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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 6. Induced CENP-T and CENP-C foci interact with microtubules and function in chromosome segregation.

Figure 6

(A) Representative immunofluorescence images of cells expressing GFP-LacI, or co-expressing RNAi resistant mCherry-CENPT-ΔC-LacI (not shown) and GFP-CENP-C-ΔC-LacI. Microtubules are shown in red. Cells were treated with 2 μM ZM447439 or 3.3 μM Nocodazole, or 48 h CENP-C and CENP-T RNAi as indicated. In all cases, cells were cold treated for 20 min prior to fixation to visualize stable kinetochore microtubule fibers. Arrows indicate the foci. The mean length/width ratio for the foci is shown below the indicated images. N >5 cells. (B) Electron micrographs of an ectopic CENP-T-LacI foci showing the presence of microtubule attachments (red arrow). Dark spots indicate immuno-labeling with anti-Hec1 antibody. This ectopic CENP-T-LacI foci was defined by the size and extensive anti-Hec1 labeling of this structure relative to an endogenous kinetochore. Direct correlative light-EM is shown to analyze ectopic kinetochore structure in Fig. S5. (C) Graph showing the percentage of cells with more than one LacI foci 72 h after expression of GFP-LacI or GFP-CENP-T-ΔC-LacI. N=200 cells (D) Graph showing quantification of LacI foci segregation in live cells expressing GFP-LacI, GFP-CENP-T-ΔC-LacI, or GFP-CENP-C-ΔC-LacI. (E) Selected images from time-lapse movies of U2OS-LacO cells expressing mCherry-histone-H2B to visualize chromatin, and GFPCENP-T-ΔC-lacI or GFP-LacI showing segregation of LacI foci at anaphase. Time in shown in minutes after NEBD. Scale bars, 5 μm. (F). Centromere replacement assay in chicken DT40 cells (see Fig. S6 for a schematic of this strategy). Left and middle panels; representative images of DT40 cells with GFP-LacI fusion protein localized to a LacO array on the Z chromosome (arrows), after Cre recombinase mediated excision of the centromeric region of the same chromosome. Left, representative images of the effected chromosome lagging at anaphase. Middle, representative images of correct segregation of the Z chromosome. Right panel; graph showing the percentage of GFP foci containing cells with lagging or equally dividing Z chromosomes 18 h after addition of tamoxifen to induce excision of the endogenous centromere cells. N=78 cells per condition. Also see Fig. S5 and movies S1–S4.