First published online 11 March 2008
doi: 10.1242/jcs.020982
Journal of Cell Science 121, 1014-1024 (2008)
Published by The Company of Biologists 2008
The integrity of a lamin-B1-dependent nucleoskeleton is a fundamental determinant of RNA synthesis in human cells
Chi W. Tang1,*,
Apolinar Maya-Mendoza1,*,
Catherine Martin1,
Kang Zeng1,
Songbi Chen1,
Dorota Feret2,
Stuart A. Wilson3 and
Dean A. Jackson1,
1 Faculty of Life Sciences, University of Manchester, MIB, 131 Princess Street, Manchester, M1 7DN, UK
2 Cancer Research UK, Paterson Institute for Cancer Research, Christie Hospital, Wilmslow Road, Manchester, M20 9BX, UK
3 Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK
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Fig. 1. Inhibiting expression of nuclear lamin proteins using RNAi. RNAi was used to inhibit lamin protein expression in HeLa cells and analysis was performed on individual cells. (A,B) RNAi was performed by transient expression and transfection monitored by co-transfection of a vector expressing YFP-tubulin (green). At selected times after transfection (48 hours in this example) cells were fixed and immunolabelling of the residual lamina performed (red). Histograms (C) show averaged data (fluorescent intensities expressed as arbitrary units; n>75) from three equivalent experiments in which the average expression of lamin B1 and A/C was reduced (KD) to 20.7 and 19.8% of untransfected cells (Control) in the same samples. As negative controls, cells were transfected with empty vector (not shown) or appropriate scrambled RNAi (C). Scale bars: 10 µm.
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Fig. 2. Reduced lamin B1 expression correlates with inhibition of RNA synthesis. HeLa cells were co-transfected with RNAi vectors and a vector expressing YFP-tubulin and the structure and activity of transcription sites monitored 48 hours later using either indirect immunofluorescence of the active form of RNA polymerase II (A) or BrUTP incorporation in permeabilised cells (B). (A) RNA polymerase (red) in transfected (expressing YFP-tubulin – green) cells after transfection with: lamin B1 RNAi vector (Kd LB1); RNAi vector co-transfected with lamin B1 rescue vector (Kb LB1 + Rescue); rescue vector alone (Rescue vector) and lamin A/C RNAi vector (Kd LA/C). (B) Nascent transcripts (BrU – red) in control cells and cells transfected with lamin A/C (Kd LA/C) and lamin B1 (Kd LB1) RNAi vectors. Lamin B1 depletion was assessed by indirect immunofluorescence (Anti-LB1, red). As a control for possible labelling artefacts, the order of antibody labelling was also reversed (Anti-LB1/BrU – merged image of lamin B1 labelled green and transcripts labelled red). Quantitative image analysis of active RNA polymerase II – H5 epitope (C) and nascent transcripts (D-F) in control and lamin-depleted cells showed that transcription was unaffected in cells with lamin A/C depleted (D,F) whereas depletion of lamin B1 (C-E) correlated with a clear decrease in transcription of both pre-mRNA and pre-rRNA (see supplementary material Fig. S2). For pre-mRNA synthesis, statistical analysis using two-tailed Student's t test showed the lamin-B1-depleted and control cell populations (E) to be unrelated. Scale bars: 10 µm (A); 5 µm (B).
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Fig. 3. Lamin B1 depletion correlates with altered morphology in nucleoli and nuclear speckles. Cells co-transfected with RNAi vectors and a vector expressing histone H2B were analysed after 48 hours to monitor the morphology of major nuclear compartments. (A) Sites of pre-rRNA synthesis in nucleoli were visualised by indirect immunolabelling of fibrillarin (green). Changes were monitored in cells expressing DsRed-histone H2B (red) and different patterns recorded; shown in cartoon form and occurrence as a frequency histogram [LB1 kd, 187 cells (four experiments); Control, 100 cells (two experiments)]. Control cells were transfected with empty vector, although untransfected cells in RNAi samples also serve as controls. (B) Nuclear speckles were visualised by indirect immunolabelling of the splicing factor SC-35 (green). Structural changes were monitored in cells expressing DsRed-histone H2B (red) and the appearance and frequency of major changes described (LB1 kd, 196 cells (five experiments); Control, 98 cells (two experiments). (C) The structure of the chromatin compartment in HeLa cells was monitored by expression of DsRed-histone H2B. Typical examples of untreated cells (control) and cells with lamin A/C or B1 depleted (Kd LA/C; Kd LB1) are shown. Scale bars: 5 µm (A,C); 10 µm (B).
