Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Summary

We describe the use of two ratiometric, genetically encoded biosensors, which are based on GFP, to monitor mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells.

Abstract

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a Förster resonance energy transfer (FRET) probe in which the ε subunit of the F_oF₁-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the ε subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.

Introduction

Mitochondria are essential organelles for ATP production, biosynthesis of amino acids, fatty acids, heme, iron sulfur clusters and pyrimidines. Mitochondria also play pivotal roles in calcium homeostasis, and in regulation of apoptosis.¹ Increasing evidence links mitochondria to aging and age-related diseases including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease.² While individuals live their entire lives with mutations in mitochondrial proteins that are associated with neurodegenerative diseases, the disease symptoms occur only later in life. This indicates that changes occur in mitochondria ....

Protocol

1. Transformation of Yeast Cells with the Biosensors

1. Transform the desired yeast strain with plasmid bearing mito-roGFP or mitGO-ATeam2 using the lithium acetate method²⁷.
2. To confirm transformation with the plasmid-borne biosensor and to prevent loss of the plasmid, select and maintain transformants on the appropriate selective synthetic complete medium (SC-Ura for mito-roGFP, or SC-Leu for mitGo-ATeam2). If the fluorescent probe has been subcloned in a different plasmid than those described here, use the appropriate selective medium. Visualize transformants by fluorescence microscopy to confirm that they express the fluorescent biosen....

Results

Measuring mitochondrial redox state with mito-roGFP

Here, we show that mito-roGFP1 has the dynamic range to detect changes in mitochondrial redox state from fully oxidized to reduced in living yeast cells, without affecting yeast cell growth or mitochondrial morphology. First, we find cells expressing mitochondria-targeted GFP and roGFP1 grow at normal rates (Figure 1A). The maximum growth rate, as measured by maximum slope of the growth curve during log-phase growth and the time.......

Discussion

Here, we describe methods to use mito-roGFP1 and mitGO-ATeam2 as biosensors to assess mitochondrial redox state and ATP levels in living yeast cells. We find that expression of plasmid-borne mito-roGFP1 or mitGO-ATeam results in quantitative targeting to mitochondria, without any obvious effect on mitochondrial morphology or distribution or on cellular growth rates.³ Mito-roGFP1 can detect changes in mitochondrial redox state from highly oxidized to highly reduced states. Similarly, mitGO-ATeam can measure cha.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
			Reagents
Antimycin A	Sigma-Aldrich (St. Louis, MO)	1397-94-0	Dissolved in ethanol to a 2 mg/ml stock solution.
SGlyc (synthetic glycerol-based) yeast growth medium *omit for SGlyc-Ura **omit for SGlyc-Leu			Dissolve in H2O. Adjust pH to 5.5 with NaHCO3. Autoclave. Ingredients: 0.67% Yeast nitrogen base without amino acids 3% Glycerol 0.05% Glucose 2 mg/ml adenine 2 mg/ml uracil* 1 mg/ml L-arginine 1 mg/ml L-histidine 1 mg/ml L-leucine** 3 mg/ml L-lysine 2 mg/ml L-methionine 4 mg/ml L-phenylalanine 2 mg/ml L-tryptophan 3 mg/ml L-tyrosine
SC (synthetic complete, glucose-based) yeast growth medium *omit for SGlyc-Ura **omit for SGlyc-Leu			Dissolve in H2O. Adjust pH to 5.5 with NaHCO3. Autoclave. Ingredients: 0.67% Yeast nitrogen base without amino acids 3% Glucose 2 mg/ml adenine 2 mg/ml uracil* 1 mg/ml L-arginine 1 mg/ml L-histidine 1 mg/ml L-leucine** 3 mg/ml L-lysine 2 mg/ml L-methionine 4 mg/ml L-phenylalanine 2 mg/ml L-tryptophan 3 mg/ml L-tyrosine
Valap			Combine ingredients in a 1:1:1 (w:w:w) ratio. Melt by submerging in a 70 °C H2O bath. Aliquot into glass petri dishes. Store at room temperature. Ingredients: Vaseline petroleum jelly, hard paraffin, lanolin
			Equipment and Software
Precleaned Gold Seal Rite-on Micro Slides	Thomas Scientific (Swedesboro, NJ)	3050	Size: 25 x 75 mm; Thickness: 0.93 to 1.05 mm
High-performance coverslips, No. 1.5, 18x18 mm	Zeiss (Thornwood, NY)	474030-9000-000	These are less variable in thickness (170±5 μm) than standard coverslips, reducing spherical aberration and improving 3D imaging performance
Fisherbrand Microscope Cover Glass, No. 1.5	Fisher Scientific (Pittsburgh, PA)	12-545E	Size: 22 x 22 mm, No. 1.5 thickness (170 μm)
A1 laser scanning confocal microscope with spectral detector and 100x/1.49 NA Apo-TIRF objective	Nikon (Melville, NY)
AxioObserver.Z1 microscope equipped with a 100x/1.3NA EC Plan-Neofluar objective (Zeiss) and Orca ER cooled CCD camera (Hamamatsu) and controlled by Axiovision software	Zeiss (Thornwood, NY); Hamamatsu (Hamamatsu City, Japan)
Volocity 3D Image Analysis software	Perkin Elmer (Waltham, MA)		Restoration module for deconvolution; Quantitation module for ratio calculation and measurement
ImageJ software	National Institutes of Health (Bethesda, MD)		http://rsb.info.nih.gov/ij/