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. 2010 Jan 19;8(1):e1000284.
doi: 10.1371/journal.pbio.1000284.

Origin of irreversibility of cell cycle start in budding yeast

Gilles Charvin  1 , Catherine Oikonomou, Eric D Siggia, Frederick R Cross
Affiliations

Origin of irreversibility of cell cycle start in budding yeast

Gilles Charvin et al. PLoS Biol. .

Erratum in

  • PLoS Biol. 2011 Jul;9(7). doi:10.1371/annotation/90916531-1b34-4eb5-92a9-a7f8c5d72318

Abstract

Budding yeast cells irreversibly commit to a new division cycle at a regulatory transition called Start. This essential decision-making step involves the activation of the SBF/MBF transcription factors. SBF/MBF promote expression of the G1 cyclins encoded by CLN1 and CLN2. Cln1,2 can activate their own expression by inactivating the Whi5 repressor of SBF/MBF. The resulting transcriptional positive feedback provides an appealing, but as yet unproven, candidate for generating irreversibility of Start. Here, we investigate the logic of the Start regulatory module by quantitative single-cell time-lapse microscopy, using strains in which expression of key regulators is efficiently controlled by changes of inducers in a microfluidic chamber. We show that Start activation is ultrasensitive to G1 cyclin. In the absence of CLN1,2-dependent positive feedback, we observe that Start transit is reversible, due to reactivation of the Whi5 transcriptional repressor. Introduction of the positive feedback loop makes Whi5 inactivation and Start activation irreversible, which therefore guarantees unidirectional entry into S phase. A simple mathematical model to describe G1 cyclin turn on at Start, entirely constrained by empirically measured parameters, shows that the experimentally measured ultrasensitivity and transcriptional positive feedback are necessary and sufficient dynamical characteristics to make the Start transition a bistable and irreversible switch. Our study thus demonstrates that Start irreversibility is a property that arises from the architecture of the system (Whi5/SBF/Cln2 loop), rather than the consequence of the regulation of a single component (e.g., irreversible protein degradation).

