Name: Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells (Video) | JoVE Journal
Uploaded: 2013-07-22T13:00:00.000+00:00
Duration: 12 min 22 s
Description: Learn ratiometric biosensor techniques to measure mitochondrial redox state and ATP levels in living yeast cells using GFP variants. This method enables real-time subcellular analysis of organelle function.

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5 minutes

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 described here, use the appropriate selective medium. Visualize transformants by fluorescence microscopy to confirm that they express the fluorescent biosensor and exhibit normal mitochondrial morphology.</li>
</ol>
2. Growth of Cells and Preparation for Imaging
Cell function and response to drug treatment are highly dependent on cell density and metabolic activity. Best results are obtained when cells are actively dividing (mid-log phase, ~0.5 - 1 x 107 cells/ml). The most reliable way to generate mid-log phase cultures of consistent density is to inoculate from a stationary-phase pre-culture.
<ol>
 <li>Prepare a stationary-phase pre-culture: pick a single colony of transformed cells from a plate and inoculate selective liquid media (5 ml in a 50 ml conical-bottom tube). Grow at 30 &deg;C with shaking at 225 rpm until the optical density of the culture at 600 nm (OD600) has reached a plateau (24-48 hr).</li>
 <li>Prepare a mid-log phase culture for observation: use the appropriate volume of pre-culture to inoculate 5 ml of selective media in a 50 ml conical-bottom tube. YPD media is autofluorescent and should not be used for these studies. Use synthetic complete, glucose based, dropout (SC-Ura) media. Grow cells at 30 &deg;C, shaking at 225 rpm, for 4-16 hr, until they reach mid-log phase (~0.5 - 1 x 107 cells/ml). Cell density can be determined by measuring OD600; calibrate the spectrophotometer to determine the correct OD600 reading. On our Beckman DU530 (Beckman Coulter, Indianapolis, IN), mid-log phase corresponds to an OD600 of 0.1-0.3.</li>
</ol>
Mito-roGFP1 senses fluctuations in the organelle in response to metabolic changes. For example, in this assay mitochondrial redox state changes when yeast grow on fermentable carbon sources (e.g. glucose, as in SC media) versus non-fermentable carbon sources (e.g. glycerol, as in SGlyc media), and even in different batches of the same media. Therefore, use the same batch of media for all experiments.
<ol start="3">
 <li>Cells are ready for concentration (see step 2.4) and imaging if no treatment is being performed. If cells are being treated, incubate cells with the appropriate treatment and continue to the next step.</li>
 <li>Concentrate 1 ml of culture by centrifugation at 6,000 x g for 15 sec and resuspension of the cell pellet in 20 &mu;l of media. These conditions maximize the number of distinguishable cells in the field of view.</li>
 <li>Apply 2 &mu;l of the resuspended cells to a slide. Cover with a coverslip (No. 1.5, preferably high-performance 170&plusmn;5 &mu;m thickness), and seal the edges of the coverslip with clear nail polish or valap (see Reagents). To seal with valap, melt a small amount on a metal spatula by holding it over a Bunsen burner flame, then spread a small amount along the edges of the coverslip.</li>
 <li>Maintain cells at 30 &deg;C during imaging. An objective heater on the 100x oil immersion lens used for imaging works well for this application. Under these conditions, mitochondrial morphology, redox state and ATP levels remain unchanged during imaging for 10-15 min.</li>
</ol>
3. Imaging Setup
<ol>
 <li>Setup for imaging mito-roGFP1 on a wide-field fluorescence microscope 
 The steps here are tailored to the AxioObserver.Z1 microscope equipped with a Colibri LED excitation source, a wide-field Orca ER camera and Axiovision acquisition software. Photobleaching of both channels and photoconversion of oxidized mito-roGFP1 is reduced significantly using LED illumination compared to mercury arc lamp illumination (see below). 
