Articles

Dual Role of Cdc42 in Spindle Orientation Control of Adherent Cells

Masaru Mitsushima, Fumiko Toyoshima, Eisuke Nishida
DOI: 10.1128/MCB.01713-08

Article Figures & Data

Figures

  • FIG. 1.
    FIG. 1.

    Cdc42 and Rac1 are required for the spindle orientation parallel to the substratum in HeLa cells. (A) Total lysates of M phase-synchronized HeLa cells transfected with GFP siRNA, RhoA siRNA, Rac1 siRNA, or Cdc42 siRNA were subjected to immunoblotting with anti-RhoA, anti-Rac1, anti-Cdc42, and anti-α-tubulin antibodies. (B) The X-Z projections of metaphase cells as prepared in panel A and stained with anti-γ-tubulin (green) and Hoechst (blue). (C) The spindle angle (left; α°) was measured in metaphase cells. Distribution (right; histogram; n = 50) and the average (right; inset; mean ± standard deviation; n = 50) of spindle angles in each condition are shown (right). **, P value of <0.001 compared with that of control GFP siRNA, analyzed by F-test.

  • FIG. 2.
    FIG. 2.

    Cdc42 is required for the activation of PI(3)K during mitosis. (A) Total lysates of M phase-synchronized HeLa cells transfected with GFP siRNA, RhoA siRNA, Rac1 siRNA, or Cdc42 siRNA were subjected to immunoblotting with anti-RhoA, anti-Rac1, anti-Cdc42, anti-phospho-Akt (Ser473), anti-Akt, anti-cyclin B1, and anti-α-tubulin antibodies. (B) Total lysates of synchronized cells transfected with GFP siRNA, Cdc42 siRNA, or β1 integrin siRNA were subjected to immunoblotting with anti-phospho-Akt (Ser473), anti-Akt, anti-Cdc42, anti-β1 integrin, anti-cyclin B1, anti-cyclin A2, anti-cyclin E, and anti-α-tubulin antibodies. (C) Total lysates of M phase-synchronized cells transfected with GFP siRNA or Cdc42 siRNA, pretreated with LY294002 for 2 h, washed with condition medium, and exposed to carrier histone or PIP3-histone for 5 min or 10 min were subjected to immunoblotting with anti-phospho-Akt (Ser473) and anti-Akt antibodies.

  • FIG. 3.
    FIG. 3.

    Cdc42 and PAK2 regulate actin reorganization during mitosis. (A) Z-stack images (2.5 μm apart) with phalloidin (green) and Hoechst (blue) in metaphase HeLa cells transfected with GFP siRNA, RhoA siRNA, Rac1 siRNA, or Cdc42 siRNA. The scale bar represents 10 μm. (B) Total lysates of M phase-synchronized cells transfected with GFP siRNA or PAK2 siRNA were subjected to immunoblotting with anti-PAK2, anti-PAK1, and anti-α-tubulin antibodies. (C) Total lysates of M phase-synchronized HeLa cells transfected with GFP siRNA or PAK2 siRNA were subjected to immunoblotting with anti-phospho-Akt (Ser473) and anti-Akt antibodies. (D) Z-stack images (2.5 μm apart) with phalloidin (green) and Hoechst (blue) in metaphase cells transfected with PAK2 siRNA. The scale bar represents 10 μm.

  • FIG. 4.
    FIG. 4.

    PAK2 is required for the proper spindle orientation. (A) The X-Z projections of metaphase cells transfected with GFP siRNA or PAK2 siRNA and stained with anti-γ-tubulin (green) and Hoechst (blue). (B) Spindle orientation analyses of the cells transfected with the indicated siRNAs. Distribution (histogram; n = 50) and the average (inset; mean ± standard deviation; n = 50) of spindle angles in each condition are shown. **, P value of <0.001 compared with that of control GFP siRNA, analyzed by F-test. (C) Total lysates of M phase-synchronized cells transfected with GFP siRNA or PAK2 siRNA together with pEGFP or pEGFP-PAK2-res were subjected to immunoblotting with anti-PAK2, anti-GFP, and anti-α-tubulin antibodies. (D) Spindle orientation analysis in the cells transfected with PAK2 siRNA together with pEGFP or pEGFP-PAK2-res. Distribution (histogram; n = 50) and the average (inset; mean ± standard deviation; n = 50) of spindle angles in each condition are shown. **, P value of <0.001 compared with that of the control pEGFP-transfected cells, analyzed by F-test.

  • FIG. 5.
    FIG. 5.

