Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy.
Abstract
Time-lapse fluorescence microscopy is an important tool for measuring in vivo gene
dynamics in single cells. However, fluorescent proteins are limited by slow chromophore
maturation times and the cellular autofluorescence or phototoxicity that arises from
light excitation. An alternative is luciferase, an enzyme that emits photons and is
active upon folding. The photon flux per luciferase is significantly lower than that
for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully
used to track gene dynamics only in larger organisms and for slower processes, for
which more total photons can be collected in one exposure. Here we tested green, yellow,
and red beetle luciferases and optimized substrate conditions for in vivo luminescence.
By combining time-lapse luminescence microscopy with a microfluidic device, we tracked
the dynamics of cell cycle genes in single yeast with subminute exposure times over
many generations. Our method was faster and in cells with much smaller volumes than
previous work. 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.
Type
Journal articleSubject
AnimalsBeetles
Cell Cycle
Cell Cycle Proteins
Fireflies
Gene Expression Regulation, Fungal
Insect Proteins
Luciferases
Luminescent Measurements
Microfluidic Analytical Techniques
Microscopy, Fluorescence
Saccharomyces cerevisiae
Saccharomyces cerevisiae Proteins
Single-Cell Analysis
Time-Lapse Imaging
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https://hdl.handle.net/10161/9353Published Version (Please cite this version)
10.1091/mbc.E14-07-1187Publication Info
Mazo-Vargas, Anyimilehidi; Park, Heungwon; Aydin, Mert; & Buchler, Nicolas E (2014). Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy.
Mol Biol Cell, 25(22). pp. 3699-3708. 10.1091/mbc.E14-07-1187. Retrieved from https://hdl.handle.net/10161/9353.This is constructed from limited available data and may be imprecise. To cite this
article, please review & use the official citation provided by the journal.
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Show full item recordScholars@Duke
Nicolas Buchler
Assistant Professor of Biology
Our lab is interested in the systems biology and evolution of epigenetic switches
(bistability) and clocks (oscillators) in gene regulatory networks, two functions
that are essential for patterning, cell proliferation, and differentiation in biological
systems. We also study biochemical oscillators such as the cell cycle, metabolic rhythms,
and circadian clocks, which co-exist in the same cells and interact with one another
through shared resources.

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