 To maximize signal and resolution, use the highest numerical aperture possible in the objective, and the lowest magnification that provides sufficient spatial resolution. In addition, for mito-roGFP imaging, verify that the objective transmits well at 365 nm. The 100x/1.3NA EC PlanNeofluar objective (Zeiss) works well for this application. 
 Configure the acquisition software to capture the oxidized and reduced mito-roGFP1 species. We use the following conditions.
 <ol>
 <li>Configure the channel for oxidized mito-roGFP to use excitation at 365 nm (100% LED power) and an emission filter suitable for GFP, such as the 38 HE filter set (Zeiss), with the included excitation filter removed from the cube. Removal of the excitation filter allows excitation at both 365 nm and 470 nm without the need to switch filters, thus increasing achievable time resolution.</li>
 <li>Configure the channel for reduced mito-roGFP to use excitation at 470 nm (100% LED power) and, as mentioned above, the same emission filter cube used for the oxidized mito-roGFP. Set the camera to 1x1 binning to optimize spatial resolution.</li>
 <li>Set software to acquire a z stack consisting of 11 slices with 0.5 &mu;m spacing, collecting both channels at each z position. This mode of acquisition is slower than acquiring each z stack in turn, but it prevents artifacts arising from mitochondrial motion between acquisition of the oxidized and reduced channels.</li>
 <li>Image several cells to determine an appropriate exposure time, producing a strong but not saturating signal. It is important to maintain the ratio of exposure times for oxidized and reduced mito-roGFP1 for all experiments. For example, if the exposure times for oxidized and reduced mito-roGFP1 are 300 and 100 msec, respectively, then the exposure time for oxidized mito-roGFP1 should be 3 times that for reduced mito-roGFP for all experiments.</li>
 </ol>
 </li>
 <li>Setup for imaging mitGO-ATeam2 on a spectral confocal microscope 
 To quantify ATP levels using mitGO-ATeam2, the fluorescence from GFP (emission maximum 510 nm) must be distinguished from that from OFP (emission maximum 560 nm). There are fluorescence filter sets that resolve the fluorescence emitted from these fluorophores. However, we find that a spectral detector, available on many laser scanning confocal microscopes, works best for this application. A spectral detector separates the fluorescence emission into many components (typically 32 or more) according to wavelength. Several adjacent wavelength bands can be combined into one image channel. The wavelengths to be combined are selected empirically so as to maximize signal from GFP and OFP while avoiding crossover of signal that would confound the FRET measurement. Use the highest numerical aperture lens available. We use a 100x/1.49 or 60x/1.49 Apo-TIRF lens. We use the Nikon A1R confocal microscope running NIS Elements software. Configure the acquisition software to capture the ATP-bound and ATP-unbound mitGO-ATeam2 species. We use the following conditions.
 <ol>
 <li>Set pinhole to 1.0 Airy units (AU), which on our system corresponds to a z resolution of about 0.42 &mu;m.</li>
 <li>Set scan zoom to approach the Nyquist sampling limit, which will maximize spatial information in the image. On our system the pixel size is 0.12 &mu;m.</li>
 <li>Because mitochondria may move during imaging, it can be helpful to increase imaging speed by cropping the field to 512 x 256 pixels. The total time required to image one frame in our system is 1.0 sec, including a 2-fold scan average. Depending on desired spatial resolution, the field can be further cropped to increase time resolution.</li>
 <li>Excite at 488 nm, and collect emission from 500 - 520 nm for GFP, and from 550 - 580 nm for OFP. Actual optimal values may vary with characteristics of the imaging system.</li>
 <li>The optimal laser power for our system is between 6.0 and 6.4%. The optimal laser power will vary for each microscope system, but will ideally be as low as possible while still producing an interpretable image. Use an internal or external power meter to monitor changes in the laser power, which occur normally over time in any optical system.</li>
