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First published online May 24, 2006
doi: 10.1242/10.1242/jcs.02959

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A novel role for microtubules in apoptotic chromatin dynamics and cellular fragmentation

David K. Moss^*, Virginie M. Betin^*, Soazig D. Malesinski and Jon D. Lane

Department of Biochemistry, University of Bristol, School of Medical and Veterinary Sciences, University Walk, Bristol, BS8 1TD, UK

View larger version (36K): [in a new window]   Fig. 1. Microtubule organisation in late-apoptotic HeLa cells. (A) Confocal image of apoptotic HeLa cells (6 hours anisomycin treatment) labelled with an anti-tubulin antibody (red) and DAPI (blue). Microtubules extend away from the body of the cell into chromatin-rich surface blebs. Bar, 10 µm. A Volocity 3-D reconstruction of the lower apoptotic cell is shown in supplementary material Movie 1. (B) Chromatin redistribution into surface blebs in apoptotic HeLa cells treated with anisomycin (Aniso, 6 hours) in the absence or presence of latrunculin A (Lat A) or nocodazole (NDZ). The proportion of apoptotic cells (cleaved PARP-positive, not shown) with condensed chromatin in surface blebs was then quantified after DAPI staining. Arrows in the images indicate chromatin-containing blebs. Bar, 10 µm. (C) Microtubules are required to maintain the dispersed status of condensed apoptotic chromatin. HeLa cells were induced into apoptosis by UV irradiation, incubated for 4 hours then for a further 40 minutes in the absence or presence of nocodazole, latrunculin A or blebbistatin (Blebb) (alone or in combination). Cells were fixed and assessed for compact or dispersed chromatin (cells defined as having dispersed chromatin contained three or more distinct, pyknotic pieces of peripheral chromatin, clearly distinguishable from the central mass; see example images). Values are mean ± s.e.m. Significant differences were observed where indicated, **P<0.001 and *P<0.01 using the Student's t-test.

View larger version (35K): [in a new window]   Fig. 2. Formation of the apoptotic microtubule array in mid-to-late apoptosis. To the right, the proportion of HeLa cells possessing microtubules at various stages of apoptosis, based on the morphological characteristics shown to the left (only cells completely lacking microtubules were scored as `negative'). Anisomycin-treated HeLa cells were stained for microtubules (green), cleaved PARP (red) and DAPI (blue). Early-apoptotic cells have cleaved PARP, but no evidence of chromatin condensation. In mid-apoptotic cells, cleaved PARP-positive nuclei are still intact but display evidence of chromatin condensation. By late apoptosis, chromatin is fragmented and dispersed. Bars, 10 µm.

View larger version (68K): [in a new window]   Fig. 3. Effects of apoptosis on centrosome integrity. (A) Confocal maximum projections of viable and apoptotic cells transiently expressing GFP-Centrin 2 and subsequently labelled with antibodies against -tubulin, ninein or pericentrin. In each zoomed panel to the right of the main image, GFP-centrin labelling is to the top, -tubulin, ninein or pericentrin in the middle, with the merged image in the bottom panel. Bars, 5 µm. (B) Cartoon showing the relative locations of each of the centrosomal markers in A, adapted from Bornens (Bornens, 2002) with permission. The grey region around the centrioles represents the pericentriolar space. (C) Quantification of the mean percentage (± s.e.m.) of apoptotic HeLa cells (UV treated) positive for -tubulin labelling. Apoptosis stage was determined using the morphological criteria described in Fig. 2.

View larger version (30K): [in a new window]   Fig. 4. Microtubules are required for apoptotic cell fragmentation. (A) Influence of cytoskeletal inhibitors on apoptotic progression in UV-treated A431 cells, measured using the fluorogenic caspase substrate, Ac.DEVD.AMC (top) and by immunoblotting for cleaved PARP (bottom: tubulin shown as a loading control). Mean values (± s.e.m.) are shown from triplicate assays. (B) Assessment of apoptotic body formation in UV-treated A431 cells. Sub-5-µm apoptotic A431 cell fragments were collected by filtration and were counted by fluorescence or phase-contrast microscopy. Experiments were performed blind and are the mean ± s.e.m. of three experiments. (C) FACs analysis of apoptotic fragmentation. The relative numbers of UV-irradiated A431 cells with sub-G1 DNA content is shown in the bar chart (bottom) as a function of the value for UV only treated cells (normalised to 100%). Values are from three independent experiments (example traces are shown). (D-F) The effect of Latrunculin A on apoptotic progression (D, PARP cleavage), cellular fragmentation by apoptotic body assay (E), and FACS (F). Statistical significance by Student's t-test in A,B,C,E,F: *P<0.5; **P<0.01; ***P<0.001 compared with levels in the UV-alone group.