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The Start transition network.
(A) Schematic of the gene network involved in the budding yeast Start transition. See text for detailed description. (B) The “manual trigger” configuration used to measure nonlinearity in Start activation. Endogenous CLN1,2,3 genes have been deleted. A copy of CLN2 driven by the regulatable MET3 promoter is used to trigger the Start transition. Colored text describes the fluorescent markers used in this study to monitor the Start transition: a Whi5-GFP fusion, a CLN2pr-Venus-deg transcriptional reporter, and Cdc10-YFP to score budding. (C) The feedback test strains. Strains are isogenic with those described in (B) except that a copy of GAL1-SIC1-4A has been added. Sic1-4A is undegradable, so cells arrest prior to S-phase. The effect of CLN1,2 feedback on the stability of activation is tested by comparing cells with or without CLN1,2 (blue text).
Figure 2
Figure 2. Cells' response to exogenous CLN2 pulses of various durations.
(A) Time series of overlaid images (phase+fluorescence) of cln1,2,3 cells undergoing Start following a pulse of MET3pr-CLN2 of duration τ; green signal shows Whi5-GFP, and false-colored red corresponds to Cdc10-YFP. Cell contours have been highlighted to mark different fates of cells: the blue contours mark some cells that undergo Start transition, budding, and subsequent cell cycle completion. The red contour marks a few cells that stay blocked in G1. The green contours mark some cells that undergo partial and reversible WHI5 exit without budding. The white bar represents 5 µm. (B) Quantification of nuclear WHI5-GFP signal (method described in Figure S1) as a function of time for the experiment described in (A). Blue, green, and red lines correspond to released, partially activated, and G1-blocked cells. A.U., arbitrary units. (C) Fraction of cells undergoing Start (released cells) as a function of the pulse duration, τ. Error bars indicate statistical error.
Figure 3
Figure 3. Measurement of the nonlinearity in Start activation.
(A) Similar experiments as in Figure 2A, but with the addition of the transcriptional reporter Met3pr-Venus and without Whi5-GFP. The images represent overlay of phase and fluorescence at indicated times. Green label corresponds to released cells, and red corresponds to blocked. The white bar represents 5 µm. (B) Quantification of cytoplasmic fluorescence signal as a function of time. Transcription rate is extracted from the rise of fluorescence occurring following the pulse. A.U., arbitrary units. (C) Histogram of transcription rates for blocked cells (top panel) and released cells (middle panel), pooling data obtained with different pulse durations as indicated (total number of data points: 342). The bottom panel shows the probability of budding as a function of the transcription rate, as computed from the two histograms (blue points). The dashed line is the best fit of a Hill function, yielding a Hill coefficient n = 4.8±0.3.
Figure 4
Figure 4. Assay for irreversibility of Start.
(A–D) Time series of cells of indicated genotypes undergoing the Start transition following various pulsing protocols (see legend under images for medium switches). The three sets of images correspond to phase, Whi5-GFP signal (green), and CLN2pr-Venus-deg and Cdc10-YFP signals (yellow). Cell contours of interest are marked in red. The bar below the images indicates the timing of medium switches. Blue arrows indicate the bud neck marker Cdc10-YFP; the white rectangle represent 5 µm. (E–H) Whi5-GFP nuclear signal as a function of time. The gray region indicates −Met pulse. Each colored trace represents a different cell. The solid black line is the average over the displayed traces. A.U., arbitrary units. (I–L) CLN2pr-Venus-deg transcriptional reporter signal as a function of time. Each color corresponds to a single cell. The black solid curve is the average over the displayed traces.
Figure 5
Figure 5. Influence of Whi5 on Start irreversibility.
(A) Time series of images (phase, Cdc10-YFP, and CLN2pr-Venus-deg fluorescence signals) of cln123 whi5 delta cells following a 15-min MET3pr-CLN2 pulse. The red contour shows a typical cell of interest. (B) CLN2pr-Venus-deg fluorescence signals observed in (A). Each color corresponds to a different cell. The black solid line is an average of the displayed cells. A.U., arbitrary units.
Figure 6
Figure 6. Polarized growth with or without feedback.
(A–D) Bud to mother size ratio (top panel) and total (mother+bud) size of cells as a function of time for cells undergoing Start activation. Each plot corresponds to a different strain background, as indicated. Each color represents a different cell, and the solid black line is an average over the displayed traces. Shaded areas indicate −Met pulses. In (D), the interval between the dashed line roughly indicates the time by which nearly all cells have completed division.
Figure 7
Figure 7. Model of Start activation.
(A) Steady-state value of X as a function of input cyclin, Xe, for different values of (n, k) as indicated on the plot, revealing different classes of behaviors: monostability, bistability, and irreversibility. The red line segments indicate possible stable states, whereas black line segments show unstable regions. The amplitude, A, of the bistability region is indicated. (B) Map of the different possible behaviors as a function of the parameters n, k. The dashed region indicates the subsection of the map where the experimental system is thought to function. (Please note that the n = 1 or k = 0 cases yield limit monostable behaviors.) (C) Left panel: simulation of the temporal response of X as a function of a transient pulse of Xe in the presence of positive feedback. Right panel: SBF transcription (assumed to be proportional to the first term in the right-hand side of Equation 2) as a function of Xe. Arrows indicate the direction of the trajectory. (D) Same as [C]), except that the positive feedback is removed (X = 0). (E) Stochastic simulation (following Gillespie's method [44]) of X activation in the absence of any input (Xe = 0) but after adding a basal transcription term l (0<l<1) to the right-hand term of Equation 2. The value of n used was 4.8, the values of other parameters are indicated. To control the amplitude of statistical fluctuations in protein number, the value of alpha was adjusted such that the maximum number of proteins is on average 20. Left, middle, and right panels show sample temporal traces for different values of l. Each trace represents a single cell.
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