Figure 4.
Figure 4. Aurora B phosphorylation of Cdt1 regulates kMT stability and mitotic progression. (A) Synchronized HeLa cells treated with siRNA against endogenous Cdt1 in G2/M phase and rescued with stably expressing siRNA-resistant Cdt1-WT, 10D (phosphomimetic), 10A (unphosphorylated) proteins or vector control (abbreviated as Vec, simulating Cdt1 depleted state); were stained with antibodies against Zwint1 in red (1:400 dilution, kinetochore marker), MTs in green (1:500 dilution, spindle marker), and DAPI in black and white marking the chromosomes. Cells were placed at 4°C for 15 min before being fixed and stained. Scale bar, 5 µm. (B) Bar graph showing the quantification of MT staining intensity in each case after background subtraction (n = 10 cells). P < 0.0001 represents statistical significance as assessed by the two-sided unpaired nonparametric Student’s t test. (C) HeLa cells treated with siRNA against endogenous Cdt1 rescued with stably expressing siRNA-resistant Cdt1-WT, 10D (phosphomimetic), 10A (phosphodefective) proteins (with C-terminal HASH tags) or vector control (Vec) were lysed using 2× Laemmli buffer and electrophoresed on 10% SDS-PAGE gel. Representative Western blots from three independent experiments of synchronized HeLa cell lysates probed for expression levels of the indicated proteins with (bottom) or without (top) Cdt1 siRNA (200 nM). Expressed, ectopically expressed (with HASH tags); endogenous, endogenous (without tag) Cdt1; * in the bottom panel, detection of a nonspecific band reactive with the Cdt1 antibody. The same blot was reprobed with GAPDH (1:8,000) as a sample recovery control. (D) Quantification of the percentage of bi-attached, bi-oriented kinetochores in each of the conditions as indicated. Kinetochore marker, Zwint1, is in red, and MT is in green. (E) Interkinetochore (K-K) stretch was calculated between the two kinetochore pairs in each case and is plotted (n = 115 pairs; 10 cells). P < 0.001 represents statistical significance as assessed by the two-sided unpaired nonparametric Student’s t test. Cropped images of representative kinetochore pairs are also provided to indicate the status of their kMT attachment and K-K stretch; scale bars, 1 µm. (F) STLC washout assay was performed in double thymidine synchronized HeLa cells, treated with siRNA against endogenous Cdt1, and rescued with either Cdt1-WT or Cdt1-10A followed by treatment with STLC for 2 h. The cells were washed with and released into medium containing MG132 and fixed either immediately after the initiation of the washout (t = 0) or after 1 h (t = 60), followed by fixation and staining. DAPI is pseudocolored in red to mark chromosomes, and tubulin is in green. (G) Bar graph showing the quantification of monopolar (early prometaphase) versus bipolar spindle (metaphase) structures at the indicated time points after STLC washout. Cells from two coverslips in two independent experiments were counted; n = 2,114 and 2,659 for WT and 10A, respectively, for t = 60; n = 565 and 785 for WT and 10A, respectively, for t = 0. The data are plotted as percentage mitotic cells with monopolar or bipolar spindle structures on the y-axis. (H) Mitotic progression (metaphase-to-anaphase transition) was assessed in HeLa cells stably expressing vector, Cdt1-WT, Aurora B Cdt1-phosphomimetic (10D) or phospho-defective (10A) mutants at 11.5 and 12.5 h after release from thymidine treatment in the presence of Cdt1 siRNA. The number of cells in each stage of mitosis was counted and plotted from three different coverslips of two independent experiments to illustrate the nature of mitotic delay/arrest in Cdt1-10D mutant–expressing cells compared with cells rescued with Cdt1-WT or Cdt1-10A expression (n = 600 cells; a and b represent the statistical significance obtained using two-sided unpaired nonparametric Student’s t test performed on the WT versus 10D in the indicated stages of mitosis). (I) Representative images are shown from H; DAPI pseudocolored in red marks the chromosomes, and antibody against tubulin stained the MTs green.

