Figure 7.
The phosphorylation of cenexin S796 drives the formation of asymmetric spindles. (A) Immunofluorescence images of 1:1 metaphase RPE1 GFP-centrin1 cells expressing either WT or S796A mScarlet-cenexin, stained with DAPI (blue), GFP-centrin1 (green), and mScarlet-cenexin (yellow). Scale bars = 10 μm. (B) Quantification of the SAI in 1:1 RPE1 GFP-centrin1 cells expressing either WT or S796A mScarlet-cenexin. WT-cenexin SAI mean: 2.0 ± 6.5%, n = 73 cells, P = 0.0044 in one-sample Wilcoxon test compared to a symmetric null hypothesis. S796A cenexin SAI mean: 0.35 ± 7.5%, n = 75 cells, P = 0.7096 in one-sample t test. (C) Immunofluorescence images of RPE1 GFP-centrin1 S796A mScarlet-cenexin prophase and metaphase cells stained with DAPI (blue), GFP-centrin1 (green), mScarlet-cenexin (yellow), and Plk1 (magenta) antibody. Scale bar = 10 μm. (D) Quantification of the relative Plk1 distribution at centrosomes versus centrosome age in prophase and metaphase in WT mScarlet-cenexin cells. Means of 9.1 ± 11.8%, n = 37 cells, P < 0.0001 and 8.4 ± 11%, n = 40 cells, P < 0.0001 in one-sample t tests. (E) Quantification of the relative Plk1 distribution at centrosomes versus centrosome age in prophase and metaphase in S796A mScarlet-cenexin cells. Means of 0.9 ± 13.9%, n = 33 cells, P = 0.7242 and 1 ± 14.9%, n = 34 cells, P = 0.6970 in prophase and metaphase, respectively, in one-sample t tests. (F) Immunofluorescence images of 1:1 metaphase RPE1 GFP-centrin1 cells expressing either WT mScarlet-cenexin or S796A mScarlet-cenexin, stained with DAPI (blue), GFP-centrin1 (green), mScarlet-cenexin (yellow), and γ-tubulin (magenta) antibody. Scale bar = 10 μm. (G) Quantification of the relative γ-tubulin distribution versus centrosome age in 1:1 RPE GFP-centrin1cells expressing either WT or S796A mScarlet-cenexin. In WT-cenexin cells relative distribution mean of 2.6 ± 7.6%, n = 40 cells, P = 0.020, and in S796A cenexin cells relative distribution mean of 1.2 ± 6.1%, n = 55 cells, P = 0.149 in one-sample Wilcoxon test. (H) Quantification of the relative pericentrin distribution versus centrosome age in 1:1 RPE GFP-centrin1 mScarlet-cenexin S796A cells. Relative distribution mean of 4.1 ± 10.6%, n = 75 cells, P = 0.0014 in one-sample t test. (I) Immunofluorescence images of 1:1 RPE GFP-centrin1 cells expressing either WT or S796A mScarlet-cenexin metaphase cells treated with nocodazole and stained with cenexin (yellow) and CENP-A (green) antibody, DAPI (blue), and GFP-centrin1 (green). (J) Quantification of the percentage of unaligned chromosomes distributed versus centrosome age. The dashed line represents a symmetric distribution (50%) of the polar chromosomes between the two poles.  Means of 69.7 ± 15.2% (from 267 polar chromosomes in 96 cells) for mScarlet-cenexin cells, and 50.2 ± 8.4% (from 235 polar chromosomes in 75 cells) for mScarlet-cenexin S796A cells. P values of < 0.0001 and 1 for mScarlet-cenexin and mScarlet-cenexin S796A cells, respectively, in a binomial test against a random distribution. P value of 0.0003 (***) using a Fisher’s exact test to compare the two conditions. ns = not significant; P ≤ 0.05 = *; P ≤ 0.01 = **; P ≤ 0.001 = ***; P ≤ 0.0001 = ****.

