Regulation of Cdc42 activation in mitosis by Ect2 and MgcRacGAP. (A) Cell cycle–associated changes in the level of the GTP-bound form of each Rho GTPase. HeLa S3 cells were synchronized, collected in G2 phase (lane 1), prometaphase (lane 2), metaphase (lane 3), telophase (lane 4), and G1 phase (lane 5), respectively, and subjected to the pull-down assay to detect GTP-Cdc42 or GTP-Rac, as described in Materials and methods. Typical immunoblots are shown on the left, and the results of the quantitative analysis are on the right. Values are shown as means ± SEM (n = 4 and 3 for Cdc42 and Rac, respectively). (B) Suppression of Cdc42 activation in metaphase by expression of a dominant-negative mutant of Ect2. Synchronized HeLa cells were transfected with pCEV32F-Ect2-N1, collected at various phases of the cell cycle, and subjected to the pull-down assay as described in Materials and methods. The top diagram shows the domain structures of full-length Ect2 and Ect2-N1 (BRCT, BRCA carboxyl terminus domain; DH, Dbl homology domain; PH, pleckstrin homology domain; NLS, nuclear localization signal). The middle panel is representative of a typical experiment, and the bottom panel represents the results of quantitative analysis of three independent experiments (open bars, experiments with Ect2-N1; closed bars, the control experiment shown in A). (C) Prevention of down-regulation of Cdc42 by expression of a catalytically inactive mutant of MgcRacGAP. Synchronized HeLa cells were transfected with pME18S-MgcRacGAP^R386A, collected at various phases of the cell cycle, and subjected to the pull-down assay as described in Materials and methods. Top diagrams show the structure of MgcRacGAP and MgcRacGAP^R386A (coiled-coil, coiled-coil domain; RhoGAP, GTPase activating homology domain). The middle panel is representative of a typical experiment, and the bottom panel represents the results of quantitative analysis of three independent experiments (open bars, experiments with MgcRacGAP^R386A; closed bars, the control experiment shown in A).

RNAi for Ect2 and its effects on accumulation of GTP-Cdc42 in metaphase. (A) Depletion of Cdc42 or Ect2 by RNAi. HeLa cells were subjected to RNAi for Ect2 (Ect2 RNAi), Cdc42 (Cdc42 RNAi), or E. coli LacZ (control RNAi). After 24 and 48 h, the cells were lysed and subjected to immunoblot with the respective antibody. (B) Effects of Ect2 depletion on accumulation of GTP-Cdc42 in metaphase. HeLa S3 cells were transfected with control or Ect2 siRNA after the first thymidine block. The cells then underwent a second thymidine block and, after release from this block, were collected at different phases of the cell cycle and subjected to the pull-down assay. One sixth of the precipitates and one twentieth of the supernatant were used to show the amount of GTP-Cdc42 and total Cdc42, respectively. The top panel represents typical immunoblots and the bottom represents the results of quantitative analysis of three independent experiments (closed bars, experiments with control RNAi; open bars, Ect2 RNAi experiment). Values are shown as means ± SEM (n = 3).

Prometaphase delay by RNAi for Cdc42 or Ect2 in HeLa cells. (A) Mitotic index. Cells subjected to RNAi were synchronized in S phase by double thymidine block, and were fixed at 12 h after release from the second block. The RNAi cells were identified by coexpression of pEGFP-histone H2Bk. The cells were stained for MTs and chromosomes and the mitotic index was calculated by examining more than 250 cells in each of four independent experiments. Results are the mean ± SEM (n = 4). *, P < 0.05 versus control RNAi cells. (B) Immunofluorescence. Cells treated as in A and expressing EGFP-H2Bk (green) were stained for chromosomes (DAPI, blue) and β-tubulin (red). Arrowheads indicate misaligned chromosomes. (C) Prometaphase delay. Mitotic cells in each population were examined and the percentages of cells in prometaphase (cells with misaligned chromosomes), metaphase (cells with chromosomes aligned at the metaphase plate), or ana/telophase (cells showing chromosome segregation) were determined. Results are mean ± SEM (n = 3). (D) Mad2 staining. Cells treated as in A were subjected to staining for Mad2 (green), CENP-A (red), and chromosomes (DAPI, blue). Insets are enlargements of the indicated regions of interest. (E) Production of aberrant multinucleate cells. Cells subjected to respective RNAi were fixed at 14 h after the release and stained for β-tubulin (red) and chromosomes (DAPI, blue and right column). Arrowheads show micronuclei. (F) mDia3 localization. HeLa cells subjected to control RNAi or RNAi for Ect2 or Cdc42 were subjected to staining for mDia3 (green), CENP-A (red), and with DAPI (blue). Bars: (B, E, and F) 10 μm; (D) 2 μm.

