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First published online 10 February 2009
doi: 10.1242/jcs.037259

Journal of Cell Science 122, 644-655 (2009)
Published by The Company of Biologists 2009
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Dynamic release of nuclear RanGTP triggers TPX2-dependent microtubule assembly during the apoptotic execution phase

David K. Moss1,*, Andrew Wilde2 and Jon D. Lane1,{ddagger}

1 Cell Biology Laboratories, Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK
2 Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada


Figure 1
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  		Fig. 1. Cytoplasmic RanGTP is required for apoptotic microtubule assembly. (A) HeLa cells transiently expressing wild-type, GDP-locked (T24N) or GTP-locked (L43E) YFP-Ran were induced into apoptosis by anisomycin treatment. YFP-expressing apoptotic cells were assessed for the presence of microtubules by immunofluorescence microscopy. T24N Ran reduced the proportion of apoptotic cells assembling a microtubule network by >50%. (B) Released nuclear Ran remains bound to GTP. (Top, left). Schematic of the Rango FRET probe. CFP and YFP fluorophores are connected by a linker region consisting of the importin β-binding domain of snuportin 1. In regions of low Ran GTP (e.g. the cytoplasm of viable cells), importin β binds and prevents FRET. RanGTP (concentrated in the nucleus of viable cells) competes for importin β and releases the flexibility constraint in Rango, thereby promoting FRET. (Bottom, left) Rango FRET in the nucleus and cytoplasm of viable and apoptotic cells. In apoptotic HeLa cells (anisomycin induced), cytoplasmic and nuclear Rango FRET levels are similar, indicative of an accumulation of RanGTP in the apoptotic cytoplasm. In viable HeLa cells transiently expressing GTP-locked (L43E) mutant Ran, Rango FRET levels are also equalised between nucleus and cytoplasm, whereas Rango FRET is reduced in the cytoplasm of viable cells transiently expressing T24N Ran (mean+s.d.). (Right) Example images of donor (CFP), acceptor (YFP) and Rango FRET (FRETeff: ratios of donor fluorescence before and after acceptor photobleaching) in viable and apoptotic cells. (C) Subcellular localisation of components of the Ran pathway in apoptotic cells. Anisomycin-treated apoptotic HeLa cells labelled with antibodies against Ran and RCC1. Ran is released into the apoptotic cytoplasm (arrowhead), whereas RCC1 remains tightly associated with apoptotic chromatin (arrows). (D) siRNA-mediated depletion of RanGAP1. To the left, immunoblots of HeLa cells silenced for RanGAP1 (using two independent oligonucleotides, each at 100 pmol/µL) or lamin (data not shown). To the right, example confocal fields of lamin-silenced (top) and RanGAP1-silenced (bottom) HeLa cells, immunolabelled for RanGAP1. Arrows depict cells that have escaped siRNA silencing and retain nuclear envelope-associated RanGAP1. (E) Confocal immunofluorescence imaging of viable and apoptotic HeLa cells (treated or not treated with the caspase-6 inhibitor, zVEID.fmk) transiently expressing GFP-lamin B, and immunolabelled for RanGAP1. To the left, confocal maximum projections and to the right, single confocal optical sections and grey level line scans demonstrating co-incident staining of GFP-lamin B and RanGAP1. Viable cells are indicated by arrows, apoptotic cells are indicated by arrowheads. Bars, 5 µm (A,B), 10 µm (C-E).

 

Figure 2
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  		Fig. 2. Temporal and kinetic aspects of apoptotic nuclear Ran release. (A) Quantitation of cytoplasmic fluorescence intensities of mCherry-NLS and YFP-Ran (mean±s.d.), expressed as a percentage of maximum obtained up to 60 minutes post-cell rounding (initiation of rounding is normalised to 0 minutes and is indicated by the hatched vertical line). Example images of a co-transfected viable cell are shown at the top. (B) Nuclear Ran release occurs as a prelude to apoptotic microtubule assembly. HeLa cells transiently co-expressing mCherry-tubulin, YFP-Ran and HMGB1-CFP induced into apoptosis by anisomycin treatment, and imaged by time-lapse microscopy. Nuclear YFP-Ran release was visualised within the first ~25 minutes of apoptotic execution, and the YFP-Ran-enriched apoptotic cytoplasm (arrows) supported the assembly of bundled microtubule arrays (arrowheads). This image sequence was obtained from supplementary material Movie 3, and zoom panels are shown at the bottom. (C,D) Effect of leptomycin B on apoptotic nuclear release. (C) Release kinetics in HeLa cells transiently co-expressing YFP-Ran and mCherry-NLS, induced into apoptosis by anisomycin in the presence of Leptomycin B (mean±s.d.) (initiation of rounding is normalised to 0 minutes and is indicated by the hatched vertical line). (D) Comparison of the maximal apoptotic cytoplasmic fluorescence intensity increase of mCherry-NLS and YFP-Ran in the absence or presence of leptomycin B (mean+s.d.). (E-H) Actin–myosin-II inhibition delays apoptotic nuclear release. (E,G) Kinetics of GFP-NLS (E) and YFP-Ran (G) release in HeLa cells treated with anisomycin in the absence or presence of Y27632, latrunculin A or blebbistatin. Standard deviations are omitted to improve clarity. (F,H) Time taken for a threefold increase in cytoplasmic GFP-NLS fluorescence (F) and a doubling in cytoplasmic YFP-Ran release (H) in the absence or presence of Y27632, latrunculin A or blebbistatin (mean+s.d.). Bars, 10 µm.

