FIGURE 3.
Phenotypic analysis of INCENPOFF cells expressing mutants of the INCENP TSS motif. A–E, analysis of mitotic parameters in INCENPOFF cells expressing INCENPTSS mutants. Cells were plated on coverslips, stained with antibodies to Aurora B, microtubules, or DAPI for DNA, and scored for the percentage of mitotic cells in prometaphase or metaphase (A and C) or the percentage of binucleated cells (B and D). E, INCENPOFF cells expressing TrAP-INCENPWT or TrAP-INCENPTSS mutants exhibit a robust spindle checkpoint response to nocodazole and 10 or 100 nm Taxol. Three independent experiments were performed. Error bars indicate S.D. F–J, immunofluorescence analysis of INCENPOFF cells expressing nonphosphorylatable mutant TrAP-INCENPTS814AS815A or phospho-mimetic mutant TrAP-INCENPTS814ES815E. Cells were harvested after incubation for 28 h in doxycycline. F, in INCENPOFF cells, exogenous TrAP-INCENPWT (panels 2 and 6, red) shown co-localized with endogenous Aurora B (panels 3 and 7, green), with a typical chromosomal passenger dynamic localization throughout mitosis. Cells were also stained with DAPI to label DNA (panels 4 and 8, blue). G–J, INCENPOFF cells expressing the indicated TrAP-INCENPTSS mutant and stained with anti-SBP to show the localization of the exogenous INCENP (panels 2 and 6, red) or with antibodies to Aurora B (F, G, and I, panels 3 and 7, green), Borealin (H, panels 3 and 7, green), or Survivin (J, panels 3 and 7, green) plus DAPI for DNA (panels 4 and 8, blue). Scale bar, 5 μm. K, measurement of the distance between poles of bipolar spindles in INCENPOFF cells and INCENPOFF cells expressing TrAP-INCENPWT or the indicated INCENP mutants. Anaphase cells were measured only when both poles of a bipolar spindle were in the same plane and in a straight line.