Microtubule Interactions with the Cell Cortex Causing Nuclear Movements in Saccharomyces cerevisiae

Abstract

During mitosis in budding yeast the nucleus first moves to the mother-bud neck and then into the neck. Both movements depend on interactions of cytoplasmic microtubules with the cortex. We investigated the mechanism of these movements in living cells using video analysis of GFP-labeled microtubules in wild-type cells and in EB1 and Arp1 mutants, which are defective in the first and second steps, respectively. We found that nuclear movement to the neck is largely mediated by the capture of microtubule ends at one cortical region at the incipient bud site or bud tip, followed by microtubule depolymerization. Efficient microtubule interactions with the capture site require that microtubules be sufficiently long and dynamic to probe the cortex. In contrast, spindle movement into the neck is mediated by microtubule sliding along the bud cortex, which requires dynein and dynactin. Free microtubules can also slide along the cortex of both bud and mother. Capture/shrinkage of microtubule ends also contributes to nuclear movement into the neck and can serve as a backup mechanism to move the nucleus into the neck when microtubule sliding is impaired. Conversely, microtubule sliding can move the nucleus into the neck even when capture/shrinkage is impaired.

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.…”

“…These results indicate that microtubule dynamics can be altered to produce either net disassembly (small-budded) or net assembly (late-anaphase) of cytoplasmic microtubules. The decrease in length of the single microtubule in small-budded cells indicates that microtubule depolymerization is responsible for nuclear migration to the bud neck (Adames and Cooper, 2000;Beach et al, 2000).…”

“…Another mechanism of microtubule localization to the bud site has been proposed that involves directed-transport of microtubule ends along polarized actin filaments to the bud (Yin et al, 2000). In wild-type cells, cytoplasmic microtubules interact transiently with the future bud site and bud cortex, but the dynamic nature of the microtubules typically results in detachment and depolymerization out of the bud (Adames and Cooper, 2000;Beach et al, 2000). However, in all the tub2-C354 mutant cells examined, the cytoplasmic microtubule stopped moving and became stationary, with its end in the vicinity of imminent bud growth before bud emergence was visible.…”

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

“…Microtubule lengths were measured in triplicate, and the average value for each time point were used for calculations. 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.…”