Figure 1.
Figure 1. Kinetochores undergo intermittent deformations. (A) Measurement of intra- and interkinetochore lengths and distances. The green and red circles represent EGFP–CENP-A and mCherry-Mis12, respectively. (B) Setting a threshold for the intra- and interkinetochore lengths/distances. Fixed cells treated with 160 ng/ml (531.6 nM) nocodazole (Noc.) were analyzed for the intra- and interkinetochore lengths and distances, and their threshold values (red dotted lines) were calculated as 0.10 µm and 0.79 µm, respectively (n = 112 kinetochores from 13 cells; bottom). These values were applied to the profile in a mock condition (n = 118 kinetochores from 12 cells; top). (C) A profile for intra- and interkinetochore lengths and distances obtained for EGFP-Ndc80 and Spc25-mCherry. The threshold values determined in the 160-ng/ml nocodazole condition (n = 24 kinetochores from 5 cells) were 0.11 µm and 0.76 µm for the intra- and interkinetochore length/distance, respectively (blue dotted lines). In a mock condition, <3% of cases had intrakinetochore length beyond the threshold level (n = 38 kinetochores from 5 cells). (D) A kymograph of a kinetochore pair. Note that mCherry-Mis12 signal (red) deviates from EGFP–CENP-A signal (green) transiently. The right panel shows representative intra- and interkinetochore lengths and distances over time. Red diamonds indicate peaks for the intrakinetochore lengths that were subjected to the analysis in E. (E) Basis for CENP-A and Mis12 behavior. Relative poleward positions of CENP-A and Mis12 are determined by averaging 76 cases. Time 0 defines the peak of the intrakinetochore length, and the position of CENP-A at −6 s is set to 0. Arrows indicate the movements of CENP-A and Mis12 during 3-s intervals, and the asterisks mark the significant movement change (t test; *, P < 0.05). Movements of CENP-A and Mis12 on one kinetochore did not significantly affect their movements on the other kinetochore (Fig. S2, available); the stretching occurs on kinetochore sisters independently from each other. (F) A cartoon illustrating kinetochore extension and recoiling. Dotted red and green arrows indicate significant poleward movements of CENP-A and Mis12, respectively. Bar, 1 µm.

Kinetochores undergo intermittent deformations. (A) Measurement of intra- and interkinetochore lengths and distances. The green and red circles represent EGFP–CENP-A and mCherry-Mis12, respectively. (B) Setting a threshold for the intra- and interkinetochore lengths/distances. Fixed cells treated with 160 ng/ml (531.6 nM) nocodazole (Noc.) were analyzed for the intra- and interkinetochore lengths and distances, and their threshold values (red dotted lines) were calculated as 0.10 µm and 0.79 µm, respectively (n = 112 kinetochores from 13 cells; bottom). These values were applied to the profile in a mock condition (n = 118 kinetochores from 12 cells; top). (C) A profile for intra- and interkinetochore lengths and distances obtained for EGFP-Ndc80 and Spc25-mCherry. The threshold values determined in the 160-ng/ml nocodazole condition (n = 24 kinetochores from 5 cells) were 0.11 µm and 0.76 µm for the intra- and interkinetochore length/distance, respectively (blue dotted lines). In a mock condition, <3% of cases had intrakinetochore length beyond the threshold level (n = 38 kinetochores from 5 cells). (D) A kymograph of a kinetochore pair. Note that mCherry-Mis12 signal (red) deviates from EGFP–CENP-A signal (green) transiently. The right panel shows representative intra- and interkinetochore lengths and distances over time. Red diamonds indicate peaks for the intrakinetochore lengths that were subjected to the analysis in E. (E) Basis for CENP-A and Mis12 behavior. Relative poleward positions of CENP-A and Mis12 are determined by averaging 76 cases. Time 0 defines the peak of the intrakinetochore length, and the position of CENP-A at −6 s is set to 0. Arrows indicate the movements of CENP-A and Mis12 during 3-s intervals, and the asterisks mark the significant movement change (t test; *, P < 0.05). Movements of CENP-A and Mis12 on one kinetochore did not significantly affect their movements on the other kinetochore (Fig. S2, available); the stretching occurs on kinetochore sisters independently from each other. (F) A cartoon illustrating kinetochore extension and recoiling. Dotted red and green arrows indicate significant poleward movements of CENP-A and Mis12, respectively. Bar, 1 µm.

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