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. Author manuscript; available in PMC: 2019 Nov 5.
Published in final edited form as: Curr Biol. 2018 Oct 25;28(21):3422–3429.e5. doi: 10.1016/j.cub.2018.10.006

Figure 1. A temporal switch in the requirements for Mad1-Mad2 targeting to kinetochores during SAC signaling.

Figure 1

(A) AAV- and CRISPR-mediated genome editing was used to modify the KNTC1 locus in HCT116 cells (Figure S1A-E). Cells expressing H2B-mCherry were treated with nocodazole or STLC and followed by epifluorescence and DIC timelapse microscopy. Images were acquired at 10-min intervals. Mitotic duration (from NEBD to chromatin decondensation) was quantified in at least 25 cells per condition per experiment (N=2). P-values were computed using Kruskal-Wallis and Dunn’s multiple comparisons tests. Error bars throughout the paper indicate s.e.m. unless stated otherwise. (B) Wildtype and KNTC1–/– RPE1 cells (Figure S1F-I) were treated with nocodazole, STLC, or taxol and followed using DIC optics. Cell rounding (mitotic entry) and cortical blebbing and flattening (mitotic exit) were used as landmarks. (C) Clonal HeLa KNTC1, ZW10, and ZWILCH knockouts [27] were treated with nocodazole and followed as in (B). (D) Mitotic timing in unperturbed wildtype and RZZ-deficient HeLa, RPE, and HCT116 cells. Where indicated Mps1 kinase was inhibited with reversine. (E and F) Wildtype and KNTC1–/– HCT116 cells expressing H2B-mCherry and FLAP-Mad1 were filmed during unperturbed mitosis using spinning disk confocal microscopy. Insets show enlarged views of FLAP-Mad1 recruitment to and dissociation from kinetochores. Scale bars throughout the paper are 10 μm unless stated otherwise. See also Videos S1 and S2. (G) Quantification of FLAP-Mad1 at misaligned chromosomes in (E) and (F). (H) Cells in (E) and (F) were filmed in the presence of nocodazole (n=6 for wildtype and n=14 for KNTC1–/–). (I and J) Wildtype and KNTC1-null RPE cells were treated with nocodazole and MG132 for 30 min or 4 hours before fixation for IFM. Mad1/CREST fluorescence intensity ratios were determined for at least 100 kinetochores in 5 cells per condition (N=3). (K) Wildtype and KNTC1-null RPE cells were treated with nocodazole in the presence or absence of hesperadin (hesp) to inhibit Aurora B kinase. Mitotic duration was determined from 30 cells per condition. See also Figure S1.