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

Abstract

Dramatic changes in cellular dynamics characterise the apoptotic execution phase, culminating in fragmentation into membrane-bound apoptotic bodies. Previous evidence suggests that actin-myosin plays a dominant role in apoptotic cellular remodelling, whereas all other cytoskeletal elements dismantle. We have used fixed cells and live-cell imaging to confirm that interphase microtubules rapidly depolymerise at the start of the execution phase. Around this time, pericentriolar components (pericentrin, ninein and gamma-tubulin) are lost from the centrosomal region. Subsequently, however, extensive non-centrosomal bundles of densely packed, dynamic microtubules rapidly assemble throughout the cytoplasm in all cell lines tested. These microtubules have an important role in the peripheral relocation of chromatin in the dying cell, because nocodazole treatment restricts the dispersal of condensed apoptotic chromatin into surface blebs, and causes the withdrawal of chromatin fragments back towards the cell centre. Importantly, nocodazole and taxol are both potent inhibitors of apoptotic fragmentation in A431 cells, implicating dynamic microtubules in apoptotic body formation. Live-cell-imaging studies indicate that fragmentation is accompanied by the extension of rigid microtubule-rich spikes that project through the cortex of the dying cell. These structures enhance interactions between apoptotic cells and phagocytes in vitro, by providing additional sites for attachment to neighbouring cells.

Figure 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 (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 quantitated after DAPI staining. Arrows in the example 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 mins 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 3 or more distinct, pyknotic pieces of peripheral chromatin, clearly distinguishable from the central mass; see example images). Students’ t-test: **p < 0.001; *p < 0.01.

Figure 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.

Figure 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 γ-tubulin, ninein or pericentrin. In each zoomed panel, GFP-centrin labelling is to the top. Bars = 5 μm. (B) Cartoon showing the relative locations of each of the centrosomal markers shown in (A), adapted from (Bornens, 2002). The grey region around the centrioles represents the pericentriolar space. (C) Quantitaion of the proportion of apoptotic HeLa cells (UV-treated) positive for γ-tubulin labelling. Apoptosis stage was determined using the morphological criteria described in figure 2.

Figure 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.) 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/phase contrast microscopy. Experiments were performed blind in triplicate. (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 students’ t-test in parts B, C, E, F: *, p < 0.5; **, p < 0.01; *** p < 0.001.

Figure 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. (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: A, B = 10 μm; B’, C = 2 μm; D = 5 μm.

Figure 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). Alexa594-Annexin V labelling is false-coloured blue. Fluorescence frames are from supplementary material Video 6. Bar = 20μm. (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.

Figure 7. TEM analysis of microtubule organisation in apoptotic A431 cells

(A) Bundles of closely packed, intersecting microtubules 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, D) A microtubule (arrowheads) running parallel to the plasma membrane of an isolated cell fragment. (E, F) Transverse section through an apoptotic spike. Microtubule profiles can be seen in the framed area (arrows in F). In each example, apoptosis was induced by UV irradiation.

Figure 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. 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. To the left, frames representing images of apoptotic spikes from A431 cells stably expressing YFP-tubulin, before and after photobleaching (box). To the right, quantitation of fluorescence recovery in the boxed region. Bar = 10 μm.

Figure 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.) 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 (green) interacting with THP-1 macrophages (*). Bars = 10 μm (zoom = 5 μm).