Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy.
Bottom Line: The photon flux per luciferase is significantly lower than that for fluorescent proteins.Fluorescence of an optimized reporter (Venus) lagged luminescence by 15-20 min, which is consistent with its known rate of chromophore maturation in yeast.Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.
Affiliation: Institute for Genome Sciences and Policy, Duke University, Durham, NC 27710 Duke Center for Systems Biology, Duke University, Durham, NC 27710 Department of Biology, Duke University, Durham, NC 27710.Show MeSH
Related in: MedlinePlus
Mentions: These results suggested that our luciferase should more faithfully track cell-cycle oscillations than fluorescence reporters. To this end, we expressed FLuc-yEVenus-PEST under the transcriptional control of two different yeast cell cycle promoters, SIC1 and RNR1. We successfully tracked cell cycle luciferase dynamics with subminute resolution (Figure 5). SIC1 transcripts are known to peak at the M/G1 border near cytokinesis, whereas RNR1 transcripts peak at the G1/S border (Spellman et al., 1998). Our data showed that the luminescence of SIC1pr-FLuc-yEVenus-PEST peaks on average 13 min before budding, whereas fluorescence peaks 6 min after budding. The luminescence of RNR1pr-FLuc-yEVenus-PEST peaks on average 7 min before budding, and fluorescence peaks 11 min after budding. Thus fluorescence lagged the luminescence signal by 15–20 min. This delay was identical to the measured in vivo chromophore maturation delay of yEVenus-PEST (Charvin et al., 2008). To verify that our protein fusion was not interfering with yEVenus folding and/or maturation, we built strains with either yEVenus-PEST or FLuc under the control of SIC1 or RNR1 promoter, respectively. Fluorescence of yEVenus-PEST alone continued to exhibit a 15–20 min delay when compared with luminescence of FLuc (Supplemental Figure S7). A two-sample Student's t test of our fluorescence peak and budding data shows no significant statistical difference between FLuc-yEVenus-PEST (Figure 5) and yEVenus-PEST (Supplemental Figure S7) with either SIC1 or RNR1 promoter; see Supplemental Table S2 for a complete statistical analysis. The same is true for luminescence peak and budding data between FLuc-yEVenus-PEST and FLuc. Supplemental Table S2 also shows that PEST has no significant effect on the timing of peak signal and budding. We conclude that FLuc-yEVenus-PEST fusion does not significantly affect the timing of either FLuc luminescence or yEVenus fluorescence.
Affiliation: Institute for Genome Sciences and Policy, Duke University, Durham, NC 27710 Duke Center for Systems Biology, Duke University, Durham, NC 27710 Department of Biology, Duke University, Durham, NC 27710.