spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 3 June 2003
doi: 10.1242/jcs.00612

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Carvalho, A.
Right arrow Articles by Wheatley, S. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Carvalho, A.
Right arrow Articles by Wheatley, S. P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Survivin is required for stable checkpoint activation in taxol-treated HeLa cells

Ana Carvalho1,2,3, Mar Carmena1, Clara Sambade2,3, William C. Earnshaw41,* and Sally P. Wheatley1

1 Chromosome Structure Group, Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, Scotland, UK
2 University of Porto, IPATIMUP Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal
3 Faculty of Medicine, University of Porto, Porto, Portugal



View larger version (46K):

[in a new window]
  		Fig. 1. Survivin can be depleted from HeLa cells using RNAi. (A) Growth curve of viable HeLa cells transfected with control siRNA (blue triangles) or Survivin siRNA (red circles). Cell number in Survivin RNAi population does not increase after 48 hours. (B) Immunoblot analysis of control (C) and Survivin-siRNA-transfected (S) cultures at 0 hours, 60 hours or 84 hours post transfection as indicated. Tubulin was monitored as a loading control. (C-E) Indirect immunofluorescence images showing Survivin in red, DNA in blue and tubulin in green. (C) Control cell transfected with rhodamine-labelled control siRNA (shown in green). Survivin (red) localises normally to the centromeres. (D) Survivin-positive cell from same culture as E, showing abundant Survivin at the centromeres. (E) Survivin-depleted cell from a Survivin siRNA-transfected population, fixed and stained at 60 hours. No Survivin (red) is detectable at the centromeres. Bars, 5 µm.

 


View larger version (74K):

[in a new window]
  		Fig. 2. Stably expressed Survivin-GFP, but not the epitope(s) recognized by the monoclonal antibody 8E2, can also be repressed by Survivin RNAi. (A,B) HeLa cells stably expressing Survivin-GFP (green) were subjected to Survivin RNAi and immunostained for tubulin (red) and counterstained with DAPI (DNA; blue). (A) Control cell with Survivin-GFP concentrated at the centromeres (arrows). (B) The Survivin-siRNA-transfected cell is depleted of Survivin-GFP signal. (C,C'-D,D') Control or Survivin-siRNA-transfected cells stained for Survivin using the polyclonal antibody (C,D in red) and centromeres (C',D' in red); the epitope(s) recognized by the monoclonal antibody 8E2 (green) and DNA (blue). Survivin-specific siRNA does not abolish spindle staining by 8E2. Bars, 5 µm.

 


View larger version (31K):

[in a new window]
  		Fig. 3. Survivin depletion causes loss of Aurora-B and INCENP from the centromeres. (A-D) Survivin-siRNA-transfected cells were fixed at 60 hours and stained for Survivin, Aurora-B, INCENP and centromeres (using ACA). (A,B) Survivin versus Aurora-B. (A) Survivin-positive cell shows Survivin (A') and Aurora-B (A") at the centromeres. (B) The Survivin-depleted cell (B') shows Aurora-B diffusely localised with some residual staining on the chromosome arms (B"). Merged images do not show Survivin staining. (C,D) INCENP versus Aurora-B. (C) Survivin-positive cell, as judged by the presence of Aurora-B staining at the centromeres (green, C') shows centromeric INCENP staining (C"). (D) The Survivin-depleted cell shows no Aurora-B (green, D') or INCENP (D") at the centromeres. (E) A plot of the intensity of Survivin versus Aurora-B at centromeres revealed a linear correlation between the abundance of the two proteins. (F) A plot of the intensity of Aurora-B (to indicate Survivin depletion) versus INCENP at centromeres revealed a linear correlation between the abundance of the two proteins. (E,F) Units indicate fluorescence intensity (x103). Bars, 5 µm.