View larger version (47K): [in a new window]   Fig. 5. Fluorescence microscopy of microtubule and actin distribution in apoptotic A431 cells. Confocal (A-C) and wide-field (D) fluorescence images of cytospin preparations of floating, UV-irradiated apoptotic A431 cells. (A,B) Apoptotic cells labelled with phalloidin (F-actin; green), anti-tubulin antibody (red) and DAPI (blue). (B') Detail of a cluster of apoptotic bodies formed at the tip of two spikes from the boxed region of the panel above. (C) Actin and microtubules co-align in apoptotic spikes. The body of the cell is located to the top left. (D) An isolated apoptotic body labelled for tubulin (red), cleaved PARP (green) and DAPI (blue). Bars, 10 µm (A,B); 2 µm (B',C); 5 µm (D).

View larger version (91K): [in a new window]   Fig. 6. Microtubule and chromatin dynamics in apoptotic A431 cells. (A) Time-lapse imaging of anisomycin-treated A431 cells transiently co-expressing YFP-tubulin (green) and HMGB1-CFP (red). Alexa Fluor 594-Annexin V labelling is false-coloured blue. Fluorescence frames are from supplementary material Movie 6. Bar, 20 µm. (B,C) Zoomed areas from the boxed regions indicated in A showing increased temporal resolution (annexin V channel omitted). Arrowheads indicate packets of condensed chromatin. Bar, 10 µm.

View larger version (143K): [in a new window]   Fig. 7. TEM analysis of microtubule organisation in apoptotic A431 cells. (A) Bundles of closely packed, intersecting microtubules (asterisks) seen in longitudinal section (LS) in the body of an apoptotic A431 cell. (B) Detail of an area of cytoplasm at the base of an apoptotic spike. A bundle of microtubules (arrowheads) can be seen in LS running into the spike. Intact mitochondria (Mch), chromatin (Ch), ribonucleoprotein granules (Rnp) and unidentified membrane-bound organelles are also apparent. (C) A microtubule (arrowheads) running parallel to the plasma membrane of an isolated cell fragment is shown in close-up in D. (E) Transverse section through an apoptotic spike. Microtubule profiles can be seen in the close-up of the boxed area (arrows in F). In each example, apoptosis was induced by UV irradiation.

View larger version (52K): [in a new window]   Fig. 8. Orientation and dynamics of apoptotic microtubules. (A) Confocal immunofluorescence imaging of EB1 distribution in an apoptotic A431 cell. EB1 puncta (red) are localised to the distal tips of microtubules (green) within apoptotic spikes. The boxed areas of the merge image are shown in close-up to the right. Bar, 10 µm. (B) Kymograph derived from a time-lapse sequence of EB1-GFP dynamics in an apoptotic A431 cell spike (supplementary material Movie 7). Puncta (arrows) move towards the spike tip. (C) FRAP analysis of microtubule polymer turnover in apoptotic spikes. Frames represent images of apoptotic spikes from A431 cells stably expressing YFP-tubulin, before and after photobleaching (boxed areas). To the right, quantification of fluorescence recovery in the boxed region. Bar, 10 µm.

View larger version (35K): [in a new window]   Fig. 9. Spikes enhance interaction between apoptotic cells and phagocytes. (A) Proportion of THP-1 macrophages interacting (bound and engulfed) and engulfing apoptotic A431 cells. Target cells were generated in the absence or presence of nocodazole. Shown are means (± s.e.m.) of triplicate samples from a single representative experiment (statistical analysis by Student's t-test). (B,C) Wide-field images of CellTracker-labelled apoptotic A431 cells (arrowheads) interacting with THP-1 macrophages (asterisks). Zoomed images of the boxed areas are shown in the bottom panels of B and C. Bars=10 µm (zoom=5 µm).