 

Figure 3
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  		Fig. 3. Blebbistatin and Y27632 treatments block apoptotic microtubule network assembly. Live-cell imaging of apoptotic microtubule network assembly in GRASP65-GFP HeLa cells (Lane et al., 2002Go) transiently co-expressing mCherry-tubulin and HMGB1-CFP. Loss of Golgi-associated GFP fluorescence occurs within 15 minutes of loss of Golgi fluorescence (asterix indicates cells depleted for Golgi fluorescence). Apoptotic microtubule networks assemble within 45 minutes of cell rounding in control cells (arrows), but microtubules are not observed up to 2 hours post execution phase initiation in blebbistatin or Y27632-treated cells (to the bottom). Frames are taken from supplementary material Movies 4, 5 and 6. Bars, 10 µm.

 

Figure 4
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  		Fig. 4. Distribution of NuMA, Kid and Mklp1 in viable and apoptotic cells. (A) Top panel: viable HeLa cells co-labelled with antibodies recognising the N- and C-termini of NuMA (NuMA N, NuMA C, respectively). In interphase cells, both antibodies label the nucleus. Middle panel: viable HeLa co-labelled for NuMA (C-terminus) and tubulin, showing enrichment of NuMA at the spindle poles (arrows). Bottom panel: apoptotic HeLa cells (anisomycin treated) co-labelled for NuMA N and NuMA C. The N-terminal region of NuMA remains condensed in discrete patches that lie adjacent to apoptotic chromatin, whereas the C-terminal domain is redistributed uniformly throughout the cytoplasm. (B) Early and late apoptotic cells labelled with Kid antisera. (C) Distribution of Mklp1 in viable and apoptotic cells. In mitotic HeLa cells (top three panels), Mklp1 is cytoplasmic, with weak kinetochore fibre labelling at metaphase (arrows), and strong midbody labelling during cytokinesis (arrows). In early apoptotic HeLa cells (anisomycin treated; bottom two panels), Mklp1 remains concentrated within chromatin-rich nuclear domains (arrows) but is redistributed into the cytoplasm of late apoptotic cells. Bars, 10 µm.

 

Figure 5
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  		Fig. 5. TPX2 is released from the nucleus during apoptosis, co-localises with apoptotic microtubules and is required for their efficient assembly. (A) TPX2 distribution in mitotic and apoptotic HeLa cells. TPX2 associates with spindle microtubules during mitosis, and decorates filamentous structures in apoptotic cells. (B) TPX2 co-localises with microtubules in apoptotic cells. Top panel: confocal maximum projection of an apoptotic HeLa cell (anisomycin treated) co-labelled for TPX2 and tubulin. Bottom panel: zoomed view of a single confocal section of the cortical region of interest as indicated by the hashed box in the panel above. (C,D) Silencing TPX2 disrupts apoptotic microtubule assembly. (C) Immunoblots of extracts of HeLa cells treated with siRNA oligonucleotides for 72 hours. Example shown is an experiment using oligonucleotide 1 at 50 pmol for TPX2; oligonucleotide 1 at 50 pmol for Mklp1; oligonucleotide 1 at 150 pmol for Kid; lamin at 40 pmol (refer to Materials and Methods for nomenclature). (D) Analysis of apoptotic microtubule assembly following silencing of Ran-coordinated spindle-assembly factors using the siRNA parameters described in part (C). Anisomycin-treated apoptotic cells were identified by chromatin morphology and were scored for the presence of `typical', `irregular' or `absent' apoptotic microtubules (mean + s.d.). Representative images of TPX2-silenced cells are shown in supplementary material Fig. S2B. (E,F) Examples of apoptotic microtubule arrays and co-incident TPX2 labelling in HeLa cells. (E) An apoptotic cell with strong microtubule TPX2 labelling (arrow), and an apoptotic cell lacking TPX2. In the former, microtubules are abundant, whereas in the latter, few, short microtubules can be seen. (F) An apoptotic cell with weak microtubule TPX2 staining (arrow), and apoptotic cells with TPX2 retained within the nucleus (arrowheads). In each case, microtubules are relatively sparse and poorly organised. Bars, 5 µm (A,E,F), 3 µm (B, top panel), 1 µm (B, bottom panel).

 

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© The Company of Biologists Ltd 2009