Oligomerization of the KI region of Spc105 recruits binding partners and is sufficient for poleward movement. (A) T-COFFEE alignments of human (Hs) KNL1 and fly (Dm) Spc105 identify putative KI motifs. (B, D–F, and J) Representative confocal Z-planes of cells coexpressing the indicated EGFP-tagged protein (green) and oligomerized clusters of the KI motif-containing region (266–384) fused to mCherry-Cry2 (magenta). (C) Colocalization of the indicated EGFP-tagged proteins with 266–384-mCherry-Cry2 clusters in cells over a range of expression. Colocalization with clusters was never observed in cells over the full range of EGFP (control) or EGFP-Bub1 expression. With the exception of the EGFP control (to establish a baseline measurement for the assay), only cells exhibiting colocalization were used to measure the fold enrichment for each binding partner. (G–I and K) Quantification of the fold enrichment of the signal of the indicated EGFP-tagged protein relative to the local background signal (EGFP; n = 53 drops from 10 cells, Bub1-EGFP; n = 20 drops from 4 cells, Zw10-EGFP; n = 80 drops from 16 cells, sfGFP-Rod; n = 65 drops from 13 cells, BubR1-EGFP; n = 70 drops from 14 cells). (L) Measurements from G–I and K are shown on the same plot. In all plots, each binding partner is compared with the same control EGFP data set. (M) Representative confocal time-lapse of sfGFP-Rod (green) colocalized with a 266–384-mCherry-Cry2 cluster (magenta) moving poleward with accompanying kymograph. Boxed regions are highlighted in the insets. The average fold enrichment for the representative cell is indicated in each image. Bar plots report mean ± SD. Different letters above each plot column indicate significant differences (P < 0.05) determined by a randomization method (PlotsOfDifferences) for all pairwise combinations. Shared letters indicate that the difference is not significant (P > 0.05). Scale bars, 10 μm; 1 μm (insets), and 1 μm (horizontal); 1 min (vertical) in kymograph. Displayed time is min:s.