Name: ratiometric biosensors that measure mitochondrial redox state and atp in living yeast cells
Uploaded: 2013-07-22T13:00:00
Description: Watch this Scientific Journal Video about Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells at JoVE.com

This work was supported by awards from HHMI 56006760 to JDV, the National Institutes of Health (NIH) (2 TL1 RR 24158-6) to DMAW, and from the Ellison Medical Foundation (AG-SS-2465) and the NIH (GM45735, GM45735S1 and GM096445) to LP. GM45735S1 was issued from the NIH under the American Recovery and Reinvestment Act of 2009. The microscopes used for these studies were supported in part through a NIH &frasl; NCI grant (5 P30 CA13696).

1. Transformation of Yeast Cells with the Biosensors
<ol>
 <li>Transform the desired yeast strain with plasmid bearing mito-roGFP or mitGO-ATeam2 using the lithium acetate method27.</li>
 <li>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 descri...

<ol>
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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...

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 with age that allow disease pathology to emerge. Indeed, mitochondrial fitness is correlated with overall cell health and lifespan in yeast and mammalian cells.3,4 Here, we describe how to use genetically encoded, ratiometric fluorescent biosensors to assess two critical features of mitochondria in living yeast cells: redox state and ATP levels.
Mitochondrial function in aerobic energy mobilization is well established. Mitochondrial redox state is a product of reducing and oxidizing species in the organelle, including NAD+/NADH, FAD/FADH2, NADP+/NADPH, glutathione/glutathione disulfide (GSH/GSSG) and reactive oxygen species (ROS). Uncoupling mitochondria or hypoxia affects mitochondrial respiratory activity and alters the ratio of NAD+ to NADH in the organelle. ROS, which are produced from inefficient electron transfer between complexes of the electron transport chain in the inner mitochondrial membrane, as well as from the deamination of amines via monoamine oxidase in the outer mitochondrial membrane 5, damage lipids, proteins, and nucleic acids and have been linked to aging and age-associated neurodegenerative diseases 6,7,8. ROS also play a role in signal transduction in mitochondria, through oxidation of GSH. For example, NADH dehydrogenase not only contributes to ROS production but also is regulated through interactions with the glutathione pool 9,10. &alpha;-Ketoglutarate dehydrogenase and aconitase, components of the TCA cycle, exhibit reduced activity in oxidizing environments11,12.
Indeed, redox-dependent regulation of aconitase activity is conserved from bacteria to mammals13,14. Thus, monitoring the redox state and ATP levels of mitochondria is crucial to understanding their function and role in disease pathology.
Biochemical methods have been used to assess the redox state or ATP levels of whole cells or isolated mitochondria. Widely used methods to assess the redox state of whole cells or isolated mitochondria are based on measuring the levels of the redox pair GSH/GSSG15. The luciferin-luciferase system is commonly used to measure mitochondrial ATP levels in either permeabilized whole cells or isolated mitochondria.16,17,18,19,20 In this assay, luciferase binds to ATP and catalyzes the oxidation of and chemiluminescence from luciferin.21 The intensity of the emitted light is proportional to the amount of ATP in the reaction mixture.22
These methods have revealed fundamental information regarding mitochondrial function, including the finding that patients with neurodegenerative diseases, such as Alzheimer's disease, have abnormally low ATP levels.23 However, they cannot be used to image living, intact cells. Moreover, methods based on whole-cell analysis provide an average of redox state or ATP levels in all compartments of the cell. Measurements in isolated organelles are potentially problematic because mitochondrial redox state or ATP levels may change during subcellular fractionation. Finally, recent studies from our laboratory and others indicate that mitochondria within individual cells are heterogeneous in function, which in turn affects the lifespan of mother and daughter cells.3 Thus, there is a need to measure mitochondrial ATP levels and redox state in living cells with subcellular resolution.
The biosensors for mitochondrial function described here are both based on GFP. Redox-sensitive GFP (roGFP)24,25 is a GFP variant in which surface-exposed cysteines are added to the molecule. roGFP, like wild-type GFP, has two excitation peaks (at ~400 nm and ~480 nm) and one emission peak at ~510 nm. Oxidation of the cysteine residues in roGFP results in an increase in excitation at ~400 nm. Reduction of those cysteines favors excitation at ~480 nm. Thus, the ratio of 510 nm emission upon excitation of roGFP at 480 nm and 400 nm reveals the relative amount of reduced and oxidized roGFP, which reflects the redox state of the fluorophore's environment.
Two versions of roGFP are widely used: roGFP1 and roGFP2. Both contain the same cysteine insertions. roGFP1 is based on wild-type GFP and roGFP2 is based on S65T GFP, which has more efficient excitation at 480 nm and less efficient excitation at 400 nm compared to wtGFP24. roGFP1 is less pH sensitive than roGFP2 and its dynamic range extends further into the reduced range. Thus, roGFP1 may be more useful for monitoring more reducing compartments such as mitochondria or the cytosol, and compartments with variable pH, such as endosomes. roGFP2 offers brighter signal and, in some studies, a greater dynamic range than roGFP124,26. Studies in Arabidopsis thaliana indicate that the time required for response to changes in redox state is similar for both sensors (t&frac12; for oxidation, 65 and 95 sec and t&frac12; for reduction, 272 and 206 sec, for roGFP1 and roGFP2, respectively).26
MitGO-ATeam2 is a minimally invasive, reliable sensor that measures mitochondrial ATP in the budding yeast Saccharomyces cerevisiae. GO-ATeam is a F&ouml;rster resonance energy transfer (FRET) probe that consists of the &epsilon; subunit of the FoF1-ATP synthase sandwiched between FRET donor and acceptor fluorescent proteins (GFP and orange fluorescent protein (OFP), respectively). 27 Binding of ATP to the &epsilon; subunit results in conformational changes in the protein that bring the FRET donor in close proximity to the acceptor and allow for energy transfer from donor to acceptor. There are two variants of GO-ATeam, GO-ATeam1 and GO-ATeam2. GO-ATeam2 has a higher affinity for MgATP than GO-ATeam1, making it more suitable for measuring the typically lower [ATP] in mitochondria compared to the cytosol.27
To probe mitochondrial redox state, we constructed a fusion protein (mito-roGFP1) consisting of roGFP1 fused to the ATP9 leader sequence and expressed from a centromere-based (low copy number) yeast expression plasmid under control of the strong glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter (p416GPD, Addgene). We used roGFP1 to probe the redox status of mitochondria in the context of aging of the model fungus Saccharomyces cerevisiae. We find that roGFP1 can detect changes in mitochondrial redox state that occur during aging and in response to nutrient availability but has no apparent detrimental effect on yeast cells. We also see variability in the redox state of mitochondria within individual living yeast cells, a finding that underscores the importance of a biosensor with subcellular spatial resolution.
MitGO-ATeam2 is a variant of GO-ATeam2, which has the mitochondrial signal sequence of cytochrome c oxidase subunit VIIIA inserted at the amino terminus of GO-ATeam2.27 We modified the mitGO-ATeam2 probe (kindly provided by the laboratory of H. Noji, Institute of Scientific and Industrial Research, Osaka University, Japan) for use in yeast by subcloning it, via Xba1 and HindIII sites, into the yeast expression vector pAG415GPD-ccdB (Addgene, Cambridge, MA, USA), which is a low-copy plasmid containing the strong constitutive GPD promoter. We expressed mitGO-ATeam2 in budding yeast, and find, by counterstaining with the DNA-binding dye DAPI, that it localizes exclusively to mitochondria, where it serves as an effective probe to measure physiological changes in mitochondrial ATP levels.
roGFP and GO-ATeam are both genetically encoded. As a result, they can be introduced and stably maintained in intact cells, and provide information on redox state or ATP levels in individual, living cells. Moreover, both biosensors monitor changes in redox state or ATP levels that occur under physiological conditions.28 Both probes are also ratiometric. As a result, measurements made with these probes are not affected by changes in biosensor concentration or sample illumination or thickness. Finally, both biosensors provide subcellular spatial resolution. Indeed, roGFP has been targeted to mitochondria, ER, endosomes and peroxisomes24, and can detect changes in redox state of each of these organelles, largely independent of pH.