Aurora B phosphorylation of Cdt1 regulates kMT stability and mitotic progression. (A) Synchronized HeLa cells treated with siRNA against endogenous Cdt1 in G2/M phase and rescued with stably expressing siRNA-resistant Cdt1-WT, 10D (phosphomimetic), 10A (unphosphorylated) proteins or vector control (abbreviated as Vec, simulating Cdt1 depleted state); were stained with antibodies against Zwint1 in red (1:400 dilution, kinetochore marker), MTs in green (1:500 dilution, spindle marker), and DAPI in black and white marking the chromosomes. Cells were placed at 4°C for 15 min before being fixed and stained. Scale bar, 5 µm. (B) Bar graph showing the quantification of MT staining intensity in each case after background subtraction (n = 10 cells). P < 0.0001 represents statistical significance as assessed by the two-sided unpaired nonparametric Student’s t test. (C) HeLa cells treated with siRNA against endogenous Cdt1 rescued with stably expressing siRNA-resistant Cdt1-WT, 10D (phosphomimetic), 10A (phosphodefective) proteins (with C-terminal HASH tags) or vector control (Vec) were lysed using 2× Laemmli buffer and electrophoresed on 10% SDS-PAGE gel. Representative Western blots from three independent experiments of synchronized HeLa cell lysates probed for expression levels of the indicated proteins with (bottom) or without (top) Cdt1 siRNA (200 nM). Expressed, ectopically expressed (with HASH tags); endogenous, endogenous (without tag) Cdt1; * in the bottom panel, detection of a nonspecific band reactive with the Cdt1 antibody. The same blot was reprobed with GAPDH (1:8,000) as a sample recovery control. (D) Quantification of the percentage of bi-attached, bi-oriented kinetochores in each of the conditions as indicated. Kinetochore marker, Zwint1, is in red, and MT is in green. (E) Interkinetochore (K-K) stretch was calculated between the two kinetochore pairs in each case and is plotted (n = 115 pairs; 10 cells). P < 0.001 represents statistical significance as assessed by the two-sided unpaired nonparametric Student’s t test. Cropped images of representative kinetochore pairs are also provided to indicate the status of their kMT attachment and K-K stretch; scale bars, 1 µm. (F) STLC washout assay was performed in double thymidine synchronized HeLa cells, treated with siRNA against endogenous Cdt1, and rescued with either Cdt1-WT or Cdt1-10A followed by treatment with STLC for 2 h. The cells were washed with and released into medium containing MG132 and fixed either immediately after the initiation of the washout (t = 0) or after 1 h (t = 60), followed by fixation and staining. DAPI is pseudocolored in red to mark chromosomes, and tubulin is in green. (G) Bar graph showing the quantification of monopolar (early prometaphase) versus bipolar spindle (metaphase) structures at the indicated time points after STLC washout. Cells from two coverslips in two independent experiments were counted; n = 2,114 and 2,659 for WT and 10A, respectively, for t = 60; n = 565 and 785 for WT and 10A, respectively, for t = 0. The data are plotted as percentage mitotic cells with monopolar or bipolar spindle structures on the y-axis. (H) Mitotic progression (metaphase-to-anaphase transition) was assessed in HeLa cells stably expressing vector, Cdt1-WT, Aurora B Cdt1-phosphomimetic (10D) or phospho-defective (10A) mutants at 11.5 and 12.5 h after release from thymidine treatment in the presence of Cdt1 siRNA. The number of cells in each stage of mitosis was counted and plotted from three different coverslips of two independent experiments to illustrate the nature of mitotic delay/arrest in Cdt1-10D mutant–expressing cells compared with cells rescued with Cdt1-WT or Cdt1-10A expression (n = 600 cells; a and b represent the statistical significance obtained using two-sided unpaired nonparametric Student’s t test performed on the WT versus 10D in the indicated stages of mitosis). (I) Representative images are shown from H; DAPI pseudocolored in red marks the chromosomes, and antibody against tubulin stained the MTs green.

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