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.

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Citation

Published Version (Please cite this version)

10.1091/mbc.E14-07-1187

Publication Info

Mazo-Vargas, Anyimilehidi, Heungwon Park, Mert Aydin and Nicolas E Buchler (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.

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Scholars@Duke

Mazo-Vargas

Anyi Mazo-Vargas

Assistant Professor of Biology

Anyi Mazo-Vargas is originally from Cali, Colombia, where she graduated from the Biology program at Universidad del Valle. She then pursued a master’s degree at the University of Puerto Rico in Mayaguez, focusing on weevil beetles and phylogenetics. After moving to the United States, Anyi worked as a laboratory manager at Duke University in Dr. Buchler’s lab, utilizing luciferases to study the cell cycle of Saccharomyces yeast. These experiences inspired her to pursue a PhD in the Entomology Department at Cornell University, where she combined her knowledge in insects, evolution, and genetics to study butterfly color patterning. At Cornell, she investigated genes involved in wing element patterning and their regulation. She later joined George Washington University as a postdoctoral researcher, working on transgenesis projects in both moths and butterflies.

The AMV Laboratory at Duke employs genetics, functional genomics, and developmental biology approaches to untangle the genetic basis of morphological adaptive traits. Our research aims to dissect the underlying developmental processes and place our findings within a micro- and macro-evolutionary framework. We seek to identify genomic and developmental properties, explaining how adaptive traits such as wing patterns and wing shapes involved in mimicry, camouflage, thermoregulation, and other defense mechanisms develop and evolve.


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