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First published online August 29, 2005
doi: 10.1242/10.1242/jcs.02529

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Active relocation of chromatin and endoplasmic reticulum into blebs in late apoptotic cells

Jon D. Lane^1,2,, Victoria J. Allan^2,* and Philip G. Woodman^2,*

¹ University of Bristol, Department of Biochemistry, School of Medicine, University Walk, Bristol, BS8 1TD, UK
² University of Manchester, Faculty of Life Sciences, The Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK

View larger version (45K): [in a new window]   Fig. 1. Biphasic plasma membrane blebbing in adherent apoptotic cells. (A) Analysis of blebbing kinetics in adherent cells. Top: phase contrast images of biphasic blebbing in an apoptotic SW13 cell. Images in this sequence are frames taken from supplementary material Movie 2. Bar, 10 µm. Bottom: kinetics of blebbing types in relation to PS exposure in adherent cell-lines. To the left: time analyses of apoptotic features obtained from time-lapse experiments of a variety of apoptotic adherent cell-types (time across the x-axis is in minutes counting back from the onset of annexin V binding). To the right: cartoon of the kinetics of morphological changes in adherent cells undergoing apoptosis. (B) Analysis of non-adherent cells. Top: phase contrast images of a Jurkat cell undergoing apoptosis. Only one blebbing phase is observed, and blebs appear similar to late blebs in adherent cell-lines (frames taken from supplementary material Movie 3). Bar, 10 µm. Bottom: time-analyses of blebbing, as for A, using non-adherent THP-1 and Jurkat cells.

View larger version (58K): [in a new window]   Fig. 2. Chromatin relocates into surface blebs during the late phase of plasma membrane blebbing in adherent cells. (Top) Phase contrast and (bottom) fluorescence images of an apoptotic HeLa cell transiently expressing HMGB1-YFP. Arrows in zoomed frames indicate chromatin moving outwards into large surface protrusions during late blebbing. Open arrowhead indicates a late plasma membrane bleb apparently lacking chromatin. Images are taken from supplementary material Movie 4 which is presented as a colour overlay of the HMGB1-YFP (red) and phase contrast frames. Bars, 10 µm.

View larger version (68K): [in a new window]   Fig. 3. Late blebs contain cortical ER but exclude other organelles. (A) Confocal slices at 1 µm intervals through anisomycin-treated apoptotic HeLa cells stained with an anti-KDEL antibody (red) and DAPI (blue). Arrowheads indicate examples of ER membranes accumulating at the cell periphery within large surface blebs that contain chromatin (arrows). Bar, 5 µm (B) Confocal maximum projections of anisomycin-treated apoptotic HeLa cells labelled with markers for the ER (anti-calnexin), the cis-Golgi (anti-GM130), mitochondria (anti-HSP60) and lysosomes (anti-LAMP-2). In merged images, DAPI is false-coloured blue, ER markers are red and other organelle markers (GM130, HSP60 and LAMP-2) are green. Bars, 5 µm.

View larger version (54K): [in a new window]   Fig. 4. Reorganisation of ER into sheets underlying the plasma membrane. (A) Confocal image of anisomycin-treated HeLa cells stained with an anti-KDEL antibody (red) and DAPI (blue). Arrowheads indicate extended areas of ER membrane at the cell periphery which are not in blebs and which are not associated with chromatin. Arrows indicate sheets of ER that end abruptly. Cell profiles are indicated by solid lines. Part of a non-apoptotic cell is present at the bottom, indicating the normal organisation of ER. Bar, 5 µm. (B-D) Magnified views from panel A, showing ER staining (left panels) and DAPI (right panels).

View larger version (104K): [in a new window]   Fig. 5. Transmission electron microscopy of late blebs in apoptotic HeLa cells. In ultrathin sections, late apoptotic cells were identified by their asymmetric profile due to the presence of few, large surface blebs containing condensed chromatin. (A) An example of a chromatin-containing surface bleb. Chromatin (Ch) is loosely enclosed by a continuous double membrane (arrowheads) that closely abuts the plasma membrane, with its outer leaflet following the surface topography of the plasma membrane (arrowhead in zoom panel [i]). On the outer leaflet of the surrounding membrane, ribosomes are apparent (arrowheads in [ii]), and a nuclear pore is also evident (arrowhead in [iii]). (B) A membrane sheet, probably ER, running close to the underside of the plasma membrane of an apoptotic bleb (its tip is marked by an arrowhead). Bars, 1 µm (A); Ai-iii, 100 nm (Ai-iii); 200 nm (B).

