoptosis. Cells grown on glass coverslips were treated with Fas antibody and PD 98059 for 16 hours and double-stained with NuMA (A) and the p85 fragment of PARP-1 (B) or NuMA (F) and cleaved caspase-3 (G) antibodies. FITC-conjugated goat-anti-mouse (A and F) and TRITC-conjugated goat-anti-rabbit (B and G) secondary antibodies were used. Samples were counterstained for DNA with Hoechst (D and I). In apoptotic cells, NuMA is condensed or localized outside the fragmented nuclei within the apoptotic bodies (A and F, arrows). Note that these cells are positive for cleaved p85 fragment of PARP-1 and cleaved caspase-3 (B and G). Bar, 10 µm.

View larger version (21K): [in a new window]   Fig. 8. The amount of apoptotic, atypical apoptotic and NuMA negative nuclei in the presence of caspase inhibitors. HeLa cells grown on glass coverslips were treated with Fas antibody and PD 98059 for 24 hours in the presence or absence of 100 µM z-DEVD-FMK, z-VEID-FMK or z-IETD-FMK and processed for immunofluorescence microscopy analyses. 800-1200 adherent cells in randomly selected areas were counted for each coverslip. The data represent mean values and standard deviations from three separate experiments. Note the correlation between the numbers of atypical apoptotic and NuMA negative nuclei.

View larger version (86K): [in a new window]   Fig. 4. Confocal microscope analysis of cultured HeLa cells during Fas-mediated apoptosis. Cells grown on glass coverslips were untreated (A-E) or treated with Fas antibody and PD 98059 for 24 hours (F-T) and prepared for immunofluorescence microscopy. Samples were double-stained with NuMA (A, F and K) and lamin B (B, G and L) or lamin A/C (P) and lamin B (Q) antibodies. FITC-conjugated rabbit-anti-mouse (A, F, K and P) and TRITC-conjugated rabbit-anti-goat (B, G, L and Q) secondary antibodies were used. Samples were counterstained for DNA with Hoechst (D, I, N and S; Hoechst stain is shown in glow color). In the beginning of apoptosis NuMA condenses in the center of the nucleus (F) and excludes the condensed chromatin in the nuclear periphery (arrows). Lamin B shows a folded staining pattern (G). Note that lamin B staining shows the upper surface of the nuclear lamina in a normal interphase cell. At the end of apoptosis, NuMA partially encircles the NuMA-negative nuclear fragments (arrows) within apoptotic bodies (K). Lamin B remains around NuMA and the fragmented nuclei (L). Lamin A/C colocalizes with lamin B (P-R). Transmission views show typical apoptotic features including shrinking of the cell and nucleus, cytoplasmic blebbing, detaching of the cells and finally formation of the apoptotic bodies (E, J, O and T). Bar 10 µm.

View larger version (45K): [in a new window]   Fig. 5. The cleavage of NuMA, lamins A/C, lamin B, PARP-1 and pro-caspase-8, -3 and -7 during Fas-mediated apoptosis in HeLa cells. Cells grown on a monolayer were treated with Fas antibody and PD 98059 and prepared for SDS-PAGE at different time points as indicated. Identically loaded gels were run and immunoblotted with NuMA, lamin A/C, lamin B, PARP-1, caspase-8, caspase-3 or caspase-7 antibody. Note the gradual cleavage of NuMA and PARP-1 after 8 hours and lamin B after 8-12 hours. Lamins A and C are only partially cleaved during apoptosis. The amounts of procaspase-8, -3 and -7 are diminished and the cleaved 42/44 and 25 kDa fragments of caspase-8 are increased during apoptosis.

View larger version (59K): [in a new window]   Fig. 6. The cleavage of NuMA, lamin A/C, lamin B, PARP-1 and procaspase-8, -3 and -7 is inhibited in the presence of z-DEVD-FMK, z-VEID-FMK or z-IETD-FMK. HeLa cells grown on a monolayer were treated with Fas antibody and PD 98059 for 24 hours in the presence or absence of 100, 10, 1 or 0.1 µM z-DEVD-FMK, z-VEID-FMK or z-IETD-FMK. Cells were prepared for SDS-PAGE and immunoblotted with NuMA, lamin A/C, lamin B, PARP-1, caspase-8, caspase-3 or caspase-7 antibody. Note, that z-DEVD-FMK, z-VEID-FMK and z-IETD-FMK inhibit the cleavage of NuMA, lamins, PARP-1 and pro-caspase-8, -3 and -7 identically and dose dependently.