    PAK2 regulates spindle orientation in a Cdc42/Rac1 binding-dependent and kinase activity-independent manner. (A) Total lysates of M phase-synchronized HeLa cells transfected with PAK2 siRNA together with pEGFP, pEGFP-PAK2-res, or pEGFP-PAK2-res-H82, 85L were subjected to immunoblotting with anti-PAK2, anti-GFP, and anti-α-tubulin antibodies. (B) Spindle orientation analysis in the cells transfected with PAK2 siRNA together with pEGFP, pEGFP-PAK2-res, or pEGFP-PAK2-res-H82, 85L. Distribution (histogram; n = 50) and the average (inset; mean ± standard deviation; n = 50) of spindle angles in each condition are shown. **, P value of <0.001 compared with that of the control pEGFP-transfected cells, analyzed by F-test. (C) Total lysates of M phase-synchronized HeLa cells transfected with GFP siRNA or PAK2 siRNA together with pEGFP, pEGFP-PAK2-res, or pEGFP-PAK2-res-K278R were subjected to immunoblotting with anti-PAK2, anti-GFP, and anti-α-tubulin antibodies. (D) Spindle orientation analysis in the cells transfected with PAK2 siRNA together with pEGFP, pEGFP-PAK2-res, or pEGFP-PAK2-res-K278R. Distribution (histogram; n = 50) and the average (inset; mean ± standard deviation; n = 50) of spindle angles in each condition are shown. *, P value of <0.01 and **, P value of <0.001 compared with those of control pEGFP-transfected cells, analyzed by F-test.

  • FIG. 6.
    FIG. 6.

    PAK2 regulates spindle orientation through binding to βPix. (A) Total lysates (80 μl) of synchronized HeLa cells were incubated with anti-PAK2 antibody or rabbit IgG. Total lysates (7.5 μl) and the precipitates were subjected to immunoblotting with anti-βPix, anti-Git1, and anti-PAK2 antibodies. (B) Total lysates of M phase-synchronized HeLa cells transfected with or without (mock) GFP siRNA, βPix siRNA, or Git1 siRNA. GFP siRNA were subjected to immunoblotting with anti-βPix, anti-Git1, and anti-α-tubulin antibodies. (C) Spindle orientation analyses in the cells transfected with GFP siRNA, βPix siRNA, or Git1 siRNA. Distribution (histogram; n = 50) and the average (inset; mean ± standard deviation; n = 50) of spindle angles in each condition are shown. **, P value of <0.001 compared with that of GFP siRNA-transfected cells, analyzed by F-test. (D) Total cell lysates of the cells transfected with pcDL-SRα-myc-PAK2-res, PAK2-res-P185A/R186A, or PAK2-res-H82, 85L were incubated with anti-Myc antibody. The precipitates were subjected to immunoblotting with anti-βPix and anti-Myc antibodies. (E) Total lysates of M phase-synchronized HeLa cells transfected with GFP siRNA or PAK2 siRNA together with or without (mock) pcDL-SRα-myc-PAK2-res or PAK2-res-P185A/R186A were subjected to immunoblotting with anti-PAK2, anti-Myc, and anti-α-tubulin antibodies. (F) Spindle orientation analyses of the cells as prepared in panel E. Distribution (histogram; n = 50) and the average (inset; mean ± standard deviation; n = 50) of spindle angles in each condition is shown. **, P value of <0.001 compared with that of control GFP siRNA-transfected (GFPsi)/mock cells, analyzed by F-test. (G) Total lysates of M phase-synchronized HeLa cells transfected with GFP siRNA or βPix siRNA were subjected to immunoblotting with anti-phospho-Akt (Ser473) and anti-Akt antibodies. (H) Z-stack images (2.5 μm apart) with phalloidin (green) and Hoechst (blue) in metaphase cells transfected with βPix siRNA. The scale bar represents 10 μm.

  • FIG. 7.
    FIG. 7.

    Cdc42, PAK2, and βPix regulate the distribution of cortical dynactin. (A) Z-stack images (2.5 μm apart) with p150Glued (green) and Hoechst (blue) in metaphase cells transfected with GFP siRNA, Cdc42 siRNA, PAK2 siRNA, or βPix siRNA. The scale bar represents 15 μm. (B) Quantification of the widths of distribution for cortical dynactin along the z axis in metaphase cells transfected with the indicated siRNAs. Values are means ± standard deviations. (n = 20). (C) A model for the two distinct pathways downstream from Cdc42 that control the spindle orientation in nonpolarized adherent cells.

Additional Files

  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 - Movies S1 to S4 (siRNA-transfected cells)
      Zipped MPG files, 2.9MB.
    • Supplemental file 2 - Fig. S1 PtdIns(3,4,5)P3-induced PI(3)K activation, S2 (GFP signals in siRNA-transfected cells), S3 (Cdc42 knockdown and misaligned chromosomes), S4 (Misoriented spindle assembly in siRNA-transfected cells), S5 (Dominant-negative Cdc42 and spindle misorientation), S6 (Cdc42 and Rac1 knockdown and cell area of metaphase HeLa cells), S7(Cdc42 or PAK2 knockdown and retraction fiber number), and S8 (Spindle formation in PAK2-depleted cells)
      Zipped PDF file, 1.8MB.
    • Supplemental file 3 - Fig. S9 and S10 (PAK2 and spindle orientation in MCF10A [S9] and HeLa [S10] cells), S11 (Cdc42-dependent PAK2 localization), S12 [PI(3)K and PAK2-βPix interaction], S13 [PAK2 and PI(3)K in spindle orientation[, S14 (Spindle orientation in Cdc42- or β1 integrin-depleted cells), S15 (PAK1 and βPix in spindle orientation control), and S16 (PAK2 depletion and Aurora A activation)
      Zipped PDF file, 2.1MB.
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KEYWORDS

cell adhesion
cell polarity
Spindle Apparatus
cdc42 GTP-Binding Protein

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