CAL1 is sufficient to recruit CENP-A, CENP-C, and Ndc80 de novo. (A) IF-FISH on metaphase chromosomes from lacO cells expressing CAL1–GFP–LacI. CAL1–GFP–LacI (green) localizes to the lacO array (FISH probe, red). DAPI is in blue. (B) lacO chromosomes showing recruitment of CENP-A, CENP-C, and Ndc80 (all in red) in the presence of CAL1–GFP–LacI but not in the presence of the control protein GFP-LacI (both green) at the lacO array. DAPI is in blue. (C) Quantification of the percentage of cells recruiting the indicated kinetochore proteins at the ectopic site by CAL1–GFP–LacI (CAL1) or GFP-LacI (GFP). nCENP-A = 89; nCENP-C = 89; nNdc80 = 51. ***, P < 0.0001. Shown are data for a single representative experiment out of 2–3 repeats per kinetochore protein. (D) CAL1–GFP–LacI (visualized with anti-LacI antibody; green) recruited Mad2 (red). CENP-A is in blue, DAPI in gray. White arrows show the location of the lacO array. Bars, 1 µm.
