A quantitative and spatial analysis of cell cycle regulators during the fission yeast cycle

Abstract

We have carried out a systems-level analysis of the spatial and temporal dynamics of cell cycle regulators in the fission yeast Schizosaccharomyces pombe . In a comprehensive single-cell analysis, we have precisely quantified the levels of 38 proteins previously identified as regulators of the G2 to mitosis transition and of 7 proteins acting at the G1- to S-phase transition. Only 2 of the 38 mitotic regulators exhibit changes in concentration at the whole-cell level: the mitotic B-type cyclin Cdc13, which accumulates continually throughout the cell cycle, and the regulatory phosphatase Cdc25, which exhibits a complex cell cycle pattern. Both proteins show similar patterns of change within the nucleus as in the whole cell but at higher concentrations. In addition, the concentrations of the major fission yeast cyclin-dependent kinase (CDK) Cdc2, the CDK regulator Suc1, and the inhibitory kinase Wee1 also increase in the nucleus, peaking at mitotic onset, but are constant in the whole cell. The significant increase in concentration with size for Cdc13 supports the view that mitotic B-type cyclin accumulation could act as a cell size sensor. We propose a two-step process for the control of mitosis. First, Cdc13 accumulates in a size-dependent manner, which drives increasing CDK activity. Second, from mid-G2, the increasing nuclear accumulation of Cdc25 and the counteracting Wee1 introduce a bistability switch that results in a rapid rise of CDK activity at the end of G2 and thus, brings about an orderly progression into mitosis.

The fission yeast cell size control system integrates pathways measuring cell surface area, volume, and time

“…We considered the alternative hypothesis that Cdc13 accumulates as a “timer” molecule, such that its concentration reflects time since cell birth as opposed to absolute cell size. Because cells increase in size over time during normal growth conditions, this mechanism could explain accumulation of Cdc13 in our initial experiments as well as in previous studies (Patterson et al, 2019; Curran et al, 2022). To test this idea, we performed timelapse microscopy of Cdc13-mNG in wild type and cdr2 Δ mutant cells (Figure S5).…”

“…Here, we focused on the Cdk1-activating phosphatase Cdc25 and the B-type mitotic cyclin Cdc13. The concentrations of both Cdc25 and Cdc13 increase as cells grow (Moreno et al, 1990; Keifenheim et al, 2017; Patterson et al, 2019; Curran et al, 2022), but it has not been known what aspect of size and/or growth this accumulation reflects. Both Cdc25 and Cdc13 accumulate in the nucleus where Cdk1 activation likely occurs, so we focused on their nuclear concentrations.…”

“…A third possible size measuring system is superscaling of Cdc25 phosphatase (Moreno et al, 1990; Keifenheim et al, 2017). A recent study found that Cdc13 and Cdc25 are the only known mitotic regulatory proteins that increase in concentration as cells grow (Curran et al, 2022). However, it has not been known what aspect of cell size or growth drives accumulation of these mitotic activators.…”

Size-Dependent Expression of the Fission Yeast Cdc13 Cyclin is Conferred by Translational Regulation

“…Based on the correlation between concentration and cell size for both Cdc13 (Creanor and Mitchison, 1996; Patterson et al, 2019; Curran et al, 2022) and Cdc25 (Moreno et al, 1990; Creanor and Mitchison, 1996; Keifenheim et al, 2017; Curran et al, 2022; Miller et al, 2022), these two positive regulators of the G2/M transition have been proposed to be involved in fission yeast G2 size control. We tested whether Cdc25 is in fact expressed in a size-dependent manner, as we had done for Cdc13, and we find it is (Figure 4A), consistent with the short half life of the Cdc25 protein and transcript (Keifenheim et al, 2017) and with a recent report (Miller et al, 2022).…”

“…The existence of accumulating activators as regulators of size control was inferred from cell biology experiments in the 1950s, ‘60s and ‘70s (Prescott, 1956; Thormar, 1959; Herring, 1974; Fantes et al, 1975; Rhind, 2018). In the fission yeast, Schizosaccharomyces pombe , two mitotic activators—Cdc13, the B-type cycle required for mitotic cyclin-dependent kinase (CDK) activity, and Cdc25, the tyrosine phosphatase required to activate CDK at the G2/M transition—have been shown to increase in concentration in G2 in correlation with cell size and have thus been proposed to be accumulating activators that regulate the G2/M transition (Moreno et al, 1990; Creanor and Mitchison, 1996; Keifenheim et al, 2017; Patterson et al, 2019; Curran et al, 2022; Miller et al, 2022; Miller et al, 2022).…”