Abstract:
Saccharomyces cerevisiae requires nutrients as fuel and also as signaling molecules to drive growth and the cell division cycle. Nutrient signaling is critical for the decision to enter the cell cycle at the G1 commitment point START. However, how post START cells respond to nutrient signaling during the S/G2/M phases of the cell cycle is poorly understood.
Here, I investigate the crosstalk between metabolism and the S G2 M cell cycle with focus on the G2/M transition. I use fluorescence microscopy to analyze single cells growing in a microfluidic cultivation platform with controlled nutrient supply. In this set up, I analyze the yeast cells' response to complete glucose removal in different cell cycle phases. For example, I examine the G2/M transition by quantifying G2 specific regulators of the cyclin dependent kinase (CDK1): B type cyclin Clb2, the inhibitory kinase Swe1 (Wee1), and its counteracting phosphatase Mih1 (Cdc25).
This study shows that cells can respond to nutrient depletion in all cell cycle phases. Starved cells frequently arrest in (S )G2, but also M phase. Surprisingly, I found arrests in late G2 phase with high levels of the major mitotic promoter: nuclear CDK Clb2. Using different mutants and novel fluorescent reporters, I found that complete glucose removal leads to Swe1 transport into the nucleus, where Swe1 inactivates and thus also stabilizes CDK Clb2. This restriction of nuclear CDK activity enables high Clb2 arrests in G2 phase. Deleting swe1 causes premature mitotic entry and more unregulated arrests in glucose starved cells. The Swe1 reporter is stabilized in the nucleus of wild type (WT) cells even under prolonged starvation, whereas Δswe1 cells rapidly degrade it after starvation entry. I conclude that the Swe1 – CDK Clb2 negative feedback loop but also the Swe1 – Mih1 balance are key regulators to enable and maintain cell cycle arrests in G2 phase.
I propose a novel starvation response mechanism that links complete glucose removal to suppression of CDK activity in order to arrest cells in (S )G2 phase. My results provide evidence for a mechanism that relies on the Swe1 dependent phosphorylation of CDK Clb2 and further that this response is independent of the established Bud Morphogenesis Checkpoint. I thus speculate that the novel starvation response mechanism constitutes a previously undescribed G2 nutrient checkpoint, restricting mitotic entry to favorable nutrient conditions.
