Organelle-Targeted GEVI for Mitochondrial Voltage Measurement
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Solution Overview
Problem
Current methods lack practical means to study the internal electrical signals of mitochondrial and endoplasmic reticulum membranes, leading to a significant gap in understanding normal and pathological cellular processes, particularly in diseases related to these organelles.
Innovation Solution
Development of genetically-encoded voltage indicators (GEVIs) comprising a transmembrane domain and a fluorescent protein, covalently linked, which target specific organelles like mitochondria and the endoplasmic reticulum, allowing for voltage measurement across these membranes using patch-clamp fluorometry.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional voltage measurement methods are used at the plasma membrane, then voltage can be readily measured, but there are no practical methods available for studying internal electrical signals of mitochondrial and ER membranes
Solution Approach 1:
The invention divides the voltage measurement function into separate modular components: a transmembrane domain for membrane insertion and a fluorescent protein domain for voltage sensing. This segmentation allows the GEVI to be adapted to different organelles by swapping transmembrane domains while retaining the voltage-sensing fluorescent protein, enabling versatile application across mitochondrial, ER, and other membranes.
Solution Approach 2:
The invention introduces genetically-encoded voltage indicators as intermediary molecules that bridge the gap between electrical signals and optical detection. These GEVIs act as mediators that convert voltage changes into fluorescence signals, enabling non-invasive optical measurement of membrane potentials in living cells without direct electrical contact.
2Measurement precision
If hybrid voltage sensors with covalently linked transmembrane domain and fluorescent protein are developed, then robust fluorescence changes in response to voltage steps are produced, but device complexity increases
Solution Approach 1:
The invention merges two previously separate functional elements into a single hybrid protein: the transmembrane domain (which anchors the sensor to the membrane) and the fluorescent protein (which reports voltage changes). This merging creates a unified GEVI molecule that simultaneously performs membrane localization and voltage sensing, simplifying the overall system while enhancing measurement capability.
Solution Approach 2:
The GEVI is constructed as a composite molecular structure combining hydrophobic transmembrane sequences with hydrophilic fluorescent protein domains. This composite design allows the molecule to function effectively at the membrane-cytosol interface, with the transmembrane domain embedding in the lipid bilayer and the fluorescent protein extending into the aqueous environment for optical detection.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
These hybrid voltage sensors efficiently target organelles, producing robust fluorescence changes in response to voltage steps, enabling detailed study of organelle function and potential drug screening for diseases such as Alzheimer's and Parkinson's.
Implementation Method 1
recording a voltage change across the organelle membrane by patch-clamp fluorometry of the fluorescent protein-FRET partner
Implementation Method 2
contacting the organelle membrane with a FRET partner for the fluorescent protein of the GEVI
Data Source
AI summary
As described herein, a hybrid voltage sensor genetically-encoded voltage indicator (GEVI) for mitochondria or endoplasmic reticulum includes a transmembrane domain, and a fluorescent protein, wherein a terminus of the transmembrane domain and a terminus of the fluorescent protein are covalently linked directly or by a linker comprising 1 to 20 amino acids, and wherein the transmembrane domain comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or a peptide with greater than 85%, 90%, 95% or 98% identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. Also described are expression vectors, expression cassettes, and organelle membranes, as well as methods of determining the voltage across an organelle using the GEVIs.