Fig. 5: Evidence of the temporal segregation of biosynthetic processes is found in NAD(P)H dynamics.

a, NAD(P)H oscillations are unperturbed (dashed rectangle) in microaerobic condition with disrupted fluorescence dynamics of mCherry fused to ATP synthase subunit Atp3. Methods describe technical solutions to attain the microaerobic condition. More cells are shown in Extended Data Fig. 9. b, NAD(P)H oscillations exist in a strain lacking two subunits of trehalose-6-phosphate synthase/phosphatase complex TPS1 and TPS2, and two paralogs encoding glycogen synthase GSY1 and GSY2. c,d, NAD(P)H oscillations are present in cells growing in various media: minimal medium containing 1% glucose (Glu), 2% pyruvate (Pyr), combination of 1% Glu with a lipid mixture (LM; seven fatty acids) or with a complete supplement mixture (CSM; 12 amino acids, two nucleobases); complex medium YPD with 1% Glu. NAD(P)H oscillations with respect to absolute time in single cells growing in indicated medium and going through several cell and metabolic cycles (c). Summarized NAD(P)H oscillations with respect to cell-cycle-relative time (phase) in multiple cells growing in indicated medium (d). Curves and shaded areas show median and its 95% CI. Numbers of individual cell cycles (and single cells going through them) in each condition are 355 (102) for 1% Glu; 268 (93) for 2% Pyr; 27 (9) for 1% Glu + LM; 258 (98) for 1% Glu + CSM; and 124 (15) for 1% Glu YPD. NAD(P)H fluorescence values were detrended and normalized by performing LOWESS in an entire single-cell trace (large window size for line fits) and dividing raw NAD(P)H values by the resulting LOWESS curve (a–d). Markers show detrended values; curves show LOWESS (small window size for line fits) smoothing of detrended values (a–c). Window sizes used in LOWESS for detrending and smoothing are shown in Supplementary Table 8. Phase shifts and cell-cycle coupling of NAD(P)H oscillations across growth conditions are shown in Extended Data Fig. 10. e, NAD(P)H oscillations cease when CYH and CER are added and when Ugp1 is depleted via NAA-induced degradation. Markers show raw (not detrended and normalized) NAD(P)H or Ugp1-mCherry fluorescence; curves show smoothing with LOWESS (six and three data points for line fitting). Vertical lines indicate budding (a–e).