Figure 1.
Katanin p60 is localized at both the spindle pole and midbody.
A. 3Y1, RL34, and FAA indicate immunostained images of 3Y1 cells, RL34 cells, and FAA-HTC1 cells, respectively. Cells were labeled for katanin p60 (green), β-tubulin (microtubules; red), and DNA (blue). Merge indicates merged images of katanin p60, ß-tubulin, and DNA, showing the localization of katanin p60 at the spindle pole (Metaphase) and midbody (Cytokinesis) during mitosis. Scale bars: 10 µm. Samples were fixed in methanol and analyzed by fluorescence microscopy (Axioskop II; Carl Zeiss).
B. Inhibition of katanin p60 by katanin siRNA was determined by quantitative RT-PCR 24 h after transfection. Katanin p60 mRNA expression levels were normalized relative to TATA Binding Protein (TBP) mRNA as an internal control.
C. Inhibition of katanin p60 by katanin siRNA was determined by Western blotting analysis 48 h after transfection. Four different siRNAs derived from the rat katanin p60 sequence were used independently (1 - 4) and mixed (Mix). IB indicates immunoblotting with anti-katanin p60 antibody (IB: Katanin p60) and with anti-actin antibody (IB: Actin).
D. 3Y1 cells labeled for katanin p60 (green), β-tubulin (red), and DNA (blue). Merge indicates merged images of katanin p60, β-tubulin, and DNA, showing the localization of katanin p60 at the spindle pole (Metaphase) and midbody (Cytokinesis) during mitosis. 3Y1 cells were transfected with control siRNA (Control) or katanin p60 siRNA (siRNA). Scale bars: 10 µm. Samples were fixed in methanol and analyzed by confocal laser scanning fluorescence microscopy (FV-1000D; Olympus).
Figure 2.
Subcellular localization of endogenous katanin p60.
A. 3Y1 cells labeled for katanin p60 (green), β-tubulin (red), and DNA (blue). Merge indicates merged images of katanin p60, β-tubulin, and DNA, showing the localization of katanin p60 during mitosis. Pro, Meta, Ana, Telo, and Cytokinesis indicate images of prophase, metaphase, anaphase, telophase, and cytokinesis, respectively. Scale bars: 10 µm. Samples were fixed in methanol and analyzed by confocal laser scanning fluorescence microscopy (FV-1000D; Olympus).
B. Higher magnification micrograph around the central spindle in Figure 2. Telophase Merged image is shown in the upper left (Central spindle). Vertical analysis images of the distribution of katanin p60 (green), β-tubulin (red), and DNA (blue). Merge indicates merged images of katanin p60, β-tubulin, and DNA, showing a cross-section of the thin yellow line in the Midbody image. Density indicates the images of the density of katanin p60 and of ß-tubulin (microtubules) in cross-section images. White arrowheads indicate the same position of images. Scale bars: 2 µm. Analyses were performed by confocal laser scanning fluorescence microscopy (FV-1000D; Olympus).
C. Micrographs around the contractile ring. 3Y1 cells labeled for katanin p60 (green), actin (red), and DNA (blue). Merge indicates merged images of katanin p60, actin, and DNA, showing the localization of katanin p60 and the contractile ring at telophase. Cross-section analysis of the region between white arrowheads is shown in the Cross-section row. Scale bars: 5 µm (Horizontal) and 2.5 µm (Cross-section). Analyses were performed by confocal laser scanning fluorescence microscopy (FV-1000D; Olympus).
Figure 3.
Katanin p80 subcellular localization was different from katanin p60 at the midbody.
3Y1 cells were labeled for katanin p60 (green), p80 (red), and DNA (blue). Merge indicates merged images of katanin p60, p80, and DNA, showing the localization at anaphase (Anaphase) and in cytokinesis (Cytokinesis) during mitosis. Katanin p60 localization at both the midzone and midbody during cytokinesis differed from katanin p80. Scale bars: 10 µm. Samples were fixed in methanol and analyzed by fluorescence microscopy (Axioskop II; Carl Zeiss).
Figure 4.
Katanin p60 was bundled with microtubules by contractility of the contractile ring and distributed in a microtubule-dependent manner.
A. 3Y1 cells were treated with 10 µM blebbistatin for 1 h and then labeled for katanin p60 (green), actin (red), and DNA (blue). Merge indicates merged images of katanin p60, actin, and DNA, showing the localization of katanin p60, microtubules, and the contractile ring at anaphase (Ana) and telophase (Telo) during mitosis. Scale bars: 10 µm. Samples were fixed in methanol and analyzed by confocal laser scanning fluorescence microscopy (FV-1000D; Olympus).
