Mitotic cell division, when a cell splits into two identical daughter cells, involves lots of moving parts that must be properly organized for accurate segregation. The main players responsible for helping to move and organize these cell parts, in addition to helping the cell physically split in two, are microtubules - dynamic filament structures that assemble and disassemble to move cargos around in the cell and dictate cell shape and movement. Unsurprisingly, disrupting these critical structural elements causes cell division to grind to a halt until the problem is fixed. While microtubule structure and function have been known to coordinate the temporal activity of cyclin dependent kinases (CDK) – proteins that play important roles in regulating the distinct phases of the cell cycle - microtubule integrity has not been tied to regulating gene expression. Dr. Sue Biggins, Senior Vice President and Director of Fred Hutch’s Basic Sciences Division explains that her lab has had a long-standing hypothesis “that the cell might have evolved a translational response to cope with microtubule defects during a mitotic arrest. Most cellular checkpoints control gene expression as well as halt the cell cycle so we speculated that the spindle checkpoint might also control gene expression, but chromosomes are condensed during mitosis so there is transcriptional repression. This caused us to hypothesize that a translational response might be the way gene expression is altered during a mitotic checkpoint arrest.” In a recently published Frontiers in Cell and Developmental Biology paper led by postdoctoral fellow Dr. Cameron Lee, the authors report the first global analysis of budding yeast translation in the presence and absence of microtubule disruption. Here, Dr. Lee uncovered that microtubule disruption inhibits the expression of genes involved in cell wall biogenesis, suggesting that this might be important to prevent premature cell separation.
To test the hypothesis that microtubule disruption might affect how expressed gene transcripts get translated into proteins, Lee first induced cell cycle arrest while also disrupting microtubules by treating cells with nocodazole, a drug that inhibits microtubule polymerization. Lee then performed ribosomal profiling, a technique that identifies mRNAs bound by translation machinery to determine whether protein translation is changed due to microtubule disruption. While protein translation in general was not affected, the researchers identified 18 genes with reduced translation. Taking a closer look at these mRNAs at the transcriptional level, they found that 14 of the 18 differentially translated genes were also significantly downregulated at the transcriptional level. Biggins explains, “it turns out the biggest targets were genes that are involved in cell septation. We expected to identify genes involved in mitosis, not post-mitosis. We later realized that we set the experiment up in a way that we identified translational control at a different time in the cell cycle [than we intended].”
After fortuitously identifying this class of genes regulated by microtubule integrity after mitotic exit but before cytokinesis, when the cell splits into two, Dr. Lee went on to investigate how microtubule disruption led to transcriptional and translational repression of cell wall genes. The researchers analyzed promoters of these genes to get insight as to what factors might regulate them and found enrichment of the transcription factor Ace2, known to be regulated by the RAM pathway. The RAM pathway mediates transcriptional control via a kinase, Cbk1 that activates Ace2-mediated transcription and also relieves translational inhibition of the protein Ssd1. Consistent with this, half of the translationally altered mRNAs are known Ssd1 targets, supporting the theory that microtubule disruption inhibits cell wall gene expression via RAM pathway transcriptional and translational control. The researchers went on to uncover that microtubule disruption downregulates RAM pathway gene targets by altering Cbk1 localization. Together these results identify a role for microtubule integrity in regulating the RAM pathway through Cbk1 signaling that help ensure proper coordination of mitotic events and prevent premature splitting of cells.
Importantly, some of the main components of this identified gene regulatory pathway in yeast, such as Cbk1, are conserved in “the mammalian Hippo signaling pathway that is critical for many processes such as cell proliferation and differentiation,” Biggins explains. She adds, “defects in Hippo signaling are associated with cancer and metastasis so understanding its regulation is important for disease research. Figuring out if any of the translational regulation of the Hippo pathway in response to microtubule defects occurs in other organisms is an important future direction.” Dr. Biggins also emphasizes that this study is “a good example of being led to something we don’t normally study because we took an unbiased and global approach.” Additionally, “this work was helped tremendously by the generosity of Drs. Andrew Hsieh and Rasi Subramanian, experts in ribosome profiling,” Biggins acknowledges.
This work was supported by the Fred Hutch/University of Washington Cancer Consortium, the Howard Hughes Medical Institute and the Helen Hay Whitney Foundation.
Fred Hutch/UW Cancer Consortium member Sue Biggins contributed to this work.
Lee CH, Biggins S. Microtubule integrity regulates budding yeast RAM pathway gene expression. Front Cell Dev Biol. 2022 Sep 12;10:989820. doi: 10.3389/fcell.2022.989820. PMID: 36172269; PMCID: PMC9511886.