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. 2013 Jan 7;200(1):45–60. doi: 10.1083/jcb.201210106

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© 2013 Hori et al.

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Figure 4. — The CENP-T and CENP-C N terminus–derived kinetochores are functional. (A) Mitotic progression of CT^1–530-LacI/ΔZcen, CC^1–643-LacI/ΔZcen, or wild-type DT40 cells observed by live-cell imaging under a microscope in the absence of IPTG. Selected images are shown. (B) Numbers of anaphase cells with normal or abnormal segregation of chromosome Z in CT^1–530-LacI/ΔZcen or CC^1–643-LacI/ΔZcen cells (in the absence of IPTG). It is rare to detect cells with abnormal segregation of chromosome Z in the absence of IPTG. GFP-LacI/Zp-ter LacO cells were used as a control. This measurement was completed once (GFP-LacI/Zp-ter LacO, n = 139; CT^1–530-LacI/ΔZcen, n = 122; CC^1–643-LacI/ΔZcen, n = 229). (C) Localization of CCAN (top), outer kinetochore (middle), and checkpoint proteins (bottom) at the LacO locus in CT^1–530-LacI/ΔZcen cells. Each tested protein is shown in red and the LacI fusion with CENP-T (1–530) is shown in green. All tested CCAN proteins were not detected at the LacO locus, but outer kinetochore and checkpoint proteins were detected. Data were also summarized in F. (D) Localization of CCAN (top), outer kinetochore (middle), and checkpoint proteins (bottom) at the LacO locus in CC^1–643-LacI/ΔZcen cells. Each tested protein is shown in red and the LacI fusion with CENP-C (1–643) is shown in green. Arrows indicate the position of the LacO sequence. Z, Z chromosome. (E) Relative signal intensities of Ndc80 or Mis12 at the LacO locus compared with those at endogenous kinetochores in CT^1–530-LacI/ΔZcen or CC^1–643-LacI/ΔZcen cells. Error bars indicate mean ± SD. (F) Summary of the localization results of various kinetochore proteins at the LacO locus in CT^1–530-LacI/ΔZcen or CC^1–643-LacI/ΔZcen cells. +, positive localization at the LacO locus; −, negative localization at the LacO locus.