Measurement of Mitochondrial Membrane Potential In Vivo using a Genetically Encoded Voltage Indicator

Summary

This protocol describes the application of mitochondria-targeted genetically encoded voltage indicators (GEVIs). These GEVIs offer a significant advantage over traditional mitochondrial membrane potential dyes by enabling specific, in vivo, and real-time monitoring of mitochondrial membrane potential.

Abstract

Mitochondrial membrane potential (MMP, ΔΨ_m) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen species (ROS) generation, and the import of proteins encoded by the nucleus. Existing methods for measuring ΔΨ_m typically use lipophilic cation dyes, such as Rhodamine 800 and tetramethylrhodamine methyl ester (TMRM), but these are limited by low specificity and are not well-suited for in vivo applications. To address these limitations, we have developed a novel protocol utilizing genetically encoded voltage indicators (GEVIs). Genetically encoded voltage indicators (GEVIs), which generate fluorescent signals in response to membrane potential changes, have demonstrated significant potential for monitoring plasma membrane and neuronal potentials. However, their application to mitochondrial membranes remains unexplored. Here, we developed protein-based mitochondrial-targeted GEVIs capable of detecting ΔΨm fluctuations in cells and the motor cortex of living animals. The mitochondrial potential indicator (MPI)offers a non-invasive approach to study ΔΨ_m dynamics in real-time, providing a method to investigate mitochondrial function under both normal and pathological conditions.

Introduction

Mitochondria are essential organelles in eukaryotic cells, serving as the primary energy suppliers through adenosine triphosphate (ATP) generation while also performing a variety of other crucial functions, such as metabolite synthesis, calcium ions buffering, heat production, and regulation of cell survival¹. Their roles are particularly critical in highly metabolic tissues like the brain and heart, where they help maintain cellular homeostasis. Mitochondrial membrane potential (MMP, Ψ_m) is central to these processes, including driving ATP synthesis via oxidative phosphorylation, facilitating the transport of metabolites and io....

Protocol

All animal care and experiments were performed in accordance with the Institutional Animal Care and Use Committee guidelines of Zhengzhou University. Sterilize all surgical instruments before use. Follow aseptic techniques to prevent infection. After all the data had been acquired, the animals were euthanized using an overdose of inhalant anesthesia followed by decapitation.

1. In vitro applications

1. Plasmid construction
   1. Obtain the accelerated sensor of action potentials 1 (ASAP1) and ASAP3 genes from Addgene or synthesizing from the sequence. (ASAP1, NCBI Accession ID: AHV90412.1, Addgene ID....

Discussion

The mitochondrial membrane voltage is maintained at -120--180 mV under resting conditions and fluctuates with changes in metabolic state. Currently, measurement of mitochondrial membrane potential can be performed using electrophysiological methods and fluorescence dye methods. Mitochondrial patch clamping requires the isolation of mitochondria and the destruction of cellular structures¹³. This approach may lead to measurements that deviate from physiological conditions. The fluorescence probe met.......

Materials

Name	Company	Catalog Number	Comments
BamHI	Thermo	FD0054
Calcium chloride	Sigma	C4901
CCCP	Sigma	C2759
Centrifuge	eppendorf	5430R
Centrifuge (cell culture)	eppendorf	5810R
CO2 cell incubator	ESCO	170L IR Sensor
Coverslips	Glaswarenfabrik Karl Hecht GmbH & Co.KG	92100100030
Dental adhensive resin cement	Sun medical company, LTD.	Super-Bond C&B Kit
D-glucose	Sigma	G7021
DNA Ligation Kit Ver. 2.1	Takara	6022
Dulbecco's modified Eagle medium	Gibco	11965092
Electric drill	RWD Instruments	78001
Fetal bovine serum	Gibco	A5670701
Fiber optic cannula	RWD Instruments	R-FOC-L200C-39NA
Fiber photometry detector	Thinker	QAXK_FPS-TC-MC-LED
Fluorescence microscope	Olympus	IX83
Glass pipette (for injection)	Drummond Scientific company	3.5" Drummond # 3-000-203-G/X
HEK293t	ATCC	Cat# CRL-3216
Hela cells	ATCC	Cat# CCL-2
HEPES	Sigma	H3375
Injection pump	Drummond Scientific company	3-000-207
Isoflurane	RWD Instruments	R510-22
Laser scanning confocal microscope	Zeiss	LSM980
MluI	Thermo	FD0564
NheI	Thermo	FD0974
Optical fibers	RWD Instruments	R-FC-L-N3-200-L1
Paraffin oil	Sangon	B500301
PCR thermal Cycler	analytik jena	Biometra Tone 96G
Pentobarbital sodium	Sinopharm Chemical Reagent Co.LTD	57-33-0
Potassium chloride	Sigma	P5405
PrimeSTAR HS DNA Polymerase	Takara	R010A
Programmable micropipette puller	Sutter Instruments	P2000
Quick self-curing acrylic resin	Yamahachi	V-PINK
Real-time PCR  thermal Cycler	analytik jena	qTOWER³ auto
Rhodamine 800	Sigma	83701
SalI	Thermo	FD0644
Sodium chloride	Sigma	S9888
Sodium phosphate dibasic	Sigma	S9763
Stereotaxic apparatus	RWD Instruments	E06354
Veterinary ophthalmic ointment	Puralube	NA
XhoI	Thermo	FD0694