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

Reagents

Unless otherwise stated, reagents were obtained from Sigma (Poole, UK). Stock solutions of anisomycin (5 mg/ml), nocodazole (5 mg/ml), taxol (paclitaxel ™ , Calbiochem, Nottingham, UK: 20 mM), zVAD.FMK (Calbiochem: 50 mM), Ac-DEVD.AMC (Calbiochem: 10 mM), Y27632 (Calbiochem: 100 mM), propidium iodide (20 μg/ml), RNaseA (15 μg/ml), DAPI (4′,6-diamidino-2-phenylindole: 1 mg/ml), latrunculin A (Molecular Probes, Eugene, OR: 10 mM) and blebbistatin (Calbiochem: 100mM) were stored at −20ºC. Alexa 594 -annexin V, Alexa 488 -phalloidin and CellTracker green (CMFDA: 10 mM) were obtained from Molecular Probes.

Antibodies

The following antibodies were used: monoclonal anti-tubulin (B5-1-2: Sigma); polyclonal anti-cleaved PARP (Promega, Southampton, UK); monoclonal anti-tyrosinated α-tubulin (YL1/2: from John Kilmartin, Cambridge, UK); monoclonal anti-acetylated α-tubulin (C3B9) and polyclonal anti-pericentrin, both from Peter March (Manchester, UK); monoclonal anti-EB1 (Transduction Labs); polyclonal anti-ninein (from Mette Mogensen, UEA, UK ( Baird et al., 2004 )).

Constructs

The human high mobility group box 1 (HMGB1)-YFP construct has been described ( Lane et al., 2005 ). HMGB1-CFP was generated by sub-cloning into pECFP-N1 (Clontech). YFP-tubulin was obtained from Clontech. EB1-GFP was from the Morrison lab (University of Leeds, UK) ( Morrison et al., 2002 ). Human GFP-Centrin 2 ( White et al., 2000 ) was obtained from Jeff Salisbury (Mayo Clinic College of Medicine, MN). GFP-γ-tubulin was a gift from Steve Doxsey (University of Massachusettes, MA). Transient transfections were carried out using Fugene 6 (Roche, Lewes, UK) according to the manufacturer’s instructions.

Cell lines

Cells were maintained either in DMEM (A431; HeLa; SW13.Cl-2) or RPMI (Meg01; Jurkat; HL60; THP-1) each containing 10% foetal bovine serum, at 37°C and 5% CO 2 . A431 cells stably expressing YFP-tubulin were obtained following transient transfection by selecting positive clones after G418 treatment ( Lane et al., 2002 ). THP-1 cells (ECACC, Salisbury, UK) were differentiated into macrophages by incubating for 72 hours with 240 nm PMA (phorbol 12-myristate 13-acetate).

Apoptosis induction and drug treatments

Cells were induced into apoptosis by treatment with 5 μg/ml anisomycin or by UV irradiation (100 Jm −2 ( Lane et al., 2002 )). Inhibitors were used at the following final concentrations: latrunculin A (1.0 μM); Y27632 (100 μM); blebbistatin (12.5μm); nocodazole (5 μg/ml); taxol (20 μM); zVAD.FMK (50 μM).

Fluorescence microscopy and live-cell imaging

Wide-field fluorescence images were obtained using an Olympus IX-71 inverted microscope (60x Uplan Fluorite objective 0.65–1.25 NA, at maximum aperture) fitted with a CoolSNAP HQ CCD camera (Photometrics, Tucson, AZ) driven by MetaMorph software (Universal Imaging Corporation, Downington, PA). Confocal images were obtained using a Leica AOBS SP2 microscope (63x PLAPO objective 1.4 NA) at 0.2 μm z-steps. For immunofluorescence, cells were fixed in 2% paraformaldehyde (PFA: methanol-free, EM grade; TAAB, Aldermaston, UK) with 0.2% gluteraldehyde (TAAB), followed by permeabilisation with 0.1% Triton X-100, or in −20ºC methanol. To analyse apoptotic spikes by immunofluorescence, floating apoptotic A431 cells were spun on to poly-L-lysine coated coverslips (using a Shandon cytospin 3: 1000 rpm, 5 mins). Live-cell imaging was carried out using the Olympus IX-71 system. Halogen lamp illumination was used for both transmitted light and for epifluorescence to extend cell viability ( Lane et al., 2002 ). Cells were maintained in CO 2 -independent DMEM (Invitrogen, Paisley, UK), at 37°C in 3 cm cell imaging dishes (MatTek Co., Ashland, MA). FRAP investigations of YFP-tubulin expressing apoptotic cells were carried out using the Leica AOBS SP2 confocal microscope.

