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Protein Dynamics in Living Cells01:19

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Developing Fast Fluorescent Protein Voltage Sensors by Optimizing FRET Interactions.

Uhna Sung1, Masoud Sepehri-Rad1, Hong Hua Piao1

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Researchers enhanced genetically encoded fluorescent protein voltage sensors for monitoring neuronal activity. Optimized FRET probes (Nabi1 and Nabi2) show improved signal size and kinetics, enabling detection of individual action potentials.

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Area of Science:

  • Neuroscience
  • Biophysics
  • Molecular Biology

Background:

  • Genetically encoded fluorescent protein (FP) voltage sensors utilize Förster Resonance Energy Transfer (FRET) for monitoring neuronal activity.
  • FRET-based sensors offer reduced noise by using signal ratios, mitigating artifacts like heartbeat.
  • Optimizing FP placement is crucial for enhancing sensor performance.

Purpose of the Study:

  • To improve the performance of FRET-based genetically encoded FP voltage sensors.
  • To optimize the positioning of donor and acceptor FPs around the voltage-sensing domain.
  • To develop novel voltage sensors with enhanced signal size, kinetics, and neuronal expression.

Main Methods:

  • Constructed 39 "Nabi1" variants by varying donor (UKG) and acceptor (mKO) FP positions relative to the Ciona intestinalis voltage-sensitive phosphatase domain.
  • Replaced the UKG/mKO FRET pair with Clover/mRuby2 to create "Nabi2" probes, aiming for improved expression in neuronal cells.
  • Tested sensor performance in HEK293 cells and primary neuronal cultures.

Main Results:

  • Several Nabi1 constructs exhibited large voltage-dependent signals (up to 11% ΔF/F for 100 mV depolarization) and fast response kinetics (~2 ms activation, ~3 ms decay).
  • Nabi2 probes demonstrated large signals and fast kinetics in HEK293 cells.
  • A Nabi2 probe successfully resolved individual action potentials at 45 Hz in primary neuronal cultures.

Conclusions:

  • Optimized FP positioning significantly enhances FRET-based voltage sensor performance.
  • The developed Nabi2 probes offer substantial improvements for monitoring neuronal electrical activity.
  • These advanced sensors hold promise for in vivo neuroscience research and understanding neural function.