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. Author manuscript; available in PMC: 2012 Apr 29.
Published in final edited form as: Cell. 2011 Apr 29;145(3):410–422. doi: 10.1016/j.cell.2011.03.031

Figure 7. Mitotic kinetochore assembly is regulated by phosphorylation of CENPT by CDK.

Figure 7

(A) Immunofluorescence images of Hela cells at the indicated cell cycle stages. Right panel shows quantification of CENP-T, phospho-CENP-T and Hec1 levels at centromeres, N ≥ 10 cells/condition, 20 kinetochores/cell, +/− SEM. Scale bars, 5 μm. (B) Immunofluorescence images showing levels of Hec1/Ndc80 at ectopic foci in mitotic and interphase U20S LacO cells expressing LacI fusions of: GFP-CENP-T, GFP-CENPT S-A (S or T to A mutations at amino acids 11, 27, 47, 85 and 195), or GFP-CENP-T S-D (S or T to D mutations at amino acids 11, 27, 85). Graph shows the relative fluorescence of Hec1/Ndc80 at kinetochores versus ectopic foci, in mitotic cells expressing the indicated CENP-T LacI fusion proteins. Quantification was done after 15 h nocodazole treatment, N ≥ 10 cells/condition, 20 kinetochores/cell, +/− SEM. Data is shown normalized to the foci/kinetochore ratio of CENP-T. (C) Left panels: Immunofluorescence images showing levels of Hec1/Ndc80 48 h after RNAi depletion of CENP-T in HeLa cells or HeLa cell lines expressing RNAi resistant GFP-CENP-T, GFP-CENP-T S-A or GFP-CENP-T S-D. Right panels: Quantification of mitotic index and Hec1/Ndc80 levels, after RNAi depletion of CENP-T and add back of the indicated GFP-CENP-T proteins, +/− SEM. (D) Model depicting interactions at endogenous and ectopic kinetochores. Also see Fig. S6.