Figure 1.
CEP192 at centriolar wall is sufficient for bipolar spindle formation.(A) Time-lapse observation of the structure of microtubules upon siRNA treatment against the indicated proteins. HeLa cells expressing EGFP-centrin1 and mCherry-NuMA were observed with a 40× objective. Gray represents SiR-tubulin. mCherry-NuMA and EGFP-centrin1 are not shown. Z-projections: 10 planes, 2.2 µm apart. Scale bar, 10 µm. Time zero corresponds to NEBD. (B) Mitotic duration, the time required from NEBD to cytokinesis, in A. Line and error bars represent the mean and SD (n ≥ 50 cells from two independent experiments). Kruskal–Wallis test was used to determine the significance of the difference. *, P < 0.05; n.s., not significant. (C) The localization of PCM proteins in mitotic spindles of the cells in which the indicated protein was depleted. Red and green represent PCM proteins (CDK5RAP2, CEP192, or pericentrin) and GT335, respectively. Z-projections of 10 sections, every 0.3 µm. Scale bar, 1 µm. (D) The signal intensity of PCM proteins on mitotic centrosomes of fixed HeLa cells was analyzed (n > 45 for each condition). Line and error bars represent median with interquartile range. Kruskal–Wallis test was used to determine the significance of the difference. *, P < 0.05; **, P < 0.0001. (E) STED images showing centriolar distribution of CEP192 in pericentrin/CDK5RAP2 double-depleted cells. HeLa cells were treated with control siRNA or pericentrin/CDK5RAP2 siRNA for 48 h and stained with the indicated antibodies. Scale bar, 1 µm. (F and G) Representative line intensity profiles (F) and measured diameters (G) of GT335 and CEP192. The line profiles were measured along the dotted lines in E. The profiles were fitted with double Gaussian curves, and the distances between the half-maximal intensity points at the far ends were measured as the diameters (schematically indicated with dotted lines and arrows in the profiles; fitted curves are not shown). Horizontal bars and error bars in the plots for the diameters represent median and interquartile range. n = 18 (for siControl) or 22 (for siPericentrin/CDK5RAP2) centrosomes; data from two independent experiments were pooled. Mann–Whitney U test was used to determine the significance of the difference. *, P < 0.0001; n.s., not significant.

CEP192 at centriolar wall is sufficient for bipolar spindle formation. (A) Time-lapse observation of the structure of microtubules upon siRNA treatment against the indicated proteins. HeLa cells expressing EGFP-centrin1 and mCherry-NuMA were observed with a 40× objective. Gray represents SiR-tubulin. mCherry-NuMA and EGFP-centrin1 are not shown. Z-projections: 10 planes, 2.2 µm apart. Scale bar, 10 µm. Time zero corresponds to NEBD. (B) Mitotic duration, the time required from NEBD to cytokinesis, in A. Line and error bars represent the mean and SD (n ≥ 50 cells from two independent experiments). Kruskal–Wallis test was used to determine the significance of the difference. *, P < 0.05; n.s., not significant. (C) The localization of PCM proteins in mitotic spindles of the cells in which the indicated protein was depleted. Red and green represent PCM proteins (CDK5RAP2, CEP192, or pericentrin) and GT335, respectively. Z-projections of 10 sections, every 0.3 µm. Scale bar, 1 µm. (D) The signal intensity of PCM proteins on mitotic centrosomes of fixed HeLa cells was analyzed (n > 45 for each condition). Line and error bars represent median with interquartile range. Kruskal–Wallis test was used to determine the significance of the difference. *, P < 0.05; **, P < 0.0001. (E) STED images showing centriolar distribution of CEP192 in pericentrin/CDK5RAP2 double-depleted cells. HeLa cells were treated with control siRNA or pericentrin/CDK5RAP2 siRNA for 48 h and stained with the indicated antibodies. Scale bar, 1 µm. (F and G) Representative line intensity profiles (F) and measured diameters (G) of GT335 and CEP192. The line profiles were measured along the dotted lines in E. The profiles were fitted with double Gaussian curves, and the distances between the half-maximal intensity points at the far ends were measured as the diameters (schematically indicated with dotted lines and arrows in the profiles; fitted curves are not shown). Horizontal bars and error bars in the plots for the diameters represent median and interquartile range. n = 18 (for siControl) or 22 (for siPericentrin/CDK5RAP2) centrosomes; data from two independent experiments were pooled. Mann–Whitney U test was used to determine the significance of the difference. *, P < 0.0001; n.s., not significant.

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