Figure 4.

Stu2 Is Sufficient to Nucleate and Extend Microtubules when It Is Tethered on a Chromosome Arm Locus

(A) A schematic diagram of Stu2 tethering on a chromosome arm locus. See text for details.

(B) Tubulin signals were often found at the Stu2-tethered site. Pgals-STU2-GFP-LacI REC102::lacOs CFP-TUB1 Pmet3-CDC20 (T4837) cells were treated with α factor in methionine drop-out medium with raffinose for 2.5 hr, and then released to YP medium containing raffinose and 2 mM methionine. After 2.5 hr, galactose was added to the medium (time 0 in graphs). As controls, cells with Pgals-GFP-LacI instead of Pgals-STU2-GFP-LacI (T4601) and cells lacking lacOs (T4840) were treated in the same way. GFP (Stu2 fused with LacI or LacI alone; green) and CFP (tubulin; red) images were acquired at indicated time points. Percentages of cells in each category (top) and representative images (at 60 min in graphs: bottom) of T4601, T4840, and T4837 cells (left to right).

(C) MTs extended from the Stu2-tethered site with distal plus ends. T4837 cells (see [B]) were treated as in (B), except that 1/10 volume of 1 M sorbitol was added to medium immediately before GFP and CFP images were acquired (left). Cells (T7184) with the same genotype as T4837, but with BIK1-4×mCherry, were treated in the same way, followed by acquisition of mCherry (Bik1; red), GFP (Stu2 fused with LacI; green), and CFP (tubulin; blue) images (right).

(D) MTs, extended from the Stu2-tethered site, showed interaction with spindle pole MTs. T4837 cells (see [B]) were treated as in (C). GFP and CFP images were acquired every 15 s (time 0: start of image acquistion). Representative time-lapse images (top) and schematic diagrams (bottom). See also Movie S4. See Figure S4 in Supplemental Information.