B. 3Y1 cells were treated with 10 µM blebbistatin for 1 h and then labeled for katanin p60 (green), β-tubulin (red), and DNA (blue). Merge indicates merged images of katanin p60, β-tubulin and DNA, showing the localization of katanin p60 and microtubules at telophase (Telo) during mitosis. Cross-section showing vertical images of the distributions of katanin p60 (green), β-tubulin (red), and DNA (blue). Green arrowheads indicate vertical analysis point corresponding to the “Center” of the plane where katanin p60 was present. Red arrowheads indicate vertical analyses points corresponding to “Right” and “Left” at both sides neighboring the plane where katanin p60 was present. “B” and “T” indicate “Bottom” and “Top” of the cell, respectively. Scale bars: 10 µm. Samples were fixed in methanol and analyzed by confocal laser scanning fluorescence microscopy (FV-1000D; Olympus).
C. 3Y1 cells were treated with 10 µM nocodazole for 30 min, and then labeled for katanin p60 (green), β-tubulin (red), and DNA (blue). Merge indicates merged images of katanin p60, β-tubulin, and DNA at prophase – metaphase (Pro – Meta) and at cytokinesis (Cytokinesis). Both katanin p60 and microtubules disappeared. Scale bars: 10 µm. Samples were fixed in methanol and analyzed by fluorescence microscopy (Axioskop II; Carl Zeiss).
Figure 5.
Katanin p60 localization was restricted in the area of non-growing microtubules during cytokinesis.
3Y1 cells labeled for katanin p60 (green), EB1 (red) (EB1), and DNA (blue). Merge indicates merged images of katanin p60, EB1, and DNA, showing the localization of katanin p60 at metaphase (Meta), telophase (Telo), and cytokinesis (Cyto) during mitosis. Scale bars: 10 µm. Samples were fixed in methanol and analyzed by confocal laser scanning fluorescence microscopy (FV-1000D; Olympus).
Figure 6.
Katanin p60 destabilized midbody microtubules and affected cytokinesis in mitosis.
A. 3Y1 cells were transfected with control siRNA (Control siRNA) or either once or twice with katanin p60 siRNA (Katanin siRNA (1×) and Katanin siRNA (2×), respectively). Forty-eight hours after transfection, cells were labeled for katanin p60 (green) and β-tubulin (red). Merge indicates merged images of katanin p60 and ß-tubulin, showing the localization of katanin p60 and β-tubulin (microtubules) on the midbody at cytokinesis. Yellow and white boxes in merged images (Merge) indicate katanin p60 localized and absent regions, respectively. Scale bars: 2 µm. Samples were fixed in methanol and analyzed by confocal laser scanning fluorescence microscopy (FV-1000D; Olympus).
B. Measured ROI value ratios were calculated from the fluorescence intensities of the target regions. Calculations were performed as follows for each merged image in Figure 6A: ROI value of katanin p60 localized region (yellow boxes)/ROI value of katanin p60 absent region (white boxes).
Gray and open boxes indicate relative ROI value ratio of katanin p60 and microtubules, respectively. ROI value ratio of control siRNA-treated cells was fixed to 1.
C. Measured ROI value ratio analyses with the same method as described in Figure 6B were performed against cells treated with control siRNA (n = 22) and katanin p60 siRNA (n = 11) in four independent transfection experiments. Obtained values against microtubule ROI values were compared by t test.
Figure 7.
Katanin p60 inhibition leads to incomplete cytokinesis.
A. 3Y1 cells labeled for katanin p60 (green), microtubules (red), and DNA (blue). Control and siRNA indicate non-treated 3Y1 cells and 3Y1 cells transfected with katanin p60 siRNA, respectively. Scale bars: 5 µm. Samples were fixed in methanol and analyzed by confocal laser scanning fluorescence microscopy (FV-1000D; Olympus).
B and C. Time courses of differential interference contrast (DIC) microscopy images at mitosis of control siRNA-treated cell (B) and katanin p60 siRNA-treated cell (C). Scale bar: 20 µm.
D. Types of cytokinesis of siRNA-treated cells are shown. Open and shaded boxes indicate normal cytokinesis completion (N) and incomplete cytokinesis by regression leading to binucleate cell formation (B), respectively. Averages and SD were calculated from three independent experiments (total 13 and 15 independent observation fields of control and katanin p60 siRNA treatment, respectively). Control: control siRNA-treated cell; Katanin p60: katanin p60 siRNA-treated cell.
Figure 8.
Model for Katanin p60 function during cytokinesis.
A. Model of katanin p60 function at the midbody.
B. Katanin p60 function through the cell cycle.