Stu2 accumulation is sufficient for MT generation, and Stu1 is not required for it. (A) Diagram showing that Stu2-GFP-LacI is tethered at lacOs at a chromosome (Chr) arm locus. rDNA, ribosomal DNA. (B) The Stu2-GFP-LacI–tethered site is associated with MT/tubulin signals but not with Stu1 signals. P_GALS-STU2-GFP-LacI REC102:lacOs STU1-4×mCherry CFP-TUB1 P_MET3-CDC20 (T11037) cells were treated with mating pheromone for 2.5 h and then released into fresh media supplemented with raffinose and methionine. From 3 h after the release, galactose was added to the media for 1 h to express Stu2-GFP-LacI, and images were acquired. White dashed outline shows the shape of a yeast cell. Graph shows the percentages of lacOs sites with tubulin and Stu1 signals. n = 28. Note that in spite of the absence of Stu1 at the Stu2-GFP-LacI–tethered site, Stu1 still showed normal localization on the spindle. (C) MT/tubulin signals appear at the Stu2-GFP-LacI–tethered site in both the presence and absence of Stu1. STU1⁺ (T11722) and stu1-aid (T11686) cells with TIR1 P_GALS-STU2-GFP-LacI REC102:lacOs CFP-TUB1 P_MET3-CDC20 cells were treated as in B, except galactose was also added to the media 2 h after the release. From 3 h after the release, auxin NAA was added to the media for 1 h (to deplete Stu1-aid), and images were acquired. Tubulin signals associated with the lacOs site were quantified (graph; n = 17 in each condition). The p-value (two tailed) was obtained by an unpaired t test. Note that the collapsed bipolar spindle (left, bottom) suggests that Stu1 was indeed depleted in this condition (Yin et al., 2002). a.u., arbitrary unit.