 <li>Adjust detector gain and illumination light intensity to maximize the detected range of pixel values but avoid saturating the signal, for as many cells as possible. Do not analyze any cells containing more than 1% saturated pixels. Image all samples using the same objective, laser power, scan zoom, pixel size, gain, and offset.</li>
 </ol>
 </li>
</ol>
4. Image Acquisition
<ol>
 <li>Locate the focal plane of cells of interest using transmitted light to minimize bleaching the fluorescent probe.</li>
 <li>After locating one or more cells of interest, collect a z-series through the whole depth of a typical cell (approx. 7 &mu;m) using a step size of 0.5 &mu;m.</li>
 <li>Image other cells of interest on the slide, but do not image a single slide for longer than 15 min. After 15 min on the slide, cells lose viability.</li>
</ol>
5. Analysis
Mitochondrial ATP level is determined by measuring the ratio of the mitGO-ATeam2 emission at 560 nm to that at 510 nm.27 The redox state of the organelle is measured as the reduced to oxidized (R/O) ratio of mito-roGFP; i.e. emission at 510 nm upon excitation at 470 nm divided by emission at 510 nm upon excitation at 365 nm. Before calculating the ratio, we subtract background and determine a threshold value for pixels belonging to the fluorescent mitochondria.
Public domain (e.g. ImageJ30) or commercially available (e.g. Volocity, Perkin-Elmer) software can be used for analysis of mito-roGFP1 or mitGO-ATeam2. Depending on the software used for image acquisition and for analysis, the images may first need to be converted to another format, such as TIFF, before opening them in the analysis software. If images are converted, it is essential to verify that pixel values are not changed during the conversion. Analysis of mito-roGFP1 data, using both programs is described below. Program menus and options to select within each menu are highlighted in bold italics.
5.1 ImageJ analysis
<ol>
 <li>Open images and change type to 32 bit: Image&rarr; Type &rarr; 32 bit.</li>
 <li>Draw a region of interest (ROI) in an area where there are no cells. Calculate the mean intensity in this ROI: Analyze &rarr; Measure.</li>
 <li>Subtract the calculated mean background from the stack: Process &rarr; Math &rarr; Subtract.</li>
 <li>Using the subtracted z-stack, find the middle slice and threshold on mitochondria: Image &rarr; Adjust &rarr; Threshold and click Apply on the Threshold window. Apply to all slices in the stack. Check Set background pixels to NaN.</li>
 <li>Create the ratio z stack: Process &rarr; Image Calculator Divide the reduced stack by the oxidized z stack for mito-roGFP1 analysis. Divide the 560 nm (ATP bound) image by the 510 nm (ATP unbound) image for mitGO-ATeam2 analysis.</li>
 <li>Draw an ROI around the area of interest. Choose Analyze &rarr; Tools &rarr; ROI Manager, and click Add to record the ROI. Multiple regions may be stored in the manager. In ROI Manager, select all ROIs, then choose More &rarr; Multi-Measure to measure all stack slices. Export data to a spreadsheet for analysis.</li>
</ol>
5.2 Volocity analysis
<ol>
 <li>Import images into a Volocity library and create an image sequence with 2 channels.</li>
 <li>Draw a region of interest (ROI) in an area where there are no cells. Choose: Tools &rarr; Ratio.</li>
 <li>Use Volocity to calculate the background: Get From ROI.</li>
 <li>Adjust the threshold to include mitochondrial structures.</li>
 <li>Check the option to apply a rainbow LUT to the ratio channel. The intensity-modulated channel may produce a less noisy image for presenta-tion, but it should not be used for quantitation of average ratio.</li>
 <li>Select the Measurement tab, select the ratio channel and draw an ROI around the area of interest. Measure the ratio channel, excluding zero values. Multiple regions may be selected and measured at the same time. Export data to a spreadsheet for analysis.</li>
</ol>

