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

Figure 1.

Figure 1

Microtubules Are Generated at Kinetochores prior to Their Interaction with Spindle Pole Microtubules

(A) Tubulin signals are found at reassembled KTs before their interaction with spindle pole MTs in S phase. CTF19-3×GFP MTW1-3×GFP YFP-TUB1 cells (T3110) were treated with α factor and subsequently released to fresh media. After 25 min, YFP (tubulin; red) and GFP (Ctf19, Mtw1; green) images were acquired. Cell shapes are outlined in white.

(B) Tubulin signals are found at CEN3 after its reactivation and showed extension in some cases. (i and iii) Pgal-CEN3-tetOs (replacing CEN3) TetR-3×CFP YFP-TUB1 Pmet3-CDC20 cells (T3828) were treated with α factor in methionine drop-out medium with raffinose for 2.5 hr, and then released to YP medium containing galactose, raffinose, and 2 mM methionine. After 3.5 hr, cells were suspended in synthetic complete medium containing glucose and methionine. Subsequently, YFP (tubulin; red) and CFP (CEN3; green) images were acquired. (ii) Pgal-CEN3-tetOs (replacing CEN15) TEL15R-tetOs TEL15L-tetOs TetR-GFP YFP-TUB1 Pmet3-CDC20 cells (T3845) were treated in the same way, and YFP (tubulin; red) and GFP (CEN, TELs on chromosome XV; green) images were acquired.

(C) Tubulin signals extended from CEN3 for a greater length, under a mild osmotic stress. T3828 cells were treated as in (B), but 1/10 volume of 1 M sorbitol was added immediately after transfer to glucose-containing medium. YFP (tubulin; red) and CFP (CEN3; green) images were acquired every 30 s (time 0, start of image acquisition). MT extensions from CEN3 are shown in (i) a representative time-lapse sequence and (ii) selected images. See also Supplemental Experimental Procedures and Movie S1. See Figure S1 in Supplemental Information.