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Fig. 4. Monitoring apoptosis in lamin-depleted HeLa cells. HeLa cells with depleted lamin expression were analysed (48 or 96 hours after transfection) to monitor binding of Annexin V. Cells were stained with Alexa-568 (red)-conjugated Annexin V and counterstained with Hoechst (blue) (A) and the percentage of Annexin-V-positive transfected cells (n=200) were determined (B). Cells on coverslips were treated as follows: (1) positive controls [Control (+)] treated with staurosporine (5 µm in medium; 4 hours); (2) negative controls [Control (–)] transfected with pYFP-tubulin using polyfect; (3) lamin-A/C-depleted cells (KD LA/C) transfected with pYFP-tubulin and pSECpuroLaminA/C; and (4) lamin-B1-depleted cells (KB LB1) transfected with pYFP-tubulin and pSEChygroLaminB1. Scale bars: 10 µm.
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Fig. 5. Reduced lamin B1 expression correlates with altered location of chromosome territories. CTs were analysed in lamin-B1-depleted cells using: (A) FISH to chromosome 19 (wide-field imaging); (B) BrdU, by indirect immunofluorescence (high-resolution 3D imaging); (C) Alexa Fluor 488 dUTP, in living cells (confocal sections). (B,C) Unsynchronised cells were labelled and grown for 5-7 days so that most labelled cells retained 1-3 discrete, labelled territories. RNAi vectors were transfected 48 hours before analysis. CTs were assigned to nuclear zones of equal area (Materials and Methods) using at least 100 territories/sample (A-C). Changes were analysed using two-tailed Student's t test as follows: (A) control vs lamin A/C depletion (t=2.396, P<0.0172*); control vs lamin B1 depletion (t=9.203. P<1.567e-17***); (B) control vs lamin A/C depletion (t=0.478, P<0.634); control vs lamin B1 depletion (t=5.780, P<2.973e-8***); (C) control vs lamin B1 depletion (t=6.442, P<1.996e-09***). Marginalisation of CTs (D, left panel, shows a single confocal section of CTs labelled with BrdU in green) correlated with collapse of open chromatin (D, centre panel, DsRed-histone H2B in red) and a corresponding expansion of the inter-chromatin compartment. Scale bars: 10 µm (A); 5 µm (C,D).
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Fig. 6. Stability of the nuclear lamina following modification by RNAi. HeLa cells were co-transfected with RNAi vectors and vector expressing the relevant GFP-laminA/C or B1 to monitor dynamics. After 48 hours, cells with low intensity reporter expression were selected and fluorescence recovery after photobleaching monitored. Panels (A) show a typical FRAP profile (from left to right): pre-bleach image; image immediately after bleaching – in each cell analysed two 2 µm zones were bleached, one is shown boxed; and two frames (15 and 60 minutes) from a time-lapse series with images taken at 15 minute intervals over 2 hours. Analysis of bleach zones (B) was used to estimate t1/2 for fluorescent recovery for cells transfected with empty RNAi vector (Control) and cells with lamin B1 (LB1 KD) and lamin A/C (LA/C KD) depleted. Data points shown in B are average intensities of ten bleach zones (five cells) for the long (300 minute) timecourse and 20 bleach zones (ten cells) for the short (50 minute) timecourse. Scale bars: 5 µm.
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Fig. 7. Changes in DNA loops in lamin-depleted cells. The structure of DNA halos from lamin-depleted cells was analysed (A-C). Cells were depleted of lamin A/C or B1 using RNAi vectors and co-transfection with a vector expressing an appropriate fluorescent-lamin A/C or B1, as reporter that marks the nuclear periphery. After 36 hours, nucleoids were prepared and samples containing ethidium bromide (A) used to define halo structure and the corresponding DNA loop length (C). Time-lapse epi-fluorescence microscopy was also used to monitor the expansion of DNA halos, using 15 second time frames over 2 minutes; two frames from typical time-lapse series at 30 and 90 seconds are shown (B). DNA halos prepared from lamin A/C depleted cells expanded to give uniform halos of DNA loops of 19.1±3.9 µm (n=58), which is equivalent to an average DNA loop, measured from the nuclear rim, of 112 kbp; this was indistinguishable from the DNA loops of untransfected controls in the same preparation (A). The average halo from lamn-B1-depleted cells was 24.0±5.5 µm (n=64), equivalent to 141 kbp. White arrows highlight abnormal structures seen in lamin-B1-depleted cells. The size of scale bars is given on individual images. Scale bars: 20 µm.
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© The Company of Biologists Ltd 2008
