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. 2013 Dec 23;203(6):943–955. doi: 10.1083/jcb.201307016

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© 2013 Vleugel et al.

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Figure 4. — Engineered KNL1 proteins reveal differential requirements for TΩ-MELT modules in the SAC and chromosome biorientation. (A) Schematic representation of synthetic LAP-KNL1 constructs, showing the microtubule- and PP1-binding domain in green and the kinetochore recruitment domain in orange. KI1 and KI2 motifs are shown as green bars, MELT-like sequences in blue, and TxxΩ-like sequences in red. See main text for details about constructs. (B and C) Representative images (B) and quantification (C) of LAP-KNL1-expressing Flp-in HeLa cells transfected with siRNAs to luciferase (siLUC) or to KNL1 (siKNL1) and treated with nocodazole. LAP-KNL1 is shown in green, BUB1 in red, centromeres (CREST) in blue, and DNA (DAPI) in white. Bars, 5 µm. Quantification in C shows total kinetochore signal intensity (+SD) of LAP-KNL1 and BUB proteins over CREST. Data are from >15 cells and representative of 3 experiments. Levels of kinetochore BUBs in control cells and of kinetochore LAP-KNL1 in KNL1-FL–expressing cells are set to 1. (D) Schematic as in A. See main text for details about constructs. (E) Time-lapse analysis of Flp-in HeLa cells expressing LAP-KNL1 variants, transfected with siLUC or siKNL1, and treated with nocodazole and 250 nM reversine. Data (n = 40 representative of 3 independent experiments) indicate cumulative fraction of cells that exit from mitosis (as scored by cell morphology using DIC) at the indicated time after NEB. (F) As in E, with the indicated constructs. (G) Time-lapse analysis of Flp-in HeLa cells expressing LAP-KNL1 variants and transfected with siLUC or siKNL1. Data (n = 40 representative of 3 independent experiments) indicate cumulative fraction of cells that exit from mitosis at the indicated time after NEB (as scored by GFP-H2B). (H) Quantification of chromosome alignment in Flp-in HeLa cells expressing LAP-KNL1 variants, transfected with siLUC or siKNL1, and treated with MG132 for 45 min. The data shown are from a single representative experiment out of three repeats. For the experiment shown, n = 40.