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A general approach to engineer positive-going eFRET voltage indicators.

Ahmed S Abdelfattah1, Rosario Valenti2, Jihong Zheng2

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Scientists engineered new voltage indicators (GEVIs) that show increased fluorescence when cells depolarize. This breakthrough allows for clearer imaging of electrical signals in genetically defined cells and neural circuits.

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Area of Science:

  • Neuroscience
  • Biotechnology
  • Molecular Biology

Background:

  • Genetically encoded voltage indicators (GEVIs) are crucial for imaging cellular electrical activity.
  • Current GEVIs often have limitations in signal polarity or dynamic range.
  • Understanding neuronal signaling requires precise tools to visualize membrane potential changes.

Purpose of the Study:

  • To develop a generalizable method for engineering GEVIs with a positive-going fluorescence response to membrane depolarization.
  • To create a novel eFRET-based GEVI with reversed fluorescence polarity compared to existing indicators.
  • To demonstrate the broad applicability of this engineering approach across different GEVI architectures.

Main Methods:

  • Rational manipulation of microbial rhodopsin proton transport pathways.
  • Engineering electrochromic fluorescence resonance energy transfer (eFRET) mechanisms within GEVIs.
  • Systematic modification of voltage-sensitive rhodopsin domains and reporter systems.

Main Results:

  • Successfully transformed the Voltron GEVI into a new indicator, Positron, with reversed fluorescence polarity.
  • Positron exhibits comparable kinetics and sensitivity to Voltron.
  • Demonstrated the generalizability of the approach by applying it to diverse rhodopsin domains and fluorescent reporters.

Conclusions:

  • A novel strategy for engineering positive-going GEVIs has been established.
  • The Positron indicator and the general approach offer enhanced capabilities for voltage imaging in neuroscience.
  • This work provides versatile tools for studying electrical signaling in biological systems.