Recruitment of CENP-A by HJURP^Scm3 mediates kinetochore formation at the LacO array. (A) Mitotic chromosome spreads from U20S-LacO/TRE cells transfected with LacI or LacI-HJURP^Scm3, arrested in nocodazole, and stained with antibodies for NDC80. 40% of LacI-HJURP arrays recruited NDC80. (B) Monastrol-arrested cells transfected with LacI or LacI-HJURP^Scm3 and immunostained for centromere marker CENP-T. Radial distribution plots describe the mean centromere position (black circle) in the cells measured (>26 cells per condition) relative to the center of the DNA mass. The array position is diagrammed relative to the center of the DNA mass as blue triangles (LacI) or red diamonds (LacI-HJURP^Scm3). The gray circle represents one standard deviation from the mean centromere position. The LacI-HJURP^Scm3 array falls within the centromere region in 69% of transfected cells versus 15% for LacI controls. (C) Selective stabilization of kinetochore-bound microtubules through cold treatment demonstrates the LacI-HJURP^Scm3 arrays form stable microtubule interactions similar to endogenous centromeres. Insets show magnified views of the boxed region. (D) LacO-SceI-TRE NIH3T3 cells were transfected with YFP–histone H2B and followed by live-cell imaging as they progress through mitosis. Times are given relative to the last frame when cells were in metaphase. Arrows indicate the array, and asterisks indicate nonchromatin-bound unspecific LacI staining. (E) Insets taken from images in D show the behavior of the array (red in merge) and YFP-H2B (green in merge) for (1 and 2) LacI-HJURP^Scm3 at 6 and 9 min into anaphase, respectively. Bars: (A–D) 5 µm; (E) 2 µm.