Ect2 and Cdc42 depletion induce abnormalities in chromosome alignment and segregation in HeLa cells. HeLa cells were cotransfected with pEGFP-EB1 and pDsRed2-histone H2Bk together with control siRNA (A), Ect2 siRNA (B), or Cdc42 siRNA (C) and time-lapse imaging was taken after 48 h. The bottom panels of each set show the EGFP-EB1 images only. The movies of these time-lapse acquisitions are available online as Videos 1–3 (available at http://www.jcb.org/cgi/content/full/jcb.200408085/DC1).

Effects of overexpression of Ect2-N1 on mitosis of HeLa cells. (A) Mitotic phenotype. HeLa cells synchronized in S phase were transfected with pEGFP (a and b) or pEGFP-Ect2-N1 (c–h). The cells were fixed 120 min after nocodazole removal and subjected to fluorescence microscopy for GFP (a, c, and e, green), anti–β-tubulin staining (g, green), and DAPI (a–h, blue). The Ect2-N1-expressing cells show the binucleate phenotype (c and d), the multinucleate phenotype with macro- and micro-nuclei (e and f), or the prometaphase arrest (g and h). (B) Mad2 staining. The Ect2-N1–expressing cells were treated as in A and stained for CENP-A (red), Mad2 (green), and chromosomes (DAPI, blue). Positive Mad2 staining was detected at some kinetochores. (C) Quantitative analysis of the mitotic phenotypes of the cells expressing Ect2-N1. These experiments were repeated seven times, and the numbers of cells showing each mitotic phenotype were determined in more than 200 cells each experiment. Results shown are the mean ± SEM (n = 7). *, P < 0.05 versus each corresponding subpopulation of the pEGFP-transfected cells. Bars: (A) 10 μm; (B) 2 μm.

Effects of overexpression of MgcRacGAP^R386A on mitosis of HeLa cells. (A) Mitotic phenotypes. HeLa cells synchronized in S phase were transfected with either pEGFP alone or pEGFP and pME18S-MgcRacGAP^R386A (a–f). At 120 min after nocodazole removal, the cells were fixed and subjected to fluorescence microscopy for GFP (a and c, green), anti–β-tubulin staining e, green), and DAPI (a–f, blue). MgcRacGAP^R386A-expressing cells show the binucleate phenotype (a and b), the multinucleated phenotype with macro- and micro-nuclei (c and d), or the prometaphase arrest (e and f). (B) Mad2 staining. The MgcRacGAP^R386A-expressing cells were treated as in A and stained for CENP-A (red), Mad2 (green), and chromosomes (DAPI, blue). Positive Mad2 staining was detected at some kinetochores. (C) Quantitative analysis of the mitotic phenotypes of the cells expressing MgcRacGAP^R386A. The mitotic phenotype of HeLa cells expressing GFP, MgcRacGAP^R386A (MRG-R386A), or Cdc42^G12V was examined in more than 200 cells for each experiment, and the percentages of cells showing prometaphase arrest, binucleate, or aberrant multinucleate phenotypes were determined. Results represent the mean ± SEM (n = 7). *, P < 0.05 versus each corresponding subpopulation of the pEGFP-transfected cells. Bars: (A) 10 μm; (B) 2 μm.

Localization of Cdc42 in mitotic cells. (A) Specificity of the antibody to Cdc42. One fiftieth of HeLa cell extracts from one 10-cm dish (lane 1) or one fourth of the pull-down precipitates of lysates from four dishes of HeLa cells at metaphase using the GST-CRIB-Pak (lane 2) were probed with the monoclonal anti-Cdc42 antibody. (B) Cdc42 localization in mitotic HeLa S3 cells. Randomly growing HeLa S3 cells were preextracted for 1 min with 0.5% Triton X-100 in PHEM, fixed, and stained for Cdc42 (green), MgcRacGAP (red), and chromosomes (DAPI, blue). Immunofluorescence images of the cells in each phase of mitosis are shown. (C) Competition experiments. The cells were treated as in B and subjected to staining for Cdc42 (red) and chromosomes (DAPI, blue) in the presence of the immunogenic peptide. A single representative cell in metaphase is shown (a, merged image; b, staining for Cdc42 alone). (D) Effects of MgcRacGAP^R386A or Ect2-N1 expression on distribution of Cdc42 on the metaphase spindle. The cells were cotransfected with MgcRacGAP^R386A together with EGFP (a–d) to allow the identification of transfected cells, or with EGFP-Ect2-N1 (e–h) and treated as in B. Chromosomes were stained with DAPI (a, c, e, and g, blue). Cdc42 was stained with monoclonal anti-Cdc42 antibody and detected with goat anti–mouse IgG coupled to Alexa-647 and pseudo-colored to green (a–d) or with donkey anti–mouse IgG coupled to Alexa-594 shown in red (e–h). MgcRacGAP is shown in red (a–d). EGFP-Ect2-N1 is shown in green (e–h). Two different optical sections of a single cell are shown each in a and b and c and d. Bars, 10 μm.