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. 2017 Jul 15;130(14):2266–2276. doi: 10.1242/jcs.203000

Fig. 4.

Fig. 4.

KT detachment is found in physiological conditions after an end-on attached KT comes into contact with a laterally attached KT. (A) Representative example of centromere detachment in a Kar3-depleted cell in physiological conditions. Cells (T11434) with kar3-aid TIR1 CEN5-lacOs GFP-LacI GFP-TUB1 MTW-4×mCherry NDC80-4×mCherry were treated with a mating pheromone for 3 h (to arrest them in G1 phase) and released into fresh medium. NAA was added for the last 30 min of the G1 arrest and also after release from the arrest (to deplete Kar3). 30 min after release from the G1 arrest, images (GFP and mCherry signals) were acquired every 10 s. See Movie 5. A representative example of a Kar3 wild-type cell is shown in Fig. S3B. (B) Diagrams explaining analyses in C and D. The ‘original capture position’, ‘contact position’ and ‘detachment position’ were measured as shown here and plotted in C. Rectangles in color represent the categorized situations shown in D in the same color. (C) In the absence of KT sliding, contact between a KT (not at CEN5) and CEN5, and subsequent detachment of CEN5 happens further from a spindle pole. Graphs show the initial capture position (distance from a spindle pole) of CEN5 only in cases where there was subsequent detachment (left; see B), the position of KTs coming into contact with CEN5 (middle; see B) and the positions of CEN5 detachment (right; see B). T11434 cells (see A) and T11435 cells (KAR3+, otherwise the same as T11434 cells) were treated and images were acquired, as in A. Graphs show individual data points and mean±s.e.m. The P-values were obtained by t-test. (D) In the absence of KT sliding, CEN5 detachment happens more frequently in physiological conditions. Images acquired in A were used for this analysis. Following the situation where a KT (not on CEN5) and CEN5 formed a lateral attachment on presumably the same MT (the KT is more distal to a spindle pole than CEN5), one of the following three events took place (see B): (1) both the KT and CEN5 reached a spindle pole without contact (blue), (2) after the KT formed end-on attachment, it came into contact with CEN5 and they were co-transported to the spindle pole (green), or (3) after the KT formed end-on attachment, it came into contact with CEN5, and CEN5 showed detachment (orange). Images acquired in A were used for this analysis. The graph shows the percentage of each event. The P-value was obtained by use of a χ-squared test for trend as in Fig. 3D.