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. 2013 Feb;42(2):460-8.
doi: 10.3892/ijo.2012.1730. Epub 2012 Dec 7.

Centrosomal localization of RhoGDIβ and its relevance to mitotic processes in cancer cells

Yong-Sheng Jiang  1 , Masayo Maeda, Mayumi Okamoto, Mikiko Fujii, Ryuichiro Fukutomi, Masato Hori, Masaaki Tatsuka, Takahide Ota
Affiliations

Centrosomal localization of RhoGDIβ and its relevance to mitotic processes in cancer cells

Yong-Sheng Jiang et al. Int J Oncol. 2013 Feb.

Abstract

Rho GDP-dissociation inhibitors (RhoGDIs) are regulators of Rho family GTPases. RhoGDIβ has been implicated in cancer progression, but its precise role remains unclear. We determined the subcellular localization of RhoGDIβ and examined the effects of its overexpression and RNAi knockdown in cancer cells. Immunofluorescence staining showed that RhoGDIβ localized to centrosomes in human cancer cells. In HeLa cells, exogenous GFP-tagged RhoGDIβ localized to centrosomes and its overexpression caused prolonged mitosis and aberrant cytokinesis in which the cell shape was distorted. RNAi knockdown of RhoGDIβ led to increased incidence of monopolar spindle mitosis resulting in polyploid cells. These results suggest that RhoGDIβ has mitotic functions, including regulation of cytokinesis and bipolar spindle formation. The dysregulated expression of RhoGDIβ may contribute to cancer progression by disrupting these processes.

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Figures

Figure 1.
Figure 1.
Protein expression levels of RhoGDIβ in mouse organs and cell lines. (A) Organs were collected from normal female mice and the levels of RhoGDIβ proteins were determined by immunoblot analysis using anti-RhoGDIβ antibody (sc-6047). Images of representative immunoblots from two independent experiments are shown. (B) Whole-cell lysates were prepared from the indicated human cell lines and the levels of RhoGDIβ protein were determined by immunoblot analysis using anti-RhoGDIβ antibody (556498). Images of representative immunoblots from at least three independent experiments are shown. RhoGDIα, which is a universally expressed RhoGDI, was stained using anti-RhoGDIα antibody (sc-360) as a loading control.
Figure 2.
Figure 2.
Colocalization of RhoGDIβ and γ-tubulin in cultured cells. (A) DLD-1 cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, fixed again with 4% paraformaldehyde, then stained simultaneously with anti-RhoGDIβ (sc-6047) (green) and anti-γ-tubulin (red) antibodies and with DAPI (blue) (upper panels). Pretreatment with antigen peptide abolished the staining by anti-RhoGDIβ antibody (lower panels). Arrows indicate centrosomes in interphase cells and arrowheads indicate centrosomes in mitotic cells. (B) LoVo, HT-29 and HCT116 human colon cancer cells were fixed and stained as in (A). Arrows indicate centrosomes. (C) OKF6/TERT-2 cells were fixed with methanol and stained as in (A) (upper panels). Pretreatment with antigen peptide abolished the staining by anti-RhoGDIβ antibody (lower panels). Arrows indicate centrosomes. (D) HeLa cells were fixed and stained as in (A) (upper panels). Pretreatment with antigen peptide abolished the staining by anti-RhoGDIβ antibody (lower panels). Arrows indicate centrosomes. Scale bars indicate 10 μm. Representative images from more than three independent experiments are shown.
Figure 3.
Figure 3.
Subcellular localization of GFP-RhoGDIβ in HeLa cells. (A) Whole-cell lysates were prepared from HeLa cells expressing GFP-RhoGDIβ. The levels of the GFP-RhoGDIβ protein (left panel) and endogenous RhoGDIβ protein (right panel) were determined by immunoblotting analysis using anti-GFP and anti-RhoGDIβ (556498) antibodies. (B) GFP fluorescence images (upper panels) and DIC images (lower panels) of HeLa cells expressing GFP-RhoGDIβ. Scale bars, 20 μm. (C) Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, fixed again with 4% paraformaldehyde, then stained simultaneously with anti-GFP (green) and anti-γ-tubulin (red) antibodies and with DAPI (blue). Images were acquired on an LSM710 confocal microscope. Arrows indicate centrosomes. Scale bars, 5 μm. Representative images are shown from at least three independent experiments.
Figure 4.
Figure 4.
Time-lapse analysis of mitosis of HeLa cells expressing GFP-RhoGDIβ. Cells were cultured in 35-mm glass-bottomed dishes. GFP fluorescence images and DIC images were acquired on an Axiovert 200 M inverted microscope every 5 min for 30 h. (A) Incidence of mitotic abnormalities. (B) Representative images of severely distorted cell shape during cytokinesis of GFP-RhoGDIβ-expressing cell. Bars, 20 μm. (C) Among the cells observed (A), the mitotic cells, in which the mitotic processes were clearly distinguished by the criteria as described in Materials and methods and the entire mitotic duration was less than 3 h, were analyzed. Horizontal columns indicate the mitotic progression of individual cells. n=43, GFP-empty; n=20, GFP-RhoGDIβ. (D) The mean values of mitotic duration of cells (C) were compared. Bars indicate SD. *p<0.005 by the Mann-Whitney U-test.
Figure 5.
Figure 5.
Effects of siRNA knockdown of RhoGDIβ in HeLa cells. (A) Stealth RNAi duplexes were transfected into HeLa cells and the protein levels of RhoGDIβ and RhoGDIα were determined by immunoblot analysis using anti-RhoGDIβ (556498) or anti-RhoGDIα/RhoGDIβ (66586E) antibodies at the indicated time after transfection. To estimate the degree of knockdown serially diluted samples of negative control were immunoblotted. Similar results were obtained from three independent experiments. (B and C) Cells were fixed with methanol 72 h after transfection of Stealth RNAi duplexes and stained with anti-γ-tubulin antibody (red) and DAPI (blue). The number of abnormal metaphases, such as (B) monopolar spindle and (C) multiple centrosomes, was counted under a fluorescence microscope. The total numbers of metaphases observed were 176, 229, 276 and 233 for the negative control, nos. 153, 469 and 800, respectively. Similar results were obtained from two independent experiments. (D) Representative images of aberrant position and number of centrosomes observed in RhoGDIβ knockdown cells. (E) DNA histograms were obtained by flow cytometry 72 h after transfection of Stealth RNAi duplexes. Similar results were obtained from three independent experiments.
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