FIGURE 5.

Phosphorylation of HsMis13 is essential for faithful assembly of kinetochore. A, HsMis13 siRNA suppressed the HsMis13 protein accumulation. Aliquots of HeLa cells were transfected with HsMis13 siRNA and scramble control as described under “Materials and Methods.” Cells were then harvested for SDS-PAGE and subsequent Western blotting analyses of the efficiency on suppression of HsMis13 protein (upper panel) and specificity of this siRNA-mediated HsMis13 depletion by assessing the protein levels of other kinetochore proteins such as Aurora B, CENP-E, CENP-F, and MAD2 (all lower panels). B, suppression of HsMis13 prevents assembly of CENP-E and CENP-F to the kinetochore. HeLa cells were transfected with HsMis13 siRNA oligonucleotide and control oligonucleotide for 48 h followed by fixation and indirect immunofluorescence staining. This set of optical images was collected from HeLa cells stained for HsMis13 (green), ACA (red, c and c′), CENP-E (red, g and g′), CENP-F (red, k and k′), and DNA (blue), respectively. As shown in g, CENP-E protein is associated with kinetochore as resolved double dots in scramble control cells (arrows). However, virtually undetectable staining of CENP-E can be seen in HsMis13-depleted cells (g′), whereas kinetochore-associated CENP-F and ACA signals were not altered by HsMis13 suppression (c′ and k′). The scale bar represents 10 μm. C, quantitation of CENP-E, CENP-F, Nuf2, Hec1, and Aurora B levels at kinetochores of control and siRNA-treated cells. The pixel intensities of HsMis13, Nuf2, CENP-E, CENP-F, Aurora B, and Hec1 (normalized to the ACA signal) in control (closed bar) and HsMis13-repressed (open bars; HsMis13 RNAi) cells were measured. Values represent the means ± S.E. of at least 100 kinetochores in 10 different cells. D, inhibition of Aurora B prevented kinetochore distribution of GFP-HsMis13. HeLa cells were transiently transfected to express GFP-HsMis13 followed by treatment of VX-680 as described under “Materials and Methods.” Cells were then harvested for immunofluorescence staining of GFP-HsMis13 (green), HsMis12 (red), and DNA (blue). Inhibition of Aurora B by VX-680 eliminated the kinetochore localization of HsMis13 (b). The scale bar represents 10 μm. E, phospho-mimicking GFP-HsMis13^EE restored the kinetochore localization of CENP-E at the kinetochore. HeLa cells were transiently transfected to express phospho-mimicking GFP-HsMis13^EE followed by treatment of VX-680 as described under “Materials and Methods.” Cells were then harvested for immunofluorescence staining of GFP-HsMis13^EE (green), CENP-E (red), and DNA (blue). Localization of HsMis13 restored the kinetochore association of CENP-E in the presence of VX-680 (a and b). The scale bar represents 10 μm. F, quantitation of CENP-E and Nuf2 levels at kinetochores of GFP-HsMis13 expressing cells in the presence of Aurora B kinase inhibitor. The pixel intensities of CENP-E levels (normalized to the ACA signal) in control GFP-HsMis13-expressing cells (open bars) and GFP-HsMis13-expressing and X-680-treated (open bars) cells were measured. The levels of Nuf2 were also measured in the aforementioned conditions (blue bars). Values represent the means ± S.E. of at least 100 kinetochores in 10 different cells.