FIGURE 4.

SKAP regulates microtubule plus-end dynamics. A and B, HeLa cells were transfected with control and SKAP-, CENP-E-, Mis13-, and Hec1-specific siRNA, respectively. 36 h after transfection, HeLa cells were fixed after incubation at 4 °C for 15 min (A) or 30 min (B) before labeling with anti-α-tubulin antibody (green), ACA serum (red), and DAPI (DNA; blue). Scale bar, 10 μm. C, examples of spindle configurations seen in the SKAP-suppressed cells. A total of four categories were assigned. D and E, statistical analyses of spindle stability of SKAP-suppressed cells in response to cold treatments. Note that knockdown of SKAP and Mis13 did not cause the collapse of the mitotic spindle, whereas Hec1 suppression resulted in spindle collapse in the presence of cold. F, examples of time lapse fluorescent images of metaphase HeLa cells in the presence of scramble, SKAP, or Mis13 siRNA before (Pre-PA) and at the indicated times after activation (Post-PA) of GFP-tubulin fluorescence. The half-life (t½) of photoactivated GFP-tubulin from the aforementioned groups was then calculated and expressed as mean ± S.E. (error bars). G, statistic analyses of normalized fluorescence intensity over time after photoactivating kinetochore microtubule from the aforementioned three groups in F (n = 15 cells/group). H, quantification of kinetochore microtubule flux rate in HeLa cells transfected with SKAP, Mis13, or scramble control siRNA. Note that depletion of SKAP significantly reduces kinetochore microtubule flux (*, p < 0.01 compared with control cells).