 


View larger version (61K):

[in a new window]
  		Fig. 4. Survivin RNAi causes accumulation in prometaphase, failure in cytokinesis and multinucleation. (A,B) Time course of mitotic staging of cells transfected with Survivin siRNA. Survivin-positive cells were scored in A and Survivin-depleted cells were scored in B. From 36 hours post transfection, the percentage of Survivin-depleted cells in prometaphase increases and the percentage of cells in later mitotic stages decreases. (C,D) Examples of Survivin-positive and Survivin-depleted cells in mitosis. Centromeres (red); tubulin (green); DNA (blue). Inset panels show the Survivin staining in the region indicated by the box. (C) Survivin-positive cell with all chromosomes aligned. (D) Survivin-depleted cells have misaligned chromosomes (arrows). (C',D') Chromosomes in C and D in greyscale. Bars, 5 µm. (E) Representative graph showing the percentage of multinucleated cells in cultures following transfection with control (red) versus Survivin (blue) siRNA. Multinucleation increases gradually in cultures transfected with Survivin siRNA. (F) Phase-contrast images of cells from cultures transfected with control or Survivin siRNAs, blocked in S-phase, released in fresh medium and followed by time-lapse since entering mitosis (which begins approximately 10 hours post-release from S-phase). Numbers in images indicate time (in hours) since entering mitosis. The two Survivin siRNA transfected cells shown spent >1 hour in mitosis and failed cytokinesis. (G) Histogram showing the percentages of cells from cultures transfected with control (n=73) or Survivin siRNAs (n=101) that spent up to 1 hour or more than 1 hour in mitosis before completing (yellow) or failing (green) cytokinesis.

 


View larger version (76K):

[in a new window]
  		Fig. 5. Survivin depletion decreases BubR1 abundance at kinetochores of maloriented chromosomes. (A,B) Immunofluorescence images of control (A) and Survivin-depleted cells 60 hours post-transfection (B). Cells are stained for BubR1 (red), Survivin (green) and DNA (blue). Panels A'-B' show the BubR1 status of the inset regions indicated in a thermal scale where blue indicates a low signal intensity, green intermediate intensity and yellow-red high. Panels A"-B" show enlargements of the lagging chromosomes shown in the insets stained with DAPI and ACA. Unattached chromosomes of control cells have a high BubR1 signal (A'), while both high (B', upper chromosome) and low signal intensities (B", lower chromosome) are found on unattached chromosomes of a Survivin-depleted cell. (C) Chromosome spreads of HeLa cells following transfection with control or Survivin siRNA. Cells were treated for 2 hours with 0.1 µg/ml colcemid to depolymerize microtubules. In all panels Survivin is shown in green and DNA in blue. Top panels show BubR1 in red and lower panels show centromeres in red. All kinetochores are potentially capable of expressing high levels of BubR1 following Survivin depression. (D) Control and Survivin-depleted cells were treated with 0.1 µg/ml colcemid for 12 hours from 60 hours post transfection. A Survivin-positive and a Survivin-depleted cell are shown. BubR1 (red, D"), Survivin (greyscale, D'), centromeres (green) and DNA (blue). Survivin-depleted cell shows no BubR1 staining at the kinetochores. Bars, 5 µm.

 


View larger version (94K):

[in a new window]
  		Fig. 6. Survivin is required for checkpoint arrest in response to taxol. (A,B) Phase-contrast images of cells from cultures transfected with control or Survivin siRNAs that were blocked in S-phase, released in medium supplemented with 33 nM taxol (A) or 0.1 µg/ml nocodazole (B) and followed every hour after entering mitosis for 8 hours. Arrows, arrowheads or asterisks indicate individual cells of interest. (C) Histogram showing the percentages of cells from cultures transfected with control (C) or Survivin siRNAs (S) that either arrested in mitosis (yellow) or exited into interphase (green) in the above experiment. (D-G) Immunofluorescence staining of cells treated as in Fig. 6A,B, stained for Aurora-B (green), DNA (blue), BubR1 (greyscale in D',E',F',G', red in D",E",F",G") and Mad2 (green in D",E",F",G"). Survivin-depleted cells (as judged by Aurora-B staining) show no little or no staining for BubR1 or Mad2 at the kinetochores in the presence of taxol. In the presence of nocodazole, Mad2 staining is present at the kinetochores of both control and Survivin-depleted cells, whereas BubR1 is either greatly depleted or absent. Bar, 5 µm.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2003