Limits...
Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy.

Mazo-Vargas A, Park H, Aydin M, Buchler NE - Mol. Biol. Cell (2014)

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.

View Article: PubMed Central - PubMed

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

Time-lapse luminescence microscopy of cell-cycle dynamics. Single-cell time-lapse luminescence and fluorescence microscopy of multicopy strains AMV163 and AMV165 with (A, B) SIC1pr-FLuc-yEVenus-PEST and (C, D) RNR1pr-FLuc-yEVenus-PEST, respectively. Filming and image segmentation were identical to Figure 4, except that each luminescence z-stack was 12 s. (A) A representative SIC1pr-FLuc-yEVenus-PEST and (C) RNR1pr-FLuc-yEVenus-PEST time course. The raw, noisy luminescence and fluorescence were smoothed with a Savitzky–Golay filter (with a span of eight data points and one polynomial degree) to reliably detect peak SIC1 or RNR1 expression. The vertical, dashed lines in individual traces correspond to budding, a visible cell cycle event. Statistical differences in timing between budding and peak times are reported in tables beneath the figures. The yeast cell division cycle exhibits significant variability in amplitude and period, a feature that leads to loss of synchrony in a population of cells (Di Talia et al., 2007). This loss of synchrony is easily observed when we align different single-cell traces of (B) SIC1pr-FLuc-yEVenus-PEST and (D) RNR1pr-FLuc-yEVenus-PEST to their second peaks. We align to the second peak because daughter cells are known to have large, variable delays in their first cell cycle (Di Talia et al., 2009).
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230627&req=5

Figure 5: Time-lapse luminescence microscopy of cell-cycle dynamics. Single-cell time-lapse luminescence and fluorescence microscopy of multicopy strains AMV163 and AMV165 with (A, B) SIC1pr-FLuc-yEVenus-PEST and (C, D) RNR1pr-FLuc-yEVenus-PEST, respectively. Filming and image segmentation were identical to Figure 4, except that each luminescence z-stack was 12 s. (A) A representative SIC1pr-FLuc-yEVenus-PEST and (C) RNR1pr-FLuc-yEVenus-PEST time course. The raw, noisy luminescence and fluorescence were smoothed with a Savitzky–Golay filter (with a span of eight data points and one polynomial degree) to reliably detect peak SIC1 or RNR1 expression. The vertical, dashed lines in individual traces correspond to budding, a visible cell cycle event. Statistical differences in timing between budding and peak times are reported in tables beneath the figures. The yeast cell division cycle exhibits significant variability in amplitude and period, a feature that leads to loss of synchrony in a population of cells (Di Talia et al., 2007). This loss of synchrony is easily observed when we align different single-cell traces of (B) SIC1pr-FLuc-yEVenus-PEST and (D) RNR1pr-FLuc-yEVenus-PEST to their second peaks. We align to the second peak because daughter cells are known to have large, variable delays in their first cell cycle (Di Talia et al., 2009).

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.


Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy.

Mazo-Vargas A, Park H, Aydin M, Buchler NE - Mol. Biol. Cell (2014)

Time-lapse luminescence microscopy of cell-cycle dynamics. Single-cell time-lapse luminescence and fluorescence microscopy of multicopy strains AMV163 and AMV165 with (A, B) SIC1pr-FLuc-yEVenus-PEST and (C, D) RNR1pr-FLuc-yEVenus-PEST, respectively. Filming and image segmentation were identical to Figure 4, except that each luminescence z-stack was 12 s. (A) A representative SIC1pr-FLuc-yEVenus-PEST and (C) RNR1pr-FLuc-yEVenus-PEST time course. The raw, noisy luminescence and fluorescence were smoothed with a Savitzky–Golay filter (with a span of eight data points and one polynomial degree) to reliably detect peak SIC1 or RNR1 expression. The vertical, dashed lines in individual traces correspond to budding, a visible cell cycle event. Statistical differences in timing between budding and peak times are reported in tables beneath the figures. The yeast cell division cycle exhibits significant variability in amplitude and period, a feature that leads to loss of synchrony in a population of cells (Di Talia et al., 2007). This loss of synchrony is easily observed when we align different single-cell traces of (B) SIC1pr-FLuc-yEVenus-PEST and (D) RNR1pr-FLuc-yEVenus-PEST to their second peaks. We align to the second peak because daughter cells are known to have large, variable delays in their first cell cycle (Di Talia et al., 2009).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230627&req=5

Figure 5: Time-lapse luminescence microscopy of cell-cycle dynamics. Single-cell time-lapse luminescence and fluorescence microscopy of multicopy strains AMV163 and AMV165 with (A, B) SIC1pr-FLuc-yEVenus-PEST and (C, D) RNR1pr-FLuc-yEVenus-PEST, respectively. Filming and image segmentation were identical to Figure 4, except that each luminescence z-stack was 12 s. (A) A representative SIC1pr-FLuc-yEVenus-PEST and (C) RNR1pr-FLuc-yEVenus-PEST time course. The raw, noisy luminescence and fluorescence were smoothed with a Savitzky–Golay filter (with a span of eight data points and one polynomial degree) to reliably detect peak SIC1 or RNR1 expression. The vertical, dashed lines in individual traces correspond to budding, a visible cell cycle event. Statistical differences in timing between budding and peak times are reported in tables beneath the figures. The yeast cell division cycle exhibits significant variability in amplitude and period, a feature that leads to loss of synchrony in a population of cells (Di Talia et al., 2007). This loss of synchrony is easily observed when we align different single-cell traces of (B) SIC1pr-FLuc-yEVenus-PEST and (D) RNR1pr-FLuc-yEVenus-PEST to their second peaks. We align to the second peak because daughter cells are known to have large, variable delays in their first cell cycle (Di Talia et al., 2009).
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.

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.

View Article: PubMed Central - PubMed

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