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No conflicts of interest declared.

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.

<table><tbody><tr><td>&amp;nbsp;</td><td></td><td></td><td>&lt;strong&gt;Reagents&lt;/strong&gt;</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&lt;br/&gt; **omit for SGlyc-Leu</td><td></td><td></td><td>Dissolve in H&lt;sub&gt;2&lt;/sub&gt;O. Adjust pH to 5.5 with NaHCO&lt;sub&gt;3&lt;/sub&gt;. Autoclave.&lt;br/&gt; &lt;br/&gt; &lt;em&gt;Ingredients:&lt;br/&gt; &lt;/em&gt;0.67% Yeast nitrogen base without amino acids&lt;br/&gt; 3% Glycerol&lt;br/&gt; 0.05% Glucose&lt;br/&gt; 2 mg/ml adenine&lt;br/&gt; 2 mg/ml uracil*&lt;br/&gt; 1 mg/ml L-arginine&lt;br/&gt; 1 mg/ml L-histidine&lt;br/&gt; 1 mg/ml L-leucine**&lt;br/&gt; 3 mg/ml L-lysine&lt;br/&gt; 2 mg/ml L-methionine&lt;br/&gt; 4 mg/ml L-phenylalanine&lt;br/&gt; 2 mg/ml L-tryptophan&lt;br/&gt; 3 mg/ml L-tyrosine</td></tr><tr><td>SC (synthetic complete, glucose-based) yeast growth medium *omit for SGlyc-Ura&lt;br/&gt; **omit for SGlyc-Leu</td><td></td><td></td><td>Dissolve in H&lt;sub&gt;2&lt;/sub&gt;O. Adjust pH to 5.5 with NaHCO&lt;sub&gt;3&lt;/sub&gt;. Autoclave. &lt;em&gt;Ingredients:&lt;/em&gt;&lt;br/&gt; 0.67% Yeast nitrogen base without amino acids&lt;br/&gt; 3% Glucose&lt;br/&gt; 2 mg/ml adenine&lt;br/&gt; 2 mg/ml uracil*&lt;br/&gt; 1 mg/ml L-arginine&lt;br/&gt; 1 mg/ml L-histidine&lt;br/&gt; 1 mg/ml L-leucine**&lt;br/&gt; 3 mg/ml L-lysine&lt;br/&gt; 2 mg/ml L-methionine&lt;br/&gt; 4 mg/ml L-phenylalanine&lt;br/&gt; 2 mg/ml L-tryptophan&lt;br/&gt; 3 mg/ml L-tyrosine</td></tr><tr><td>Valap</td><td></td><td></td><td>Combine ingredients in a 1:1:1 (w:w:w) ratio. Melt by submerging in a 70 &amp;deg;C H&lt;sub&gt;2&lt;/sub&gt;O bath. Aliquot into glass petri dishes. Store at room temperature. &lt;em&gt;Ingredients:&lt;/em&gt;&lt;br/&gt; Vaseline petroleum jelly, hard paraffin, lanolin</td></tr><tr><td>&amp;nbsp;</td><td></td><td></td><td>&lt;strong&gt;Equipment and Software&lt;/strong&gt;</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&amp;plusmn;5 &amp;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 &amp;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></td><td>&amp;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></td><td>&amp;nbsp;</td></tr><tr><td>Volocity 3D Image Analysis software</td><td>Perkin Elmer (Waltham, MA)</td><td></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></td><td>&lt;a href=&quot;http://rsb.info.nih.gov/ij/&quot; target=&quot;_blank&quot;&gt;http://rsb.info.nih.gov/ij/&lt;/a&gt;</td></tr></tbody></table>

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

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...

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 ...

