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. 2016 Mar 14;212(6):647–659. doi: 10.1083/jcb.201408089

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© 2016 Isokane et al.

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Figure 6. — ARHGEF17 is required for targeting of Mps1 at kinetochores, and constitutive tethering of Mps1 to the kinetochore replaces ARHGEF17’s SAC function. (A and B, left) Exemplary prometaphase cells with LAP-Mps1 and phospho-KNL1 labeled at kinetochores. Overlay shows LAP-Mps1 (A) or ph-KNL1 (Thr875; B; green) and CENP-A (red) after knockdown of ARHGEF17. (insets) High magnification of kinetochores. (right) Quantitative ratiometric comparison of LAP-Mps1 (A) or ph-KNL1 (B). Box plot comparing the mean intensity ratio between LAP-Mps1/CENP-A or ph-KNL1/CENP-A ratio of >600 individual sister kinetochores/three independent experiments. Bars: (main) 5 µm; (insets) 0.5 µm. (C and D) Phenotypic rescue by artificial kinetochore tethering of Mps1 in the absence of ARHGEF17. (C) Mitotic events were automatically extracted after knockdown in cells stably expressing H2B-mCherry and mEGFP-CENP-B-Mps1 with or without 0.5 µM reversine treatment. Colors indicate H2B-mCherry morphology classes. (D) Comparison of early mitotic duration. Box plot comparing the duration of prometaphase and metaphase in each condition. Mean and standard deviation of >120 mitotic events (n indicated for each condition)/three independent experiments. Boxes show median, 25–75%; whiskers show 1.5× interquartile range. **, P < 0.01 by two-tailed unpaired Student’s t test, compared with si(Scrambled) (A and B), si(Scrambled) versus si(ARHGEF17); si(hARHGEF17) versus mEGFP-CENP-B-Mps1 expression with or without reversine (D). Sc, Scrambled; KD, knockdown.