Figure 7. Importin-β stabilizes SAFs during mitosis.

A. Stabilization by importin-β is required for accumulation of HURP on spindle microtubules. ^HAHURP and mutants were expressed in HeLa cells, and their intracellular location was determined in pre-anaphase cells by immunofluorescence using αHA-antibodies (green). The spindle was stained with antibodies against β-tubulin (red), DNA was detected by DAPI (blue). MG132 was added as indicated. B. APC/C-inhibition stabilizes HURP^KR2 in prometaphase. HeLa cells expressing ^HAHURP or ^HAHURP^KR2 were synchronized in prometaphase by nocodazole treatment. As indicated, UbcH10^C114S or ^mycEmi1, which inhibit the APC/C, were co-expressed. The levels of HURP or HURP^KR2 were determined by Western blot. C. Some endogenous HURP is degraded in an APC/C-dependent manner before metaphase. HeLa cells were treated with siRNA against UbcH10, Ube2S, and p31^comet, which inhibits the APC/C and arrests cells in prometaphase. Endogenous HURP was detected by fluorescence microscopy using αHURP-antibodies (left panel). The fluorescence intensity in pre-anaphase cells was measured using ImageJ. The quantification of three independent experiments is shown on the right. D. Stabilization by importin-β is required for HURP-function in early mitosis. HURP was depleted from HeLa cells by siRNA, and cells were arrested in mitosis by nocodazole. As indicated, depleted HeLa cells were transfected with siRNA-resistant HURP mutants. 1h after release into new medium, the number of cells that had initiated anaphase was determined. The bottom panel shows HURP depletion by siRNA and rescue by siRNA-resistant HURP*. E. Degradation of HURP is important for spindle formation. ^HAHURP or ^HAHURP^{ΔD1ΔKΔTΔD2} were expressed in HeLa cells and detected by immunofluorescence against HA (green). The percentage of HURP-positive pre-anaphase cells with incomplete chromosome alignment was determined 48h post transfection in three independent experiments (p-value < 0.05; left). Most spindles in cells expressing the stable ^HAHURP^{ΔD1ΔKΔTΔD2} were defective, examples for which are shown on the right.