PUBLICATION
TimerQuant: A modelling approach to tandem fluorescent timer design and data interpretation for measuring protein turnover in embryos
- Authors
- Barry, J.D., Donà, E., Gilmour, D., Huber, W.
- ID
- ZDB-PUB-151126-6
- Date
- 2016
- Source
- Development (Cambridge, England) 143(1): 174-9 (Journal)
- Registered Authors
- Gilmour, Darren
- Keywords
- Protein turnover, Fluorescent timer, Mathematical modelling
- MeSH Terms
-
- Animals
- Computer Simulation*
- Luminescent Proteins/metabolism
- Microscopy, Fluorescence/methods
- Models, Theoretical*
- Protein Stability
- Signal Transduction/physiology
- Zebrafish/embryology*
- Zebrafish Proteins/metabolism*
- PubMed
- 26603383 Full text @ Development
Citation
Barry, J.D., Donà, E., Gilmour, D., Huber, W. (2016) TimerQuant: A modelling approach to tandem fluorescent timer design and data interpretation for measuring protein turnover in embryos. Development (Cambridge, England). 143(1):174-9.
Abstract
Studies on signalling dynamics in living embryos have been limited by a scarcity of in vivo reporters. Tandem fluorescent protein timers provide a generic method for detecting changes in protein population age and thus provide readouts for signalling events that lead to changes in protein stability or location. When imaged with quantitative dual-colour fluorescence microscopy, tandem timers offer detailed 'snapshot' readouts of signalling activity from subcellular to organismal scales, and therefore have the potential to revolutionize studies in developing embryos. Here we use computer modelling and embryo experiments to explore the behaviour of tandem timers in developing systems. We present a mathematical model of timer kinetics and provide software tools that will allow experimentalists to select the most appropriate timer designs for their biological question, and guide interpretation of the obtained readouts. Through the generation of a series of novel zebrafish reporter lines, we confirm experimentally that our quantitative model can accurately predict different timer responses in developing embryos and explain some less expected findings. For example, increasing the FRET efficiency of a tandem timer actually increases the ability of the timer to detect differences in protein half-life. Finally, while previous studies have used timers to monitor changes in protein turnover, our model shows that timers can also be used to facilitate the monitoring of gene expression kinetics in vivo.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping