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. 2022 Sep 12:10:989820.
doi: 10.3389/fcell.2022.989820. eCollection 2022.

Microtubule integrity regulates budding yeast RAM pathway gene expression

Cameron Howard Lee  1 , Sue Biggins  1
Affiliations

Microtubule integrity regulates budding yeast RAM pathway gene expression

Cameron Howard Lee et al. Front Cell Dev Biol. .

Abstract

During mitosis, cells must spatiotemporally regulate gene expression programs to ensure accurate cellular division. Failures to properly regulate mitotic progression result in aneuploidy, a hallmark of cancer. Entry and exit from mitosis is largely controlled by waves of cyclin-dependent kinase (CDK) activity coupled to targeted protein degradation. The correct timing of CDK-based mitotic regulation is coordinated with the structure and function of microtubules. To determine whether mitotic gene expression is also regulated by the integrity of microtubules, we performed ribosome profiling and mRNA-sequencing in the presence and absence of microtubules in the budding yeast Saccharomyces cerevisiae. We discovered a coordinated translational and transcriptional repression of genes involved in cell wall biology processes when microtubules are disrupted. The genes targeted for repression in the absence of microtubules are enriched for downstream targets of a feed-forward pathway that controls cytokinesis and septum degradation and is regulated by the Cbk1 kinase, the Regulation of Ace2 Morphogenesis (RAM) pathway. We demonstrate that microtubule disruption leads to aberrant subcellular localization of Cbk1 in a manner that partially depends on the spindle position checkpoint. Furthermore, constitutive activation of the RAM pathway in the absence of microtubules leads to growth defects. Taken together, these results uncover a previously unknown link between microtubule function and the proper execution of mitotic gene expression programs to ensure that cell division does not occur prematurely.

