Figure 5: Nature Communications

Fig. 5

From: A regulatory circuit of two lncRNAs and a master regulator directs cell fate in yeast

Fig. 5

Single-cell analysis of the IME1, IRT2, and IRT1 feedback cascade. a Schematic overview of IME1 locus used for time-lapse microscopy (top). Diploid cells were heterozygous for the IME1 locus: one copy of GFP fused to N-terminus of IME1 (pIME1-GFP-IME1) and one copy expressing the IME1 promoter fused to mCherry lacking the IRT2 sequence (pIME1-Δirt2-mCherry). Cells were grown to log phase in synthetic complete media loaded into a microfluidic device, induced to enter meiosis and imaged for up till 50 h (see supplemental information for details). Example images of phase, GFP, and mCherry of a single cell taken at start and end of a time-lapse experiment (bottom). The scale bar in right bottom corner represents 3.2 μm. b Example of traces of a single cell. GFP and mCherry fluorescence signals were monitored and overall signals were scaled (see supplemental information for details). The graph also displays the time of initiation of expression (onset time), the rate of accumulation, and maximum levels (max intensity) of pIME1-GFP-IME1 and pIME1-Δirt2-mCherry. c Summary of data obtained from time-lapse microscopy experiments. All strains harbored one copy of pIME1-GFP-IME1 and one copy of pIME1-Δirt2-mCherry, combined with either wild-type RME1 (FW4843), pRME1-Δaa1 (FW4844), or pRME1-Δaa1/pCUP-IRT2 (FW5051). Cells were imaged and GFP and mCherry fluorescence signals were quantified. Mean onset times, rate of accumulation, and max intensity were determined. The means ± SEM of n = 58 (FW4843), n = 88 (FW4844), and n = 64 (FW5051) cells are shown. All pairwise differences, except between FW4843 and FW4844 (left panel only), are statistically significant; p < 0.001 (Kolmogorov–Smirnov test)