Figure 8.
Clathrin promotes chromosome congression and timely mitosis by antagonizing MCAK activity via GTSE1 recruitment. (A) Immunoblots of cell lysates (input) and anti-GFP IPs of GTSE1-GFP or GTSE15xLID-GFP under conditions illustrated. Immunoblots with anti-GFP, anti-CHC, or anti-MCAK antibodies. (B) Fraction of cells that entered anaphase as a function of the time (minutes) after NEB in GTSE15xLID cells transfected with control (Ctrl), GTSE1, MCAK, or GTSE1 + MCAK siRNA. The mean time between NEB and anaphase onset is indicated in the inset (± standard error, n ≥ 312 per condition over n = 4 experiments pooled for analysis and representation). In the Stat. columns, conditions with the same letter are not statistically different (statistics from Wilcoxon test adjusted for multiple comparisons [false discovery rate], P < 0.05). (C) Immunofluorescence images (EB1 and tubulin staining) showing STLC-induced monopolar spindles in GTSE15xLID cells after the labeled siRNA transfections. Dashed yellow lines indicate cell borders. (D) Mean growing MT length per cell (EB1 comet to monopole distance) in 3D reconstruction of cells from C (control n = 19, GTSE1 n = 20, GTSE1 + MCAK n = 20, MCAK n = 12, n = 1 experiment, P values from ANOVA followed by Tukey’s test, data presented as box and whisker plots). n.s., not significant. (E) CHC and GTSE1 depletion in U2OS cells lead to similar and nonadditive mitotic delays. Data plotted as described in A. (F) Codepletion of MCAK partially rescues the mitotic delay induced by CHC depletion. Data plotted as in A. Data in E and F were obtained concomitantly (control and CHC siRNAs conditions are shared; n ≥ 255 over n = 3 experiments pooled for analysis and representation). Plots in E and F are cropped on the time abscissa at t = 150 min for representation purposes. In the Stat. columns, conditions with the same letter are not statistically different (statistics from Wilcoxon test adjusted for multiple comparisons [false discovery rate], P < 0.05). Scale bar: 10 µm. Numeric data is shown in Table S1.

Clathrin promotes chromosome congression and timely mitosis by antagonizing MCAK activity via GTSE1 recruitment. (A) Immunoblots of cell lysates (input) and anti-GFP IPs of GTSE1-GFP or GTSE15xLID-GFP under conditions illustrated. Immunoblots with anti-GFP, anti-CHC, or anti-MCAK antibodies. (B) Fraction of cells that entered anaphase as a function of the time (minutes) after NEB in GTSE15xLID cells transfected with control (Ctrl), GTSE1, MCAK, or GTSE1 + MCAK siRNA. The mean time between NEB and anaphase onset is indicated in the inset (± standard error, n ≥ 312 per condition over n = 4 experiments pooled for analysis and representation). In the Stat. columns, conditions with the same letter are not statistically different (statistics from Wilcoxon test adjusted for multiple comparisons [false discovery rate], P < 0.05). (C) Immunofluorescence images (EB1 and tubulin staining) showing STLC-induced monopolar spindles in GTSE15xLID cells after the labeled siRNA transfections. Dashed yellow lines indicate cell borders. (D) Mean growing MT length per cell (EB1 comet to monopole distance) in 3D reconstruction of cells from C (control n = 19, GTSE1 n = 20, GTSE1 + MCAK n = 20, MCAK n = 12, n = 1 experiment, P values from ANOVA followed by Tukey’s test, data presented as box and whisker plots). n.s., not significant. (E) CHC and GTSE1 depletion in U2OS cells lead to similar and nonadditive mitotic delays. Data plotted as described in A. (F) Codepletion of MCAK partially rescues the mitotic delay induced by CHC depletion. Data plotted as in A. Data in E and F were obtained concomitantly (control and CHC siRNAs conditions are shared; n ≥ 255 over n = 3 experiments pooled for analysis and representation). Plots in E and F are cropped on the time abscissa at t = 150 min for representation purposes. In the Stat. columns, conditions with the same letter are not statistically different (statistics from Wilcoxon test adjusted for multiple comparisons [false discovery rate], P < 0.05). Scale bar: 10 µm. Numeric data is shown in Table S1.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal