Mutation of Ser172 in yeast β tubulin induces defects in microtubule dynamics and cell division

Abstract

Ser172 of β tubulin is an important residue that is mutated in a human brain disease and phosphorylated by the cyclin-dependent kinase Cdk1 in mammalian cells. To examine the role of this residue, we used the yeast S. cerevisiae as a model and produced two different mutations (S172A and S172E) of the conserved Ser172 in the yeast β tubulin Tub2p. The two mutants showed impaired cell growth on benomyl-containing medium and at cold temperatures, altered microtubule (MT) dynamics, and altered nucleus positioning and segregation. When cytoplasmic MT effectors Dyn1p or Kar9p were deleted in S172A and S172E mutants, cells were viable but presented increased ploidy. Furthermore, the two β tubulin mutations exhibited synthetic lethal interactions with Bik1p, Bim1p or Kar3p, which are effectors of cytoplasmic and spindle MTs. In the absence of Mad2p-dependent spindle checkpoint, both mutations are deleterious. These findings show the importance of Ser172 for the correct function of both cytoplasmic and spindle MTs and for normal cell division.

Figure 1. SA and SE cells are benomyl-supersensitive; SE cells are cold-sensitive.

(A) Partial protein sequence of S. cerevisiae Tub2p (WT), and Tub2p with S172A or S172E mutations (SA or SE mutants), aligned with human β1 tubulin sequence. (B) Growth at 30°C of sequential dilutions of WT, SA and SE cells spotted on YPD media without benomyl (control) or containing 15 µg/ml of benomyl. (C) Measurement of optical density (OD) at 600 nm of cultures of 2 clones of WT, SA and SE cells in liquid YPD during several hours at 30°C or 37°C, or during several days at 10°C. Values are mean ± SEM.

Figure 2. At the restrictive temperature of 10°C, SE cells undergo a mitotic block, and nuclei are mis-located and mis-segregated in mitotic SA and SE cells.

(A, B) WT, SA, and SE diploid cells grown overnight in liquid medium at 30°C were either counted or shifted at 10°C for 24 h before counting. For each condition tested, 300 to 400 cells from 2 independent clones were scored. (A) Large-budded, M, small-budded, S/G2, and unbudded, G1 cells were scored. Percent of cells in M phase was significantly different in SA and SE strains as compared to WT strain. *** p<0.001, χ² test comparisons. (B) Nuclei of cells were stained with Hoechst. The percentages of four types of nuclear morphology in large-budded mitotic cells are indicated: an undivided nucleus in one cell body, an undivided nucleus at the bud neck, divided nuclei properly segregated into each cell body, and divided nuclei both located in one cell body. Distributions were significantly different in SA and SE cells as compared to WT cells: p<0.001 using χ² test comparisons.

Figure 3. Nuclear movements are inhibited in SA and SE cells prior to anaphase.

WT, SA and SE cells were transformed with a plasmid expressing GFP-Tub1p, and were analyzed by time-lapse microscopy. (A) Representative WT and SA pre-anaphase cells for which the bud-directed SPB was tracked at each time point of the time-lapse experiment. The track is overlaid in red on an image of the movie. Scale bar, 3 µm. Movies available in supplementary Figure S1. (B) Measurement of the travel distance covered by the bud-directed SPB in pre-anaphase cells over a period of 15–30 min (results are normalized in µm/min). Comparing to WT strain, spindle movements were reduced in both mutant strains. Number of analyzed cells: WT, n = 19; SA, n = 15; SE, n = 17. Error bars are SEM. ** p<0.01, *** p<0.001, t test comparisons of mutant cells vs. WT cells.

Figure 4. Nucleation and/or elongation activities of cMTs are modified in SA and SE cells.

WT, SA and SE cells were transformed with a plasmid expressing Bik1p-GFP, and were analyzed by time-lapse microscopy. (A) Representative WT, SA and SE pre-anaphase cells for which the plus-end of each cMT was tracked at each time point of the time-lapse experiment. Tracks are overlaid on an image of the movie. Scale bar, 3 µm. Movies are available in supplementary Figure S2. (B) The number of cMTs that appeared in 15 min in pre-anaphase cells was counted. Comparing to WT cells, this number was reduced in SA cells, but was greatly increased in SE cells. Number of analyzed cells: WT, n = 10; SA, n = 11; SE, n = 11; error bars are SEM. ** p<0.01, *** p<0.001, t test comparisons of mutant cells vs. WT cells.

Figure 5. Bud-directed cMT dynamics in WT, SA and SE cells.

Values are taken from Table 1 and only data for which significant differences between a mutant strain and the WT strain were found were represented here. (A) Frequencies of catastrophes and rescues, (B) growth, shrinkage and pause mean durations, (C) mean length change during growth and shrinkage, (D) total time spent while growing, shrinking and pausing, and (E) mean life time. Comparing to WT cells, many parameters of cMT dynamics varied in mutant cells, with SA cMTs behaving very differently from SE cMTs. Error bars are SEM. * p<0.05, ** p<0.01, *** p<0.001, t test comparisons of mutant cells vs. WT cells.

Figure 6. SA mutation worsens kar9Δ or dyn1Δ deletion phenotypes, with a number of double-mutant mitotic cells exhibiting polyploidy.

SA, kar9Δ, dyn1Δ simple mutant cells or SA/kar9Δ and SA/dyn1Δ double mutant cells, were grown in liquid medium overnight at 30°C and shifted for 24 h at 10°C. Cells were then fixed, nuclei stained with Hoechst and nuclear number and position in large-budded mitotic cells were scored. Results are presented as percentages of a total of 400 mitotic cells from 2 independent clones, for each condition tested. Only abnormal mitotic patterns are shown in the graph. Error bars are SEM.