TimerQuant: a modelling approach to tandem fluorescent timer design and data interpretation for measuring protein...

Area of Science:

• Developmental Biology
• Biophysics
• Molecular Biology

Background:

• Studying signaling dynamics in living embryos is challenging due to a lack of effective in vivo reporters.
• Tandem fluorescent protein timers offer a generic method to assess protein population age, indicating signaling events affecting protein stability or location.

Purpose of the Study:

• To explore the behavior of tandem fluorescent protein timers in developing systems using computer modeling and embryo experiments.
• To provide a mathematical model and software tools for selecting and interpreting tandem timer designs in biological research.

Main Methods:

• Development of a mathematical model for tandem timer kinetics.
• Computer simulations and experimental validation using novel zebrafish reporter lines.
• Quantitative dual-color fluorescence microscopy for imaging timer readouts.

Main Results:

• The quantitative model accurately predicts tandem timer responses in developing embryos.
• Increased Förster resonance energy transfer (FRET) efficiency enhances a timer's ability to detect protein half-life differences.
• Tandem timers can monitor not only protein turnover but also gene expression kinetics in vivo.

Conclusions:

• Tandem fluorescent protein timers, guided by quantitative modeling, can revolutionize in vivo studies of embryonic development.
• The developed tools will aid researchers in designing and interpreting experiments using these timers.
• This work expands the application of tandem timers to include the monitoring of gene expression dynamics.