Fig. 1: Protein biosynthesis has two activity waves during the cell cycle.

a,b, Heterologous promoter (tetO₇)-expressed sfGFP production rate computed from dynamic single-cell fluorescence and volume measurements, incorporating sfGFP maturation (Extended Data Fig. 1a–d). Cell-cycle traces (line-connected markers) of sfGFP production rate are summarized by posterior mean (thick solid curve) and region of high posterior probability (shaded area, mean ± s.d.) of a Gaussian process regression model with a radial basis function (RBF) kernel (a). AU, arbitrary units; b/w, between. Dashed and dotted thick curves indicate posterior means obtained via the same data analysis pipeline in two additional replicate experiments (number and average duration of analyzed cell cycles indicated). To align cell-cycle traces and calculate cell-cycle phases, we used as reference points mitotic exit (ME), START, budding (BUD) and next ME, whose average timing in three replicates is indicated. sfGFP production rate in cell cycles presented separately (b). Cell-cycle traces from the first replicate in a were interpolated, sampled at 17 evenly spaced phase points and min–max normalized. c, Measuring protein biosynthesis activity with the stop-and-respond method by determining single-cell NAD(P)H response to CYH, which is the difference between NAD(P)H derivative upon CYH addition and the median NAD(P)H derivative at the same phase (\(\theta _{\mathrm{BUD}}^c\)) in the unperturbed condition. In CYH experiments, this difference is multiplied by −1 so that metabolic response is on average non-negative. Markers indicate raw mother-cell NAD(P)H fluorescence; curve indicates smoothing (Savitzky–Golay filter). d, NAD(P)H response to CYH has two peaks during the cell cycle. Markers indicate single cells analyzed as in c from one replicate experiment. Solid curve and shaded area indicate posterior mean and region of high posterior probability (mean ± s.d.) of a Gaussian process regression summarizing the marker values via an RBF kernel. Dashed curve indicates posterior mean obtained via the same data analysis pipeline in the second replicate experiment (number of analyzed cells indicated). Vertical lines indicate mean phases of ME, START and BUD in two replicate experiments. The phase of expected ME is the mean cell-cycle duration before CYH addition. We analyzed cells that had produced at least two buds before the perturbation (not newborn cells).