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

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Date
2014-11-05
Authors
Mazo-Vargas, Anyimilehidi
Park, Heungwon
Aydin, Mert
Buchler, Nicolas E
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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 article
Subject
Animals
Beetles
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
Permalink
https://hdl.handle.net/10161/9353
Published Version (Please cite this version)
10.1091/mbc.E14-07-1187
Publication 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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Scholars@Duke

Buchler

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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