Abstract
Firefly luciferase (Fluc) is frequently used to report circadian gene expression rhythms in mammalian cells and tissues. During longitudinal assays it is generally assumed that enzymatic substrates are in saturating excess, such that total bioluminescence is directly proportional to Fluc protein level. To test this assumption, we compared the enzyme kinetics of purified luciferase with its activity in mammalian cells. We found that Fluc activity in solution has a lower Michaelis constant (Km) for luciferin, lower temperature dependence, and lower catalytic half-life than Fluc in cells. In consequence, extracellular luciferin concentration significantly affects the apparent circadian amplitude and phase of the widely used PER2::LUC reporter in cultured fibroblasts, but not in SCN, and we suggest that this arises from differences in plasma membrane luciferin transporter activity. We found that at very high concentrations (>1 mM), luciferin lengthens circadian period, in both fibroblasts and organotypic SCN slices. We conclude that the amplitude and phase of circadian gene expression inferred from bioluminescence recordings should be treated with some caution, and we suggest that optimal luciferin concentration should be determined empirically for each luciferase reporter and cell type.
Highlights
Luciferase (Fluc) is frequently used to report circadian gene expression rhythms in mammalian cells and tissues
Mg2+ is not strictly a substrate, we measured the Km for Mg2+ since it is an essential cofactor for ATP and its intracellular availability is dynamically regulated (Feeney et al, 2016). We performed these assays at 27 °C and 37 °C to reflect the range of temperatures over which Firefly luciferase (Fluc) has been used in mammalian cells
Our experiments revealed that the half-life of luciferase in solution and in cells is predominantly determined by substrate turnover, as well as by thermal instability, and that physiologically relevant changes in the intracellular availability of PPi, CoASH, and Mg·ATP are unlikely to significantly affect the activity of Fluc expressed in the cytosol
Summary
Luciferase (Fluc) is frequently used to report circadian gene expression rhythms in mammalian cells and tissues. During longitudinal assays it is generally assumed that enzymatic substrates are in saturating excess, such that total bioluminescence is directly proportional to Fluc protein level To test this assumption, we compared the enzyme kinetics of purified luciferase with its activity in mammalian cells. Seminal publications by Kay and Millar (Millar et al, 1992; Millar et al, 1995) identified the utility of Fluc as a genetically encoded reporter for circadian rhythms and revealed that, critically, the catalytic stability of Fluc is significantly lower than its protein stability, being sufficiently short (
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
