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, 17 (1), 85-91

Cyclin A2 Regulates Nuclear-Envelope Breakdown and the Nuclear Accumulation of Cyclin B1

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Cyclin A2 Regulates Nuclear-Envelope Breakdown and the Nuclear Accumulation of Cyclin B1

Delquin Gong et al. Curr Biol.

Abstract

Mitosis is thought to be triggered by the activation of Cdk-cyclin complexes. Here we have used RNA interference (RNAi) to assess the roles of three mitotic cyclins, cyclins A2, B1, and B2, in the regulation of centrosome separation and nuclear-envelope breakdown (NEB) in HeLa cells. We found that the timing of NEB was affected very little by knocking down cyclins B1 and B2 alone or in combination. However, knocking down cyclin A2 markedly delayed NEB, and knocking down both cyclins A2 and B1 delayed NEB further. The timing of cyclin B1-Cdk1 activation was normal in cyclin A2 knockdown cells, and there was no delay in centrosome separation, an event apparently controlled by the activation of cytoplasmic cyclin B1-Cdk1. However, nuclear accumulation of cyclin B1-Cdk1 was markedly delayed in cyclin A2 knockdown cells. Finally, a constitutively nuclear cyclin B1, but not wild-type cyclin B1, restored normal NEB timing in cyclin A2 knockdown cells. These findings show that cyclin A2 is required for timely NEB, whereas cyclins B1 and B2 are not. Nevertheless cyclin B1 translocates to the nucleus just prior to NEB in a cyclin A2-dependent fashion and is capable of supporting NEB if rendered constitutively nuclear.

Figures

Figure 1
Figure 1
The timing of mitotic entry in HeLa cells treated with cyclin d-siRNAs. (A–D) Cells were synchronized by double thymidine block and transfected with a mitotic biosensor (MBS) and various diced siRNAs. (A) Cyclin levels of d-siRNA-treated HeLa cells as assessed by immunoblotting. (B) Scoring NEB with the MBS. Four frames from a 14 h time-lapse video are shown. The time stamps (in hours and minutes) indicate the time after release from double thymidine block. (C) The timing of NEB in d-siRNA-treated cells released from double thymidine block. Approximately 500 cells (442–540) were counted for each treatment. (D) Final cumulative % NEB 19 h after release from double thymidine block. Data are expressed as means ± S.E. from six movies (two movies per well, three wells per d-siRNA treatment). (E, F) Rescue of normal mitotic timing with an RNAi-resistant cyclin A2 construct. Cells were synchronized by double thymidine block and transfected with the MBS plus one of two diced siRNAs (GL3 d-siRNAs or d-siRNAs derived from the cyclin A2 3’-UTR) and one of two rescue constructs (a YFP “sham” rescue construct or a cyclin A2-YFP rescue construct lacking the cyclin A2 3’-UTR). (E) Cyclin levels of d-siRNA-treated HeLa cells at two time points, 5 h and 20 h after release from double thymidine block, as assessed by immunoblotting. α-Tubulin was used as a loading control. (F) The timing of NEB in cells treated with d-siRNAs plus a cyclin A2 or a YFP sham rescue construct. Between 279 and 405 cells were counted for each treatment. NEB was assessed by epifluroescence time-lapse microscopy. The bar graph (inset) shows the final cumulative % NEB at 19 h, expressed as means ±S.E. for data from three individual wells. Note that mitotic entry was consistently slower in cells transfected with 10 ng DNA (panels E, F) than with 5 ng (panels A–D).
Figure 2
Figure 2
DNA content in d-siRNA-treated HeLa cells released from double thymidine block. (A) Cyclin levels 20 h after release, as assessed by immunoblotting. (B) DNA content. Cells treated with various cyclin d-siRNAs were harvested at various times after release from double thymidine block, fixed and stained with propidium iodide, and subjected to flow cytometry to assess DNA content.
Figure 3
Figure 3
Cyclin-Cdk activities and localization in cells treated with cyclin d-siRNAs. (A, B) Timing of cyclin A2-Cdk and cyclin B1-Cdk activation. Cells were treated with the indicated d-siRNAs, lysed at various times after release from double thymidine block, and subjected to immunoprecipitation with cyclin A2 or cyclin B1 antibodies followed by histone H1 kinase assays. Activities were quantified by phosphorimaging. Data are expressed as means ±S.E. from four or five independent experiments. (C) Montages of centrosome separation, cyclin B1 nuclear accumulation, and NEB in a GL3-knockdown cell and a cyclin A2 knockdown cell. Movies of these cells are provided as Supplemental Data. (D) Cumulative data on the timing of centrosome separation, nuclear accumulation of cyclin B1, and NEB in synchronized cells treated with GL3 or cyclin A2 d-siRNAs. Data are shown from 92 GL3-treated cells and 159 cyclin A2 knockdown cells. (E) The interval between centrosome separation and NEB in GL3- and cyclin A2-knockdown cells. We included all cells that carried out centrosome separation during the first 11 hours after release from double thymidine block (37 GL3-knockdown cells and 41 cyclin A2-knockdown cells) so that we could follow each cell for at least an additional 8 hours.
Figure 4
Figure 4
Rescue of normal NEB timing by expression of a constitutively-nuclear cyclin B1. (A) Expression levels of endogenous cyclin B1, WT-cyclin B1-YFP and NLS-cyclin B1-YFP in GL3-treated and cyclin A2-knockdown cells (top blots). The efficacy of the cyclin A2 knockdown was assessed by cyclin A2 immunoblotting (bottom blots). (B) Localization of WT-cyclin B1-YFP and NLS-cyclin B1 in interphase cells. The localization of the mitotic biosensor (MBS) is shown for comparison. (C) The timing of NEB in cells treated with GL3 or cyclin A2 d-siRNAs plus one of three rescue constructs: YFP (sham), WT-cyclin B1-YFP, or NLS-cyclin B1-YFP. Between 345 and 474 cells were counted for each treatment. NEB was assessed by epifluroescence time-lapse microscopy. The bar graph (inset) shows the final cumulative % NEB at 19 h, expressed as means ± S.E. for data from three individual wells. (D) Schematic depiction of the regulation of centrosome separation and NEB.

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