Analysis of apoptosis and cellular fragmentation UV-irradiated

A431 cells were incubated for 8 hours in the absence or presence of inhibitors. Apoptosis was assessed by measuring Ac.DEVD.AMC fluorescence in cell lysates (according to the manufacturer’s instructions). In parallel experiments, A431 cells were processed for immunoblotting using anti-cleaved PARP and anti-tubulin antibodies. To quantitate apoptotic bodies, CellTracker green-labelled A431 cells were fixed by adding a one third volume of 6% PFA to the culture medium, and passed through a 5 μm pore filter (Millipore, Watford, UK) by gravity flow ( Cline and Radic, 2004 ). Apoptotic cell fragments were cytospun on to poly-L-lysine coated coverslips as described above. The number of fragments in 10 random fields was assessed, in triplicate, in blind experiments by fluorescence and phase contrast microscopy. For flow cytometry, cells were fixed in ice-cold 70% ethanol, then incubated with 20 μg/ml propidium iodide in the presence of 15 μg/ml RNaseA for 1 hour at 37°C before being analysed using a FACScan (Becton Dickinson). Cellular fragmentation was expressed as the percentage of cells with sub-G1 DNA content. Phagocytosis assays PMA-differentiated THP-1 monocytes were adhered to glass coverslips in 6-well plates (750 000 cells/well). CellTracker-labelled apoptotic A431 cells were irradiated then incubated for 8 hours in the absence or presence of 5 μM nocodazole. Floating apoptotic cells were washed and 120 000 cells added to the THP-1 culture in 1 ml serum-free DMEM. After 30 mins co-incubation at 37°C, coverslips were washed extensively in PBS, and cells fixed in 2% PFA. The number of THP-1 macrophages interacting (bound and engulfed) and engulfing cell fragments was calculated in ten random fields in triplicate by fluorescence and phase contrast microscopy.

Electron microscopy Apoptotic

A431 cells were obtained by aspiration of culture medium from a dish of UV-irradiated cells, fixed by adding an equal volume of 4% gluteraldehyde, then processed for transmission electron microscopy as described previously ( Lane et al., 2005 ).

Analysis of apoptosis and cellular fragmentation UV-irradiated

A431 cells were incubated for 8 hours in the absence or presence of inhibitors. Apoptosis was assessed by measuring Ac.DEVD.AMC fluorescence in cell lysates (according to the manufacturer’s instructions). In parallel experiments, A431 cells were processed for immunoblotting using anti-cleaved PARP and anti-tubulin antibodies. To quantitate apoptotic bodies, CellTracker green-labelled A431 cells were fixed by adding a one third volume of 6% PFA to the culture medium, and passed through a 5 μm pore filter (Millipore, Watford, UK) by gravity flow ( Cline and Radic, 2004 ). Apoptotic cell fragments were cytospun on to poly-L-lysine coated coverslips as described above. The number of fragments in 10 random fields was assessed, in triplicate, in blind experiments by fluorescence and phase contrast microscopy. For flow cytometry, cells were fixed in ice-cold 70% ethanol, then incubated with 20 μg/ml propidium iodide in the presence of 15 μg/ml RNaseA for 1 hour at 37°C before being analysed using a FACScan (Becton Dickinson). Cellular fragmentation was expressed as the percentage of cells with sub-G1 DNA content.

Supplementary Material M1 M2 M3 M4 M5 M6 M7 S1 S2 S3 S4