Figure 3.
Figure 3. After Stu1 depletion, KT-derived MTs are abolished, and KT capture by spindle MTs is delayed in physiological conditions. (A) Two examples of KT capture by spindle MTs in STU1 wild-type cells. STU1+ MTW1-4×mCherry NDC80-4×mCherry YFP-TUB1 cells (T11242) were treated with a mating pheromone for 3 h and released to fresh media. 30 min before the release, rapamycin was added to the media (it was present after the release as well). From 20 min after the release, images were acquired every 10 s. The first time point when a noncaptured KT was detected is denoted as time 0. On the left, a KT-associated tubulin signal was visible at 0 s, and the KT was captured by a spindle MT at 10 s. On the right, an MT extended from the KT at 50–100 s, and the KT was captured by a spindle MT at 110 s. White dashed outlines show the shape of yeast cells. KT capture was discerned when an overlap of a KT signal with spindle MTs was followed by KT motion toward a spindle pole. (B) The percentage of noncaptured KTs (100% at time 0; time 0 is defined as in A) was plotted and fitted to a simple exponential decay curve in wild-type cells (left, black dots and line) based on the result in Fig. S3 A, Stu1 wild-type (n = 60). A KT-associated MT/tubulin signal appeared at a single or consecutive time points, and the first time point of each appearance was defined as the new time 0 for replotting the percentage of noncaptured KTs (left, green squares and line). Half-lives were calculated based on fitted exponential decay curves. (C) Two examples of the KT capture by spindle MTs with Stu1 depletion. stu1–anchor-away MTW1-4×mCherry NDC80-4×mCherry YFP-TUB1 cells (T11230) were treated, and images were acquired as in A. Time 0 is defined as in A. A KT was observed first at time 0 and captured by a spindle MT at 80 s (left) and 240 s (right), and no tubulin signal was associated with noncaptured KTs. (D) The percentage of noncaptured KTs in Stu1-depleted cells (100% at time 0; time 0 is defined as in A) was plotted and fitted to a simple exponential decay curve (left, magenta triangles and line) based on the result in Fig. S3 A, Stu1 depletion (n = 74). The result for Stu1 wild-type cells in A is shown for comparison (black dots and line). Error bars represent a standard error of proportion for the data points (right). P-values (two tailed) were obtained by log-rank tests for survival curves.

After Stu1 depletion, KT-derived MTs are abolished, and KT capture by spindle MTs is delayed in physiological conditions. (A) Two examples of KT capture by spindle MTs in STU1 wild-type cells. STU1+ MTW1-4×mCherry NDC80-4×mCherry YFP-TUB1 cells (T11242) were treated with a mating pheromone for 3 h and released to fresh media. 30 min before the release, rapamycin was added to the media (it was present after the release as well). From 20 min after the release, images were acquired every 10 s. The first time point when a noncaptured KT was detected is denoted as time 0. On the left, a KT-associated tubulin signal was visible at 0 s, and the KT was captured by a spindle MT at 10 s. On the right, an MT extended from the KT at 50–100 s, and the KT was captured by a spindle MT at 110 s. White dashed outlines show the shape of yeast cells. KT capture was discerned when an overlap of a KT signal with spindle MTs was followed by KT motion toward a spindle pole. (B) The percentage of noncaptured KTs (100% at time 0; time 0 is defined as in A) was plotted and fitted to a simple exponential decay curve in wild-type cells (left, black dots and line) based on the result in Fig. S3 A, Stu1 wild-type (n = 60). A KT-associated MT/tubulin signal appeared at a single or consecutive time points, and the first time point of each appearance was defined as the new time 0 for replotting the percentage of noncaptured KTs (left, green squares and line). Half-lives were calculated based on fitted exponential decay curves. (C) Two examples of the KT capture by spindle MTs with Stu1 depletion. stu1–anchor-away MTW1-4×mCherry NDC80-4×mCherry YFP-TUB1 cells (T11230) were treated, and images were acquired as in A. Time 0 is defined as in A. A KT was observed first at time 0 and captured by a spindle MT at 80 s (left) and 240 s (right), and no tubulin signal was associated with noncaptured KTs. (D) The percentage of noncaptured KTs in Stu1-depleted cells (100% at time 0; time 0 is defined as in A) was plotted and fitted to a simple exponential decay curve (left, magenta triangles and line) based on the result in Fig. S3 A, Stu1 depletion (n = 74). The result for Stu1 wild-type cells in A is shown for comparison (black dots and line). Error bars represent a standard error of proportion for the data points (right). P-values (two tailed) were obtained by log-rank tests for survival curves.

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