Keywords: Cbk1; RAM pathway; microtubule; mitosis; ribosome profiling; transcription; translation.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Ribosome profiling reveals a nocodazole induced translational response. (A) CDC20-AID cells (SBY14004) were split into two conditions and simultaneously treated with auxin to arrest cells in metaphase and either nocodazole or DMSO. Lysates from these populations were used for ribosome profiling and mRNA sequencing. (B) Polysome profiles generated from DMSO (blue) and nocodazole (red) treated cells. y-axis is absorbance at 254 nm and x-axis is millimeters (mm) from top of gradient. (C) Volcano plot of ribosome profiling data. Log2 fold changes of total mRNA-normalized footprint abundance (nocodazole vs. DMSO) are plotted on the x-axis, and negative log10 multiple-test corrected p-values are plotted on the y-axis. Individual genes are represented as black dots, and significantly translationally regulated genes (Wald test, Benjamini Hochberg adjusted p < 0.05) are colored in red. (D) Genome browser tracks of ribosome profiling and mRNA sequencing data for two representative genes. Data from the DMSO control condition is shown in blue and nocodazole condition in shown in red. Aggregate (across all replicates) counts per million are plotted on the y-axis. Genomic coordinates are plotted along the x-axis.
FIGURE 2
FIGURE 2
Nocodazole treatment induces transcriptional regulation of Ace2 target genes. (A) mRNA-seq data from cdc20-AID (SBY14004) cells treated with DMSO or nocodazole. Altered expression of genes upon nocodazole treatment. Log2 mean counts across all replicates and conditions plotted on the y-axis, with log2 fold change (nocodazole vs. DMSO) plotted on the y-axis. Individual genes are represented as black dots, and differentially expressed genes (Wald test, Benjamini Hochberg adjusted p < 0.05 and fold-change > 2) are colored in red. (B) Motif discovery analysis (HOMER) identification of the top 5 significant transcription factor motifs enriched within promoters of differentially expressed genes. (C) Binding profile and heatmap of Swi5, Ace2, and Swi4 ChIP-seq (Rossi et al., 2021) across the promoters of differentially expressed genes from (A). Each row represents a 1.2 kb window (−1 kb to +0.2 kb) around a given gene’s start codon and heatmap is sorted by maximum ChIP signal for each promoter. (D) Nocodazole induced decrease in gene expression in CDC20-AID cells (SBY14004) treated with auxin and either nocodazole (10 μg/ml; Red) or DMSO (Blue). Cells were simultaneously treated with auxin and drug (left) or were sequentially treated (right). N = 3 independent replicates for each treatment. qRT-PCR quantification of individual gene expression values (SVS1, CLN2, DSE4) were normalized to PGK1 expression values within the same sample and fold change is relative to DMSO controls. Error bars represent standard deviation.
FIGURE 3
FIGURE 3
Cbk1 localization is altered in nocodazole treated cells. (A) Model of Cbk1 signaling pathway controlling transcription and translation of genes involved in cell wall remodeling. (B) Immunoblot with anti-GFP antibodies from whole cell extracts of CDC20-AID arrested cells (SBY20970) treated with DMSO or nocodazole for 2.5 h to analyze Cbk1-GFP levels. Pgk1 is a loading control. (C) Nocodazole diminishes accumulation of Cbk1-3GFP in daughter cells. CDC20-AID cells (SBY20970) were arrested in mitosis and treated with DMSO or nocodazole (10 μg/ml) for 2.5 h and live imaged on agarose pads. Scale bar, 5 μm. Two representative images. (D) Binning of Cbk1-3GFP localization phenotype. N = 3 independent replicates, error bars represent standard deviation. p-value derived from two-tailed Student’s t-test. (E) Nocodazole prevents accumulation of Cbk1-3GFP at the bud neck in late mitosis. CDC20-AID mad3∆ (SBY21032) or CDC2020-AID mad3∆ bub2∆ (SBY21080) strains were arrested in mitosis (auxin) for 2.5 h and then released into media containing DMSO or nocodazole and live-imaged on agarose pads containing DMSO or nocodazole. Scale bar, 5 μm. Two representative images 80 min post-release. (F) Cumulative accumulation of Cbk1-3GFP at bud neck within the population. N = 3 independent replicates, error bars represent standard deviation. Blue = DMSO, Red = nocodazole. p-values derived from two-tailed Student’s t-test.
FIGURE 4
FIGURE 4
Nocodazole induced regulation of gene expression is mediated by Cbk1 signaling pathway. (A) A dominant allele of ACE2 is not sufficient to rescue nocodazole induced changes in gene expression. WT (SBY21046) and ACE2-F127V (SBY21047) cells were treated with DMSO (Blue) or nocodazole (10 μg/ml; Red). N = 3 independent replicates for each treatment. qRT-PCR quantification of individual gene expression values (SVS1, CLN2, DSE4) were normalized to PGK1 expression values within the same sample and fold change is relative to genotype-matched DMSO controls. Error bars represent standard deviation. Expression levels are not elevated in ACE2-F127V strains compared to WT upon nocodazole treatment (p = 0.99, 0.97, 0.99 for SVS1, CLN2, and DSE4 respectively; two-sample t-test). (B) Deletion of SSD1 partially rescues nocodazole-induced changes in gene expression. WT (SBY21118), ssd1∆ (SBY21138), and ssd1∆ ACE2-F127V (SBY21137) were treated with DMSO (Blue) or nocodazole (10 μg/ml; Red). N = 3 independent replicates for each treatment. qRT-PCR quantification of individual gene expression values (SVS1, CLN2, DSE4) were normalized to PGK1 expression values within the same sample and fold change is relative to genotype-matched DMSO controls. Error bars represent standard deviation. Expression levels are elevated in strains compared to WT upon nocodazole treatment (p < 0.05 for SVS1, CLN2, and DSE4 in both mutant backgrounds; two-sample t-test). (C) Modulation of downstream Cbk1 targets sensitizes cells to the microtubule destabilizing drug benomyl. 5-fold serial dilutions of WT (SBY21118), ACE2-F127V (SBY21119), ssd1∆ (SBY21138), and ssd1∆ ACE2-F127V (SBY21137) were plated onto YPD or YPD benomyl (15 μg/mL) plates and incubated at 23°C for 3 days.
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