Figure 1.
Figure 1. Stu1 is required for Stu2 recruitment to KTs, which promotes MT generation at KTs. (A) Stu1 depletion leads to a reduction of Stu2 signals at KTs. STU1+ (T10699) and Stu1-aid (T10697) cells with TIR1 PGAL-CEN3-tetOs TetR-3×CFP mCherry-TUB1 STU2-3×GFP PMET3-CDC20 were treated with mating pheromone for 2.5 h (to arrest in G1 phase) and released to fresh media with methionine (for Cdc20 depletion) in the presence of galactose (for CEN3 inactivation). From 3 h after the release, auxin NAA was added to the media for 1 h to deplete Stu1-aid. Cells were then suspended in medium with methionine and with glucose (for CEN3 reactivation) in the presence of NAA, and images were acquired every 2 min. Stu2 signals associated with CEN3 were quantified at the time point before CEN3 capture by a spindle MT (right; n = 20 in each condition), and representative images are shown (left). White dashed outlines show the shape of yeast cells. The p-value (two tailed) was obtained by an unpaired t test. Note that, after Stu1 depletion, Stu2 was significantly reduced at CEN3 (white arrow) but not at the MT plus end (yellow arrow). (B) Stu1 depletion leads to the reduction of MT/tubulin signals at KTs. STU1+ (T10594) and stu1-aid (T10501) cells with TIR1 PGAL-CEN3-tetOs TetR-3×CFP GFP-TUB1 PMET3-CDC20 were treated, and their images were acquired as in A (left). Tubulin signals associated with CEN3 were analyzed as in A (right; n = 20 in each condition). Insets show magnification of the area in which CEN3 localizes. Glucose was added at time 0. (C) Stu2 depletion leads to a loss of MT/tubulin signal at KTs but does not change the amount of Stu1 there. STU2+ (T10833) and stu2-aid (T10834) cells with TIR1 PGAL-CEN3-tetOs TetR-3×CFP mCherry-TUB1 STU1-2×GFP PMET3-CDC20 were treated, and their images were acquired as in A. Stu1 signals associated with CEN3 were analyzed as in A (n = 18 and 15 for Stu2 wild-type and Stu2 depletion, respectively). Note that after depletion of Stu2, KT-derived MTs were abolished and MT signals were reduced on the spindle, as shown in this example of Stu2 depletion and previously (Kitamura et al., 2010). a.u., arbitrary unit. (D) Stu1 and Stu2 are closely associated at noncaptured KTs. The diagram illustrates that a close association between Stu1-VC and Stu2-VN leads to the generation of Venus signals. STU2-VN STU1-VC (T12563), STU2-VN (T12633), and STU1-VC (T12731) cells with mCherry-TUB1 MTW1-3×CFP NDC80-3×CFP (where VN and VC are N- and C-terminal fragments of Venus fluorescent protein) were treated with mating pheromone for 3 h and then released into fresh media supplemented with nocodazole. 60 min after the release, images were acquired. Numbers of bright KT clusters (not associated with a spindle pole; denominators) and those with Venus signals (numerators) are shown on the right. P-values (two tailed) were obtained by Fisher’s exact test.

Stu1 is required for Stu2 recruitment to KTs, which promotes MT generation at KTs. (A) Stu1 depletion leads to a reduction of Stu2 signals at KTs. STU1+ (T10699) and Stu1-aid (T10697) cells with TIR1 PGAL-CEN3-tetOs TetR-3×CFP mCherry-TUB1 STU2-3×GFP PMET3-CDC20 were treated with mating pheromone for 2.5 h (to arrest in G1 phase) and released to fresh media with methionine (for Cdc20 depletion) in the presence of galactose (for CEN3 inactivation). From 3 h after the release, auxin NAA was added to the media for 1 h to deplete Stu1-aid. Cells were then suspended in medium with methionine and with glucose (for CEN3 reactivation) in the presence of NAA, and images were acquired every 2 min. Stu2 signals associated with CEN3 were quantified at the time point before CEN3 capture by a spindle MT (right; n = 20 in each condition), and representative images are shown (left). White dashed outlines show the shape of yeast cells. The p-value (two tailed) was obtained by an unpaired t test. Note that, after Stu1 depletion, Stu2 was significantly reduced at CEN3 (white arrow) but not at the MT plus end (yellow arrow). (B) Stu1 depletion leads to the reduction of MT/tubulin signals at KTs. STU1+ (T10594) and stu1-aid (T10501) cells with TIR1 PGAL-CEN3-tetOs TetR-3×CFP GFP-TUB1 PMET3-CDC20 were treated, and their images were acquired as in A (left). Tubulin signals associated with CEN3 were analyzed as in A (right; n = 20 in each condition). Insets show magnification of the area in which CEN3 localizes. Glucose was added at time 0. (C) Stu2 depletion leads to a loss of MT/tubulin signal at KTs but does not change the amount of Stu1 there. STU2+ (T10833) and stu2-aid (T10834) cells with TIR1 PGAL-CEN3-tetOs TetR-3×CFP mCherry-TUB1 STU1-2×GFP PMET3-CDC20 were treated, and their images were acquired as in A. Stu1 signals associated with CEN3 were analyzed as in A (n = 18 and 15 for Stu2 wild-type and Stu2 depletion, respectively). Note that after depletion of Stu2, KT-derived MTs were abolished and MT signals were reduced on the spindle, as shown in this example of Stu2 depletion and previously (Kitamura et al., 2010). a.u., arbitrary unit. (D) Stu1 and Stu2 are closely associated at noncaptured KTs. The diagram illustrates that a close association between Stu1-VC and Stu2-VN leads to the generation of Venus signals. STU2-VN STU1-VC (T12563), STU2-VN (T12633), and STU1-VC (T12731) cells with mCherry-TUB1 MTW1-3×CFP NDC80-3×CFP (where VN and VC are N- and C-terminal fragments of Venus fluorescent protein) were treated with mating pheromone for 3 h and then released into fresh media supplemented with nocodazole. 60 min after the release, images were acquired. Numbers of bright KT clusters (not associated with a spindle pole; denominators) and those with Venus signals (numerators) are shown on the right. P-values (two tailed) were obtained by Fisher’s exact test.

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