Figure 5. Aurora B phospho mutants of... | Rockefeller University Press

Figure 5.

$Figure 5. Aurora B phospho mutants of Cdt1 at the four sites identified by mass spectroscopic analysis partially affect kMT stability and mitotic progression. Synchronized HeLa cells treated with siRNA against endogenous Cdt1 in G2/M phase and rescued with stably expressing siRNA-resistant Cdt1-WT, 4D (phosphomimetic), 4A (unphosphorylated) proteins or vector control (simulating Cdt1 depleted state) were subjected to 4°C treatment for 15 min before being fixed and stained. (A) Representative images of cells from C, immunostained with antibodies against Zwint1 (kinetochore marker) in red, tubulin (MTs) in green or in black and white on the left of the merged images, and DAPI in blue marking the chromosomes. Scale bar, 5 µm. (B) Bar graph showing the quantification of spindle MT staining intensity after cold treatment in each case after background subtraction (n = 7). P < 0.05 represents statistical significance as assessed by the two-sided unpaired nonparametric Student’s t test. (C) Mitotic progression (metaphase-to-anaphase transition) was gauged in HeLa cells stably expressing Cdt1-WT, Cdt1-10D, Cdt1-10A, Cdt1-4D, and Cdt1-4A mutants at 11.5 and 12.5 h after release from thymidine treatment in the presence of Cdt1 siRNA (200 nM). Representative images are shown from E; DAPI pseudocolored in red marks the chromosomes, and antibody against tubulin stained the MTs green; scale bar, 10 µm. (D) The number of cells in each stage of mitosis was counted and plotted from three different coverslips to illustrate the difference in mitotic delay in Cdt1-4A/4D mutant–expressing cells compared with cells rescued with Cdt1-WT or Cdt1-10A/10D expression (n = 300); * and #, P values showing the statistical significance among the indicated groups obtained using two-sided unpaired nonparametric Student’s t test. (E) Schematic representation showing the three indicated Aurora B sites mutated to generate a 3D phosphomimetic mutant in the Cdt192–546 parental background. (F) Representative Western blot from a MT-cosedimentation experiment performed with Cdt192–546 and Cdt1-3D mutant is also shown. Samples fractionated as supernatant (S) and pellet (P) analyzed by Western blot probed with antibody against 6×-His tag to detect Cdt1 (WT or 3D mutant) and stained with Ponceau S for tubulin. (G) Quantification (mean ± SD, n = 3) showing MT cosedimentation of purified Cdt192–546 or the mutant proteins (1 µM each) with the indicated concentrations of taxol-stabilized MTs (in μM). The 95% CI values obtained for each fit were: Bmax = 0.8–1.0, Kd = 1.22–2.90 for Cdt192–546; Bmax = 0.67–0.85, Kd = 2.25–4.9 for 3D mutant.$

Aurora B phospho mutants of Cdt1 at the four sites identified by mass spectroscopic analysis partially affect kMT stability and mitotic progression. Synchronized HeLa cells treated with siRNA against endogenous Cdt1 in G2/M phase and rescued with stably expressing siRNA-resistant Cdt1-WT, 4D (phosphomimetic), 4A (unphosphorylated) proteins or vector control (simulating Cdt1 depleted state) were subjected to 4°C treatment for 15 min before being fixed and stained. (A) Representative images of cells from C, immunostained with antibodies against Zwint1 (kinetochore marker) in red, tubulin (MTs) in green or in black and white on the left of the merged images, and DAPI in blue marking the chromosomes. Scale bar, 5 µm. (B) Bar graph showing the quantification of spindle MT staining intensity after cold treatment in each case after background subtraction (n = 7). P < 0.05 represents statistical significance as assessed by the two-sided unpaired nonparametric Student’s t test. (C) Mitotic progression (metaphase-to-anaphase transition) was gauged in HeLa cells stably expressing Cdt1-WT, Cdt1-10D, Cdt1-10A, Cdt1-4D, and Cdt1-4A mutants at 11.5 and 12.5 h after release from thymidine treatment in the presence of Cdt1 siRNA (200 nM). Representative images are shown from E; DAPI pseudocolored in red marks the chromosomes, and antibody against tubulin stained the MTs green; scale bar, 10 µm. (D) The number of cells in each stage of mitosis was counted and plotted from three different coverslips to illustrate the difference in mitotic delay in Cdt1-4A/4D mutant–expressing cells compared with cells rescued with Cdt1-WT or Cdt1-10A/10D expression (n = 300); * and ^#, P values showing the statistical significance among the indicated groups obtained using two-sided unpaired nonparametric Student’s t test. (E) Schematic representation showing the three indicated Aurora B sites mutated to generate a 3D phosphomimetic mutant in the Cdt1^92–546 parental background. (F) Representative Western blot from a MT-cosedimentation experiment performed with Cdt1^92–546 and Cdt1-3D mutant is also shown. Samples fractionated as supernatant (S) and pellet (P) analyzed by Western blot probed with antibody against 6×-His tag to detect Cdt1 (WT or 3D mutant) and stained with Ponceau S for tubulin. (G) Quantification (mean ± SD, n = 3) showing MT cosedimentation of purified Cdt1^92–546 or the mutant proteins (1 µM each) with the indicated concentrations of taxol-stabilized MTs (in μM). The 95% CI values obtained for each fit were: B_max = 0.8–1.0, K_d = 1.22–2.90 for Cdt1^92–546; B_max = 0.67–0.85, K_d = 2.25–4.9 for 3D mutant.