Cell Cycle Commitment in Saccharomyces cerevisiae is a Multi-Step Process and Start is Reversible

Abstract

Cells need to coordinate their metabolism with the cell division cycle to survive un der changing nutrient conditions. In eukaryotic cells, nutrient signalling is integrated into the cell cycle during the cell cycle commitment at the end of G1. This point of irreversible commitment is determined by a positive feedback loop of CDK activation and is called Restriction Point in mammals and Start in yeast. Under unfavourable nutrient conditions, yeast cells arrest at Start until the conditions improve. However, once the yeast cells past Start the effect of nutrient signalling on the cell cycle machinery, is poorly understood. A comprehensive picture of how the metabolism interacts with the cell cycle and through which regulators the nutrient signals are received is still lacking. Thus, in this project, we aimed to understand how the yeast cell cycle responds to nutrient signalling, by imposing acute nutrient deprivation. Using live cell imaging and single cell analysis, I tracked the nuclear localization of the Start inhibitor Whi5, whose phosphorylation by CDK leads to its export and determines Start. I detected that post-Start cells respond to nutrient deprivation by delaying their cell cycle, or by going into a stable arrest until glucose is replenished. Cells that were more progressed mostly only delayed their cell cycle, whereas most of the cells that were exposed to starvation within the first 20 minutes permanently arrested. When I further characterized these early permanent arrests, we found that many of the cells can re-import Whi5 when faced with acute starvation. I demon strate that, for the cells that were within the first 25 minutes after passing Start, re-importing Whi5 corresponds to an interruption of the CDK positive feedback loop. I show that, this group of cells become sensitive to the mating pheromones just like regular pre-Start cells. Thus, upon nutrient starvation, cells can functionally reverse Start. We next sought to identify the mechanism responsible for reversing Start. We tested several regulatory mechanisms including Msn2,4, Msa1,2, Xbp1, Sic1, Cip1, Snf1, Rim15 and the non-CDK phosphorylation of Whi5. While we could not unveil the complete mechanism, we found that neither cyclin repression, nor Whi5 phosphorylation is responsible for interrupting the feedback loop. We therefore suggest that the CDK-cyclin complex itself is target of nutrient signalling. With these findings, we show that the textbook model of the one-step irreversible cell cycle commitment point in budding yeast does not hold true under nutrient deprivation. In mammals, the idea of a single restriction point has been previously challenged. Since cell cycle regulation is well conserved among eukaryotes, our findings can help understand cell cycle commitment as a multi-step process beyond yeast. Even though the proteins that the networks comprise of may be structurally different, the mechanism of the cell cycle commitment is very similar. As a result, our findings will lead to a better understanding of cell cycle control related disease states such as cancer. untranslated