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. 2010 Feb 16;18(2):248–259. doi: 10.1016/j.devcel.2009.12.018

Figure 6.

Figure 6

Kinetochore-Derived Microtubules Appear More Frequently When Kinetochore Interaction with Spindle Pole Microtubules Is Delayed, and then Seem to Facilitate This Interaction

(A–E) MTW1-4×mCherry NDC80-4×mCherry YFP-TUB1 cells (T6444) were treated as in Figure 1A. From 20 min after washout of α factor, mCherry (KTs; green) and YFP (tubulin; red) images were collected every 10 s for 10 min. During this time window in S phase, KTs were often reassembled and reassociated with spindle pole MTs (Kitamura et al., 2007).

(A) Time points in each event (75 events from 71 cells), in which KT signals were detected (first detection at time 0) away from a spindle pole and subsequently interacted with spindle pole MTs (gray box). Time points showing KT-associated MT/tubulin signals (not connected to spindle pole MTs) are indicated by cyan boxes, whereas time points showing brighter KT signals are marked with orange dots.

(B) Representative time-lapse images (Event #74 in [A]) showing Mtw1-4×mCherry/Ndc80-4×mCherry signals in green and YFP-Tub1 in red. Time is shown in seconds (time 0 is as defined in [A]).

(C) Percentages of time points (in [A]) within each time window, showing (i) MT/tubulin signals at KTs (cyan) or (iii) brighter KT signals (orange), as well as those showing such signals for two or more consecutive time points (ii, magenta; iv, blue).

(D) Percentages of free KTs (shown in [A]), which were not yet associated with spindle pole MTs, are plotted (black dots) against the time after the first detection of KT signals (time 0 in [A]). A regression curve was drawn based on a one-phase exponential decay curve. The total number of free KTs at time 0 was predicted from this exponential decay curve, including those that escaped our observation due to a rapid interaction with spindle pole MTs after KT assembly (see Supplemental Experimental Procedures).

(E) Percentages of free KTs (shown in [A]) are plotted against the time (i) after the first appearance of MT/tubulin signals at KTs (cyan) and (ii) after their first appearance for two or more sequential points (time was measured from the first time point of consecutive ones: magenta). Percentages of free KTs are also plotted against the time (iii) after the first appearance of brighter KT signals (orange) and (iv) after their first appearance for two or more sequential points (blue). In each figure, a black line links the black dots shown in (D), providing a control for comparison with each colored line. See also Supplemental Experimental Procedures. See Figure S6 in Supplemental Information.