Figure 9.
Centrosome age also breaks spindle symmetry in human fibroblast cells. (A) Immunofluorescence image of 2:2 BJ metaphase cell stained with DAPI (blue), cenexin (green), and pericentrin (magenta) antibodies. (B) Quantification of the SAI in 2:2 BJ cells. SAI mean of 3.1 ± 7.6%, n = 106 cells, P < 0.0001 in one-sample t test. (C) Immunofluorescence images of 1:1 BJ late telophase cell stained with DAPI (blue), centrin (green), cenexin (yellow) antibodies and the cell mask (magenta). (D) Quantification of the daughter cell area asymmetry of 1:1 BJ cells. Daughter cell size asymmetry of 4.2 ± 7.8%, n = 47 cells, P = 0.0007 in one-sample t test. (E) Quantification of the relative protein distribution of pericentrin and TPX2 versus centrosome age in 2:2 BJ cells. Relative pericentrin distribution of 8.7 ± 12.1%, n = 56 cells, P < 0.0001. Relative TPX2 distribution of 1.7 ± 9.9%, n = 51 cells, P = 0.4329. A one-sample t test was used for pericentrin statistical analysis and a one-sample Wilcoxon-test was used for TPX2 statistical analysis. (F) Correlation between the relative pericentrin/TPX2 distribution (x axis) and the SAI (y axis) in 2:2 BJ cells. Each dot represents a cell for which the relative pericentrin/TPX2 distribution and the SAI were measured. The Pearson correlation coefficient and its associated P value, the number of cells analyzed as well as the slope of the linear regression (light green line) are indicated for each condition. (G) Immunofluorescence images of siCtrl- and siPCNT-treated 2:2 BJ cells, stained with DAPI (blue), cenexin (green), and pericentrin (magenta) antibodies. (H) Quantification of pericentrin depletion efficiency of siCtrl- and siPCNT-treated 2:2 BJ cells. Pericentrin fluorescence intensity means of 100%, n = 33 cells in siCtrl and 4.2 ± 1.4%, n = 30 cells in siPCNT cells. (I) Quantification of the SAI of siCtrl- and siPCNT-treated 2:2 BJ cells. SAI means of 2.4 ± 6.8%, n = 43 cells, P = 0.0250, in siCtrl cells and 0.1 ± 5.6%, n = 43 cells, P = 0.8988 in siPCNT-treated cells. One-sample t tests were used for statistical analyses. All scale bars = 10 μm. ns = not significant; P ≤ 0.05 = *; P ≤ 0.001 = ***; P ≤ 0.0001 = ****. (J) Schematic model for the control of spindle asymmetry in RPE1 cells. We postulate that spindle size asymmetry is controlled in a centrosome-age-dependent manner via the cenexin-bound pool of Plk1 and centriolin, which both regulate the abundance of pericentriolar material required for microtubule (MT) nucleation, which itself regulates the abundance of TPX2 and ch-TOG.

Centrosome age also breaks spindle symmetry in human fibroblast cells. (A) Immunofluorescence image of 2:2 BJ metaphase cell stained with DAPI (blue), cenexin (green), and pericentrin (magenta) antibodies. (B) Quantification of the SAI in 2:2 BJ cells. SAI mean of 3.1 ± 7.6%, n = 106 cells, P < 0.0001 in one-sample t test. (C) Immunofluorescence images of 1:1 BJ late telophase cell stained with DAPI (blue), centrin (green), cenexin (yellow) antibodies and the cell mask (magenta). (D) Quantification of the daughter cell area asymmetry of 1:1 BJ cells. Daughter cell size asymmetry of 4.2 ± 7.8%, n = 47 cells, P = 0.0007 in one-sample t test. (E) Quantification of the relative protein distribution of pericentrin and TPX2 versus centrosome age in 2:2 BJ cells. Relative pericentrin distribution of 8.7 ± 12.1%, n = 56 cells, P < 0.0001. Relative TPX2 distribution of 1.7 ± 9.9%, n = 51 cells, P = 0.4329. A one-sample t test was used for pericentrin statistical analysis and a one-sample Wilcoxon-test was used for TPX2 statistical analysis. (F) Correlation between the relative pericentrin/TPX2 distribution (x axis) and the SAI (y axis) in 2:2 BJ cells. Each dot represents a cell for which the relative pericentrin/TPX2 distribution and the SAI were measured. The Pearson correlation coefficient and its associated P value, the number of cells analyzed as well as the slope of the linear regression (light green line) are indicated for each condition. (G) Immunofluorescence images of siCtrl- and siPCNT-treated 2:2 BJ cells, stained with DAPI (blue), cenexin (green), and pericentrin (magenta) antibodies. (H) Quantification of pericentrin depletion efficiency of siCtrl- and siPCNT-treated 2:2 BJ cells. Pericentrin fluorescence intensity means of 100%, n = 33 cells in siCtrl and 4.2 ± 1.4%, n = 30 cells in siPCNT cells. (I) Quantification of the SAI of siCtrl- and siPCNT-treated 2:2 BJ cells. SAI means of 2.4 ± 6.8%, n = 43 cells, P = 0.0250, in siCtrl cells and 0.1 ± 5.6%, n = 43 cells, P = 0.8988 in siPCNT-treated cells. One-sample t tests were used for statistical analyses. All scale bars = 10 μm. ns = not significant; P ≤ 0.05 = *; P ≤ 0.001 = ***; P ≤ 0.0001 = ****. (J) Schematic model for the control of spindle asymmetry in RPE1 cells. We postulate that spindle size asymmetry is controlled in a centrosome-age-dependent manner via the cenexin-bound pool of Plk1 and centriolin, which both regulate the abundance of pericentriolar material required for microtubule (MT) nucleation, which itself regulates the abundance of TPX2 and ch-TOG.

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