FIGURE 5.

Phosphorylation of CENP-U by Aurora-B leads to reduced kinetochore-microtubule interaction. A–C, shown is real-time imaging of chromosome movements in HeLa cells co-transfected with mcherry-H2B and GFP-CENP-U WT, AA, or DD. Chromosomes were marked by mcherry-H2B. Arrows indicate lagging chromosomes during alignment. Bar, 10 μm. D, shown is a graphic representation of chromosome alignment errors in GFP-CENP-U WT-, AA-, and DD-expressing cells. 24 GFP-CENP-U WT-expressing cells, 29 GFP-CENP-U AA-expressing cells, and 30 GFP-CENP-U DD-expressing cells were analyzed. E, overexpression of CENP-U DD results in destabilization of spindle microtubules. HeLa cells were transfected with GFP-CENP-U wild type, AA, and DD mutant for 36 h followed by a 2-h treatment of MG132 and examined for spindle stability in response to cold treatment. Cold-treated wild type (a), AA (b), and DD (c) mutant-expressing cells were stained with tubulin (red) and DAPI (blue). Bar, 10 μm. F, shown is the mitotic index of HeLa cells transfected with the indicated GFP-CENP-U plasmids and immunostained with DAPI 36 h after transfection. An average of 200 GFP-CENP-U-positive cells from three separate experiments was surveyed. Error bars represent S.E.; n = 3 preparations.