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	<tbody>
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			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma-Aldrich (St. Louis, MO)</td>
			<td>1397-94-0</td>
			<td>Dissolved in ethanol to a 2 mg/ml stock solution.</td>
		</tr>
		<tr>
			<td>SGlyc (synthetic glycerol-based) yeast growth medium *omit for SGlyc-Ura 
			**omit for SGlyc-Leu</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>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</td>
		</tr>
		<tr>
			<td>SC (synthetic complete, glucose-based) yeast growth medium *omit for SGlyc-Ura 
			**omit for SGlyc-Leu</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Valap</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Combine ingredients in a 1:1:1 (w:w:w) ratio. Melt by submerging in a 70 &deg;C H2O bath. Aliquot into glass petri dishes. Store at room temperature. Ingredients: 
			Vaseline petroleum jelly, hard paraffin, lanolin</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment and Software</td>
		</tr>
		<tr>
			<td>Precleaned Gold Seal Rite-on Micro Slides</td>
			<td>Thomas Scientific (Swedesboro, NJ)</td>
			<td>3050</td>
			<td>Size: 25 x 75 mm; Thickness: 0.93 to 1.05 mm</td>
		</tr>
		<tr>
			<td>High-performance coverslips, No. 1.5, 18x18 mm</td>
			<td>Zeiss (Thornwood, NY)</td>
			<td>474030-9000-000</td>
			<td>These are less variable in thickness (170&plusmn;5 &mu;m) than standard coverslips, reducing spherical aberration and improving 3D imaging performance</td>
		</tr>
		<tr>
			<td>Fisherbrand Microscope Cover Glass, No. 1.5</td>
			<td>Fisher Scientific (Pittsburgh, PA)</td>
			<td>12-545E</td>
			<td>Size: 22 x 22 mm, No. 1.5 thickness (170 &mu;m)</td>
		</tr>
		<tr>
			<td>A1 laser scanning confocal microscope with spectral detector and 100x/1.49 NA Apo-TIRF objective</td>
			<td>Nikon (Melville, NY)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>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</td>
			<td>Zeiss (Thornwood, NY); Hamamatsu (Hamamatsu City, Japan)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Volocity 3D Image Analysis software</td>
			<td>Perkin Elmer (Waltham, MA)</td>
			<td>&nbsp;</td>
			<td>Restoration module for deconvolution; Quantitation module for ratio calculation and measurement</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>National Institutes of Health (Bethesda, MD)</td>
			<td>&nbsp;</td>
			<td><a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a></td>
		</tr>
	</tbody>
</table>

ratiometric biosensors that measure mitochondrial redox state and atp in living yeast cells

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 F&ouml;rster resonance energy transfer (FRET) probe in which the &epsilon; subunit of the FoF1-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the &epsilon; 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.

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...

Watch this Scientific Journal Video about Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells at JoVE.com

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

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.

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell ...

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