Decreased Sli15/INCENP phosphorylation by Ipl1/Aurora promotes direct CPC binding to bundled MTs. (A) Gel filtration profile of recombinant WT CPC or sli15-17A (17A) CPC. Void volume was ∼8.0 ml. (B) Reduced Sli15 phosphorylation by Ipl1 increases CPC affinity for MTs. MT cosedimentation assay using WT, 17A, and alkaline phosphatase–treated WT CPCs. The supernatant (S) and pellet (P) were analyzed by Western blot using anti-Bir1 antibody. (C) Reduced Sli15 phosphorylation by Ipl1 increases CPC affinity for bundled MTs. Segmented MTs (see Materials and methods) were bundled with Cin8 before incubation with Alexa 488–labeled WT or 17A CPC. Bar, 5 µm. (D) Quantification of CPC association with bundled MTs represented in C. See Materials and methods for details. Significance was determined using the two-sample Kolmogorov–Smirnov test (D = 1; P = 4.9 × 10⁻¹⁰). (E) MTs facilitate CPC multimerization. Box plot showing fluorescence intensities for 17A CPC spots represented in C that were not associated (off MTs) or associated (on MTs) with MTs. The distributions’ medians differed significantly (Mann-Whitney U test = 322910, n_{off_MTs} = 445, n_{on_MTs} = 993, P < 2.2 × 10⁻¹⁶, two-tailed test; ρ = 0.73). (F) Plot of total fluorescence intensity versus spot diameter for 17A CPC that was found off MTs or on MTs. (G) 17A CPC accumulation on bundled MTs is not due to enhanced interaction with Cin8. After incubation with WT CPC or 17A CPC, Dynabeads-Cin8-7myc conjugate (BB) was separated from the supernatant (S). Cin8, Sli15, and Bir1 were detected by Western blot.