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

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.