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

Figure 3.

Figure 3

Kinetochore Components and +TIPs, but Not the γ-Tubulin Complex, Facilitate the Generation of Microtubules from Kinetochores

(A) Summary of mutant phenotypes. Using the CEN reactivation assay (Figure S1A), (i and iii) the extension of spindle pole and KT-derived MTs (original data, not shown and in Figure S3B, respectively) as well as (ii and iv) tubulin and Stu2 signals at KTs (original data in Figures S3A and S3E, respectively) were studied in wild-type and various mutant cells. NA, not applicable; ND, not determined.

(B) ndc10-1 mutant cells were defective in the nucleation of MTs at KTs and CEN3 interaction with spindle pole MTs. Wild-type NDC10+ (T3828) and ndc10-1 (T4230) cells with Pgal-CEN3-tetOs TetR-3×CFP YFP-TUB1 Pmet3-CDC20 were treated as in Figure 1B, but the temperature was raised to 35°C 30 min before cells were suspended in synthetic complete medium containing glucose and methionine. YFP (tubulin; red) and CFP (CEN3; green) images were acquired every 5 min at 35°C. Top: representative time-lapse images (time 0: transfer to glucose-containing medium). Bottom: changes in percentages of cells in which CEN3 was (yellow) and was not (magenta) associated with spindle pole MTs. The latter cases were further divided into two, in which tubulin signals were found (red) or not (blue) at CEN3. n, number of observed cells.

(C) The stu2-10 mutant was defective in the nucleation of MTs at CEN3, but showed normal KT reassembly. Wild-type STU2+ (T6670) and stu2-10 (T7285) cells with Pgal-CEN3-tetOs TetR-3×CFP YFP-TUB1 MTW1-4×mCherry NDC80-4×mCherry Pmet3-CDC20 were treated as in (B). mCherry (KTs; Mtw1, Ndc80; green), YFP (tubulin; red), and CFP (CEN3; blue) images were acquired every 5 min. Data are presented as in (B) (the presence of KT signals is shown in green stripes).

(D) Localization of Stu2 at CEN3 and the plus ends of KT-derived MTs. Pgal-CEN3-tetOs TetR-3×CFP YFP-TUB1 STU2-4×mCherry Pmet3-CDC20 cells (T6987) were treated as in Figure 1C. mCherry (Stu2; green), YFP (tubulin; red), and CFP (CEN3; blue) images were acquired every 20 s (time 0; arbitrary). See also Movie S2. See Figure S3 in Supplemental Information.