Metabolic cycling in single yeast cells from unsynchronized steady-state populations limited on glucose or phosphate

Abstract

Oscillations in patterns of expression of a large fraction of yeast genes are associated with the "metabolic cycle," usually seen only in prestarved, continuous cultures of yeast. We used FISH of mRNA in individual cells to test the hypothesis that these oscillations happen in single cells drawn from unsynchronized cultures growing exponentially in chemostats. Gene-expression data from synchronized cultures were used to predict coincident appearance of mRNAs from pairs of genes in the unsynchronized cells. Quantitative analysis of the FISH results shows that individual unsynchronized cells growing slowly because of glucose limitation or phosphate limitation show the predicted oscillations. We conclude that the yeast metabolic cycle is an intrinsic property of yeast metabolism and does not depend on either synchronization or external limitation of growth by the carbon source.

Fig. 1.

Principle of the FISH assay. (A) mRNA abundance (vertical axis) as a function of time (horizontal axis) for two anticorrelated, oscillating genes in a synchronized culture. (B) Expected mRNA abundance using FISH (colored spots) as a function of time for two anticorrelated, oscillating genes in a single cell.

Fig. 2.

FISH of mRNA from genes periodically expressed during the standard cell-division cycle. Maximum projection (z dimension) composite image from a portion of a single field in the Cy3, Cy3.5, and DAPI emission channels: SUR7 Cy 3.5 (red), POL30 Cy3 (green), and MCD1 Cy 3.5. DAPI (blue) stains the nucleus. (Inset) The joint distribution is plotted as a heat-map matrix plot. To improve the visibility while accurately representing the dynamical range, the frequency counts were shifted by 1 and were log₂ transformed.

Fig. 3.

FISH of mRNA from genes periodically expressed during the metabolic cycle. Maximum projection as in Fig. 2. (Upper Left) GAS1 Cy3 (green), HXK2 Cy3.5 (red). (Upper Right) Dye labels are reversed on these probes. (Lower Left) CTS1 Cy3, HXK2 Cy3.5. (Lower Right) Dye labels are reversed.

Fig. 4.

Joint and marginal distributions for gene pairs from the experiments shown in Fig.3. The joint distribution for each gene pair is plotted as a heat-map matrix plot (as in Fig. 2), and the corresponding marginal distributions for each mRNA are plotted above and to the right of the heat-map plot.

Fig. 5.

Correspondence of single-cell and synchronized population correlation coefficients (Full Data). (A) Metabolic (MC) single-cell correlations (black, phosphate; blue, glucose) compared with metabolic population correlations (4) and cell division (CC; red) compared with cell-division populations (15). (B) MC and CC compared with metabolic population correlations. (C) MC and CC compared with cell-division population correlations. Points with P > 0.05 are shown in muted colors.