Control of Microtubule Dynamics by Stu2p Is Essential for Spindle Orientation and Metaphase Chromosome Alignment in Yeast

Abstract

Stu2p is a member of a conserved family of microtubule-binding proteins and an essential protein in yeast. Here, we report the first in vivo analysis of microtubule dynamics in cells lacking a member of this protein family. For these studies, we have used a conditional Stu2p depletion strain expressing alpha-tubulin fused to green fluorescent protein. Depletion of Stu2p leads to fewer and less dynamic cytoplasmic microtubules in both G1 and preanaphase cells. The reduction in cytoplasmic microtubule dynamics is due primarily to decreases in both the catastrophe and rescue frequencies and an increase in the fraction of time microtubules spend pausing. These changes have significant consequences for the cell because they impede the ability of cytoplasmic microtubules to orient the spindle. In addition, recovery of fluorescence after photobleaching indicates that kinetochore microtubules are no longer dynamic in the absence of Stu2p. This deficiency is correlated with a failure to properly align chromosomes at metaphase. Overall, we provide evidence that Stu2p promotes the dynamics of microtubule plus-ends in vivo and that these dynamics are critical for microtubule interactions with kinetochores and cortical sites in the cytoplasm.

β-Tubulin C354 Mutations that Severely Decrease Microtubule Dynamics Do Not Prevent Nuclear Migration in Yeast

“…To examine the effects of these mutations on microtubule stability in more detail, we analyzed microtubule dynamics in both wild-type and tub2-C354 mutant cells using a GFP-Tub1p fusion protein. The wild-type values we obtained for the microtubule dynamic parameters of growth rate, shrinkage rate, catastrophe frequency, and rescue frequency in wild-type cells were in agreement with those reported by others (Carminati and Stearns, 1997;Shaw et al, 1997b;Tirnauer et al, 1999;Adames and Cooper, 2000;Vogel et al, 2001;Kosco et al, 2001). However, all aspects of microtubule dynamics were drastically reduced in both tub2-C354 mutants.…”

“…Thus, at least in the tub2-C354 mutants, the directed-transport mechanism appears responsible for locating the microtubule structure at the bud site, possibly with the help of actin filaments (Theesfeld et al, 1999;Yin et al, 2000). Defects in spindle position and orientation can be caused by changes in microtubule dynamics (Tirnauer et al, 1999;Kosco et al 2001). In the tub2-C354 mutants, the spindle was positioned near the bud neck at the onset of anaphase.…”

Poleward Tubulin Flux in Spindles: Regulation and Function in Mitotic Cells

“…Interestingly, Msps has been found to inhibit the paused state of microtubules in interphase S2 cells, by promoting switching from pause to growth and possibly to shrinkage (Brittle and Ohkura, 2005). In addition, the budding yeast orthologue of Msps/XMAP215, Stu2p, was found to increase the dynamics of kinetochore microtubules, by increasing both the rescue and catastrophe frequencies and reducing the pause duration of cytoplasmic microtubules (Kosco et al, 2001). If Msps increases microtubule dynamicity by forcing transitions from the paused state, then Msps could serve as a facilitator to other factors stimulating either growth or shrinkage ( Figure 5, A and B).…”

γ-Tubulin Is Required for Proper Recruitment and Assembly of Kar9–Bim1 Complexes in Budding Yeast

“…Elongation were defined as three consecutive points on a regression line in which the increased change in length of a microtubule (⌬length increase ) was Ն0.3 m. Shrinkage was defined as three consecutive points on a regression line in which the decreased change in length of a microtubule (⌬length decrease ) was Ն0.3 m. Microtubule pausing was defined as a change in length spanning three points on a regression line that was Յ0.3 m. Microtubule catastrophes were defined as shrinkage after growth or pause, whereas microtubule rescues were defined as transitions to growth after a shrinkage or pause (Adames and Cooper, 2000). The frequencies of catastrophe and rescue were calculated as described previously (Kosco et al, 2001). …”

“…To test these possibili- ties, we investigated the impact of Kar9 on the dynamics of astral microtubules in the bud in TUB4, tub4-⌬dsyl, kar9⌬, and tub4-⌬dsyl kar9⌬ cells expressing GFP-Tub1. The rate of elongation and shrinkage ( m/min), frequencies (event/s) of catastrophe and rescue, and the duration of elongation, shrinkage and pauses (minutes) were calculated for astral microtubule targeted to the bud as described previously (Adames and Cooper, 2000;Kosco et al, 2001; for experimental details, see Materials and Methods). The analysis was restricted to small-budded cells.…”