The phosphorylation of cenexin S796 drives the formation of asymmetric spindles. (A) Immunofluorescence images of 1:1 metaphase RPE1 GFP-centrin1 cells expressing either WT or S796A mScarlet-cenexin, stained with DAPI (blue), GFP-centrin1 (green), and mScarlet-cenexin (yellow). Scale bars = 10 μm. (B) Quantification of the SAI in 1:1 RPE1 GFP-centrin1 cells expressing either WT or S796A mScarlet-cenexin. WT-cenexin SAI mean: 2.0 ± 6.5%, n = 73 cells, P = 0.0044 in one-sample Wilcoxon test compared to a symmetric null hypothesis. S796A cenexin SAI mean: 0.35 ± 7.5%, n = 75 cells, P = 0.7096 in one-sample t test. (C) Immunofluorescence images of RPE1 GFP-centrin1 S796A mScarlet-cenexin prophase and metaphase cells stained with DAPI (blue), GFP-centrin1 (green), mScarlet-cenexin (yellow), and Plk1 (magenta) antibody. Scale bar = 10 μm. (D) Quantification of the relative Plk1 distribution at centrosomes versus centrosome age in prophase and metaphase in WT mScarlet-cenexin cells. Means of 9.1 ± 11.8%, n = 37 cells, P < 0.0001 and 8.4 ± 11%, n = 40 cells, P < 0.0001 in one-sample t tests. (E) Quantification of the relative Plk1 distribution at centrosomes versus centrosome age in prophase and metaphase in S796A mScarlet-cenexin cells. Means of 0.9 ± 13.9%, n = 33 cells, P = 0.7242 and 1 ± 14.9%, n = 34 cells, P = 0.6970 in prophase and metaphase, respectively, in one-sample t tests. (F) Immunofluorescence images of 1:1 metaphase RPE1 GFP-centrin1 cells expressing either WT mScarlet-cenexin or S796A mScarlet-cenexin, stained with DAPI (blue), GFP-centrin1 (green), mScarlet-cenexin (yellow), and γ-tubulin (magenta) antibody. Scale bar = 10 μm. (G) Quantification of the relative γ-tubulin distribution versus centrosome age in 1:1 RPE GFP-centrin1cells expressing either WT or S796A mScarlet-cenexin. In WT-cenexin cells relative distribution mean of 2.6 ± 7.6%, n = 40 cells, P = 0.020, and in S796A cenexin cells relative distribution mean of 1.2 ± 6.1%, n = 55 cells, P = 0.149 in one-sample Wilcoxon test. (H) Quantification of the relative pericentrin distribution versus centrosome age in 1:1 RPE GFP-centrin1 mScarlet-cenexin S796A cells. Relative distribution mean of 4.1 ± 10.6%, n = 75 cells, P = 0.0014 in one-sample t test. (I) Immunofluorescence images of 1:1 RPE GFP-centrin1 cells expressing either WT or S796A mScarlet-cenexin metaphase cells treated with nocodazole and stained with cenexin (yellow) and CENP-A (green) antibody, DAPI (blue), and GFP-centrin1 (green). (J) Quantification of the percentage of unaligned chromosomes distributed versus centrosome age. The dashed line represents a symmetric distribution (50%) of the polar chromosomes between the two poles.  Means of 69.7 ± 15.2% (from 267 polar chromosomes in 96 cells) for mScarlet-cenexin cells, and 50.2 ± 8.4% (from 235 polar chromosomes in 75 cells) for mScarlet-cenexin S796A cells. P values of < 0.0001 and 1 for mScarlet-cenexin and mScarlet-cenexin S796A cells, respectively, in a binomial test against a random distribution. P value of 0.0003 (***) using a Fisher’s exact test to compare the two conditions. ns = not significant; P ≤ 0.05 = *; P ≤ 0.01 = **; P ≤ 0.001 = ***; P ≤ 0.0001 = ****.

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