View larger version (75K): [in a new window]   Fig. 7. Immunofluorescence microscopy of Fas-treated HeLa cells in the presence of z-DEVD-FMK. Cells grown on glass coverslips were treated with Fas antibody and PD 98059 for 24 hours in the presence of 100 µM (A-D) or 30 µM (E and F) z-DEVD-FMK and stained with NuMA (A and E) or lamin A/C primary antibody (C) and with FITC-conjugated goat-anti-mouse secondary antibody. Samples were counterstained for DNA with Hoechst (B, C and F). Note the atypical apoptotic cells (arrows) in the presence of 100 µM z-DEVD-FMK, in which chromatin is partially condenced and the nuclear outer border is convoluted. These cells are negative for NuMA (A) and lamin A/C (C). In the presence of 30 µM z-DEVD-FMK, chromatin is slightly condenced in atypical apoptotic cells (F, arrows) and the nuclei have lost their normal round shape. NuMA staining shows highly irregular distribution inside the nucleus (E). Bar 10 µm.

View larger version (78K): [in a new window]   Fig. 9. Atypical apoptotic cells are cleaved caspase-3, p85 of PARP-1 and TUNEL negative. HeLa cells grown on glass coverslips were treated with Fas antibody and PD 98059 in the presence of 30 µM z-DEVD-FMK for 24 hours and double-stained with NuMA (A) and cleaved caspase-3 (B) or NuMA (F) and p85 fragment of PARP-1 (G) antibodies or processed for TUNEL-assay (K). FITC-conjugated goat-anti-mouse (A and F) and TRITC-conjugated goat-anti-rabbit (B and G) secondary antibodies were used. Samples were counterstained for DNA with Hoechst (D, I and L). Atypical apoptotic cells (arrowheads) are NuMA and TUNEL negative and do not stain with cleaved caspase-3 or p85 fragment of PARP-1 antibodies. Typical apoptotic cells (arrows) stain with all studied antibodies and are TUNEL positive. Note the single cell (star, A-E) which stains weakly for NuMA and cleaved caspase-3 and shows abnormal chromatin structure. The phase contrast view shows that both typical and atypical apoptotic cells have shrunken and partially detached. Bar 10 µm.

View larger version (71K): [in a new window]   Fig. 10. Immunofluorescence microscopy of MCF-7 cells during staurosporine-induced apoptosis. Cells grown on the coverslips were untreated (A-E) or treated (F-O) with 2 µM staurosporine for 12 hours, fixed and double-stained with NuMA (A and F) and lamin B (B and G) or NuMA (K) and p85 fragment of PARP-1 (L) primary antibodies and FITC-conjugated rabbit-anti-mouse (A and F) and TRITC-conjugated rabbit-anti-goat (B and G) or FITC-conjugated goat-anti-mouse (K) and TRITC-conjugated goat-anti-rabbit (L) secondary antibodies. Samples were counterstained for DNA with Hoechst (D, I and N). Note the partially condensed distribution of NuMA and wrinkled distribution of lamin B in shrunken apoptotic cells. Hoechst stain shows that DNA is partially condensed. These cells are p85 fragment of PARP-1 positive (arrows). Bar, 10 µm.

View larger version (30K): [in a new window]   Fig. 11. NuMA and lamins are not degraded in MCF-7 cells during staurosporine-induced apoptosis. Cells grown on a monolayer were untreated (lane 1) or treated (lane 2) with 2 µM staurosporine for 12 hours and prepared for SDS-PAGE. Identically loaded gels were run and immunoblotted with NuMA, lamin A/C, lamin B, PARP-1, caspase-8 or caspase-7 antibody. Note the partial cleavage of PARP-1, procaspase-8 and disappearance of pro-caspase-7 in staurosporine-treated cells. NuMA and lamins are not cleaved during the apoptosis.

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