Figure 5.

Kinetochore-Derived Microtubules Interact with Spindle Pole Microtubules, onto which Kinetochores Are Subsequently Loaded and Cease Generating Microtubules

(A) KT-derived MTs interacted with spindle pole MTs, on which KTs were subsequently loaded. T3828 cells (Figure 1B) were treated as in Figure 1C. Images were acquired every 15 s. Representative time-lapse images (top) and schematic diagrams (bottom), showing interaction between a KT-derived MT and a spindle pole MT in an (i) anti-parallel and (ii) parallel manner. See also Movies S5 and S6.

(B) (i) BIK1-4×mCherry (T6718) and (ii) STU2-4×mCherry (T6987) cells with Pgal-CEN3-tetOs TetR-3×CFP YFP-TUB1 Pmet3-CDC20 were treated as in Figures 1C and 1B, respectively. Images were acquired (i) every 30 s for 35 min and (ii) every 20 s for 30 min. (i) MTs did not show new growth from KTs after KTs interacted with spindle pole MTs. Graphs show the percentages of time intervals, during which new MTs started extension from CEN3 before and after CEN3 association with spindle pole MTs. n, number of observed time intervals. (ii) The amount of Stu2 at CEN3 decreased immediately after CEN3 interacted with the surface of spindle pole MTs. Representative time-lapse images showing Stu2 signals at CEN3 (arrows) in cells where CEN3s interacted with spindle pole MTs (top). The mean (and SEM) of quantified Stu2 signal intensity at CEN3 from 8 such cells (red line, bottom). Time is shown relative to CEN3 encounter with spindle pole MTs. As a control for each cell, Stu2 intensity was measured at the same time points in a neighboring cell, in which CEN3 did not interact with spindle pole MTs (blue line). n.s., not significant. See Figure S5 in Supplemental Information.