View larger version (17K): [in a new window]   Fig. 6. Actin and microtubule cytoskeletons contribute to the generation of late apoptotic blebs. (A) HeLa cells were induced to undergo apoptosis by anisomycin treatment (6 hours) in the absence or presence of 1 µM latrunculin A, 100 µM Y27632, or 5 µg ml-1 nocodazole. Cells were stained with anti-KDEL antibodies and DAPI, and apoptotic cells were identified by chromatin morphology. Apoptotic cells were scored for the presence of chromatin-containing, late blebs containing cortical ER. Data points represent means±s.e.m. of three experiments (*P<0.05; **P<0.01; ***P<0.001). (B) HeLa cells were treated for 6 hours with anisomycin in the absence or presence of blebbistatin (12.5 µM). Cells were fixed, DAPI stained, then scored for the presence of late blebs (top). Values are means of three experiments±s.e.m. (***P<0.001).

View larger version (55K): [in a new window]   Fig. 7. Organisation of the actin and microtubule cytoskeletons in apoptotic cells. (A) Confocal sections, at 1 µm intervals, through an anisomycin-treated, apoptotic HeLa cell stained with anti-KDEL antibody (red), phalloidin (green) and DAPI (blue). Fluorescence overlays are positioned above their corresponding phase contrast images. ER membranes are concentrated at the cell cortex, and are notable within chromatin-containing surface blebs. Often, chromatin-free loops of ER can be observed within surface blebs (arrow). Actin is found in retraction cables, aggregates and is associated with the cell cortex. Occasionally, actin can be found at the base of chromatin-containing blebs (arrowhead). (B-D) Confocal maximum projections of anisomycin-treated apoptotic HeLa cells labelled with phalloidin (green), anti-myosin II (red) and DAPI (blue) in (B), with anti-phospho-myosin light chain (MLC) II (red) and DAPI (blue) in (C), and with anti-tubulin (green) and DAPI (blue) in (D). Myosin II is found in late blebs, but is relatively evenly distributed throughout the cell. Phospho-MLC II is concentrated with chromatin-containing surface blebs (arrows). Microtubules are an abundant feature of late apoptotic HeLa cells, and extend around the cortex of a chromatin-containing late bleb (arrow). Bars, 5 µm.

View larger version (170K): [in a new window]   Fig. 8. Analysis of ER and chromatin organisation in zVEID-treated apoptotic cells. HeLa cells were induced into apoptosis in the presence of zVEID.FMK, then processed for fluorescence microscopy (A) or EM (B-D). (A) A confocal optical section through the centre of a zVIED-treated apoptotic HeLa cell, labelled with anti-KDEL antibodies (red), phalloidin (green) and DAPI (blue). Condensed chromatin remains enclosed within an intact nuclear envelope that labels strongly with anti-KDEL (arrowheads). Actin is located principally around the cell periphery [abundant actin is also found in retraction cables at the cell base (data not shown)]. (B) A section through the centre of a zVEID-treated apoptotic HeLa cell. Chromatin (ch) is closely associated with the inner leaflet of the intact nuclear envelope (arrows), and does not relocate to the cell periphery. Surface blebs remain relatively small. (C) High magnification of a zVEID-treated apoptotic HeLa cell showing chromatin (Ch) abutting a dilated nuclear envelope [in zVEID-treated apoptotic HeLa cells, nuclear envelope lumen diameter was significantly (P=0.001) greater than in standard apoptotic cells, increasing from 19.4 nm (s.e.m.=0.9) to 34.4 nm (s.e.m.=3.3)]. Well-preserved mitochondria (m) are found throughout the cytoplasm. (D) ER membranes form whorls and interconnecting tubular arrays in zVEID-treated apoptotic HeLa cells. ER tubules are also significantly dilated [mean diameter in standard apoptotic HeLa cells was 23.7 nm (s.e.m.=2.8), increasing to 44.9 nm (s.e.m.=4.9; P=0.001) in zVEID-treated apoptotic HeLa cells]. Bars: 5 µm (A,B) and 500 nm (C,D).

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