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

dc.contributor.author

Mazo-Vargas, Anyimilehidi

dc.contributor.author

Park, Heungwon

dc.contributor.author

Aydin, Mert

dc.contributor.author

Buchler, Nicolas E

dc.contributor.editor

Weis, Karsten

dc.contributor.editor

Lippincott-Schwartz, Jennifer

dc.coverage.spatial

United States

dc.date.accessioned

2015-01-08T18:11:22Z

dc.date.issued

2014-11-05

dc.description.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.

dc.identifier

http://www.ncbi.nlm.nih.gov/pubmed/25232010

dc.identifier

mbc.E14-07-1187

dc.identifier.eissn

1939-4586

dc.identifier.uri

https://hdl.handle.net/10161/9353

dc.language

eng

dc.publisher

American Society for Cell Biology (ASCB)

dc.relation.ispartof

Mol Biol Cell

dc.relation.isversionof

10.1091/mbc.E14-07-1187

dc.subject

Animals

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Beetles

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Cell Cycle

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Cell Cycle Proteins

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Fireflies

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Gene Expression Regulation, Fungal

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Insect Proteins

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Luciferases

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Luminescent Measurements

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Microfluidic Analytical Techniques

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Microscopy, Fluorescence

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Saccharomyces cerevisiae

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Saccharomyces cerevisiae Proteins

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Single-Cell Analysis

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Time-Lapse Imaging

dc.title

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

dc.type

Journal article

duke.contributor.orcid

Mazo-Vargas, Anyimilehidi|0000-0001-9644-2871

duke.contributor.orcid

Buchler, Nicolas E|0000-0003-3940-3432

pubs.author-url

http://www.ncbi.nlm.nih.gov/pubmed/25232010

pubs.begin-page

3699

pubs.end-page

3708

pubs.issue

22

pubs.organisational-group

Biology

pubs.organisational-group

Duke

pubs.organisational-group

Physics

pubs.organisational-group

Trinity College of Arts & Sciences

pubs.publication-status

Published

pubs.volume

25

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