JCS037259 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Effect of monastrol on apoptotic microtubule assembly. Cells were induced into apoptosis by anisomycin treatment in the presence of monastrol. Cells were labelled with anti-tubulin antibodies and DAPI, and imaged by confocal microscopy. Monastrol causes prometaphase arrest and monoasters in viable cells (top), but has negligible influence upon the apoptotic microtubule array (bottom panels). Bars, 10 µm.

• Supplemental Figure S2 -

  Fig. S2. TPX2 siRNA: impact on mitotic and apoptotic microtubule network assembly. (A) Microtubule organisation in mitotic cells silenced for TPX2. Cells were fixed and labelled with antibodies against tubulin and TPX2, and with DAPI. Mitotic cells were identified by chromatin morphology, and are indicated as follows: silenced cells lacking TPX2 staining and with disorganised microtubule arrays with arrows; a cell retaining TPX2 with wild-type spindle microtubules with arrowheads. Bar, 10 µm. (B) Confocal images of examples of apoptotic HeLa cells with typical, irregular or absent microtubule arrays. These cells were silenced for TPX2 and induced into apoptosis by anisomycin treatment. Arrow shows a cell lacking apoptotic microtubules; arrowheads indicate cells with irregular apoptotic microtubules. Bar, 5 µm.

• Movie 1 -

  Movie 1. Dynamics of nuclear dextran release during apoptotic execution. An A431 cell microinjected with 70 kDa rhodamine dextran in the nucleus, and induced into apoptosis by UV irradiation. Dextran is shown in red superimposed upon the corresponding phase contrast image (false-coloured cyan).

• Movie 2 -

  Movie 2. GFP-NLS release during apoptosis. A HeLa cell transiently expressing HMGB1-CFP (to the left) and GFP-NLS (centre) induced into apoptosis by anisomycin treatment.

• Movie 3 -

  Movie 3. Coordination of microtubule assembly with apoptotic YFP-Ran release. HeLa cells transiently co-expressing YFP-Ran (green), HMGB1-CFP (red) and mCherry-tubulin were induced into apoptosis by anisomycin treatment. Frames from this movie are shown in Fig. 2B.

• Movie 4 -

  Movie 4. GRASP65 cleavage and microtubule assembly − control. HeLa cells stably expressing GRASP65-GFP were transiently transfected with mCherry-tubulin and HMGB1-CFP and induced into apoptosis by anisomycin treatment. GRASP65 cleavage is indicated by a rapid loss of Golgi ribbon-associated GFP fluorescence and this takes place within ∼15 minutes of apoptotic cell retraction. Apoptotic microtubule assembly takes place during the subsequent 30-40 minutes, and is associated with active chromatin redistribution.

• Movie 5 -

  Movie 5. GRASP65 cleavage and microtubule assembly − blebbistatin treatment. HeLa cells stably expressing GRASP65-GFP were transiently transfected with mCherry-tubulin and HMGB1-CFP and induced into apoptosis by anisomycin treatment in the presence of blebbistatin. GRASP65 cleavage is indicated by a rapid loss of Golgi ribbon-associated GFP fluorescence and this takes place within ∼15 minutes of apoptotic cell retraction. Apoptotic microtubules are not observed.

• Movie 6 -

  Movie 6. GRASP65 cleavage and microtubule assembly − Y27632 treatment. HeLa cells stably expressing GRASP65-GFP were transiently transfected with mCherry-tubulin and HMGB1-CFP and induced into apoptosis by anisomycin treatment in the presence of Y27632. GRASP65 cleavage is indicated by a rapid loss of Golgi ribbon-associated GFP fluorescence and this takes place within ∼15 minutes of apoptotic cell retraction. Apoptotic microtubules are not observed.