The overall goal of this procedure is to measure mitochondrial function in living yeast cells using two green fluorescent protein or GFP variants. Redox sensing GFP contains surface exposed cystines, which undergo reversible and environment dependent oxidation and reduction altering the excitation spectrum of the protein reduced row. GFP has an excitation maximum at 470 nanometers, whereas oxidized row GFP has an increased deficiency of excitation at 365 nanometers.The redoc state is measured as RO GFP emission at 470 nanometers excitation over the RO GFP emission at 365 nanometers excitation go A team is a fret probe in which the epsilon subunit of the F zero F1 A TP synthase is sandwiched between a fret donor GFP and a fret acceptor orange fluorescent protein or OFP binding of a TP to the epsilon Subunit results in confirmation changes in the protein that bring the fret donor and acceptor in close proximity, allowing for energy transfer from the donor to the acceptor. A TP levels are then measured as the OFP emission upon excitation at 488 nanometers over GFP emission upon excitation at 488 nanometers. Results are obtained that monitor changes in redox state or a TP levels that occur under physiological conditions.The main advantage of these methods over existing methods is that road GFP and go. A team are genetically encoded and can be introduced and stably maintained in living cells. As a result, they can be used to measure mitochondrial redox state and a TP in living cells without affecting the fitness of either the cells or the organelles.Both probes monitor changes in redox state or a TP levels under physiological conditions. Both probes are also ratio metric as a result. Measurements made with these probes are not affected by changes in biosensor concentration or sample illumination or thickness.Both biosensors provide resolution at the level of individual organelles. In fact, RO GFP variants have been targeted to mitochondria, er, endosomes, and peroxisomes where they have detected changes in redox state, largely independent of pH. These methods may help answer key questions in fields regarding mitochondrial quality control and how mitochondria change with age.Visual demonstration of this method is useful as the thresholding and ratio steps during image analysis is difficult and need to be carried out using consistent defined guidelines To begin this procedure. Perform transformation of yeast with biosensors as described in the text protocol. Grow cells to mid log phase in five milliliters of selective complete dropout media in a 15 milliliter conical bottom tube.Use the same batch of media for all experiments. After concentrating one milliliter of culture to 20 microliters, apply two microliters of the resuspended cells to a slide and cover the cells with a cover slip. Then melt a small amount of VAP on a metal spatula and spread along the cover slip edges to seal.Alternatively, clear nail polish can be used for mito RO GFP one imaging on a wide field fluorescent microscope. Use an objective with the highest numerical aperture possible that transmits light at 365 nanometers such as the 100 x 1.3 NA EEC plan. Neo Fluor objective lens.Configure the microscope to excite at 365 and 470 nanometers to detect oxidized and reduced mitter row GFP respectively. Use LEDs to change the excitation wavelength and an emission filter cube suitable for GFP. With the excitation filter removed, set the camera to one by one benning for optimal spatial resolution.Next, set the software to acquire a Zack consisting of 11 slices with 0.5 micrometer spacing, collecting both channels at each Z position. Locate the focal plane of cells using transmitted light to minimize bleaching of the fluorescent probe. Then collect a Z series through the whole depth of a typical cell using a step size of 0.5 micrometers to set up for imaging.Might go a team two on a spectral confocal microscope. Use the highest numerical aperture lens available here. A 100 x 1.49 APO turf lens is used.Configure the microscope to excite migo a team two at 488 nanometers and collect emission from 500 to 520 nanometers for a TP bound and from 550 to 580 nanometers. For a TP unbound forms. Actual optimal values may vary with characteristics of the imaging system.Set the pinhole to 1.0 area units, then set. Scan zoom to approach the nyquist sampling limit. To increase imaging speed, crop the field to 512 by 256 pixels.Use scan averaging if needed to reduce noise. Use as little laser power as possible while still producing an interpretable image. Use an internal or external power meter to monitor changes in the laser power which occur normally over time in any optical system.Adjust detector gain and illumination intensity to maximize the dynamic range. But avoid saturation. Do not analyze any cells containing more than 1%saturated pixels.Locate the focal plane of cells using transmitted light before collecting a Z series through the hold depth of a typical cell using a step size of 0.5 micrometers image all samples using the same objective laser power scan, zoom pixel size, gain, and offset. Open the acquired images and change type to 32 bit to determine the background. Draw a region of interest or ROI in an area where there are no cells.Choose, analyze, measure to add the mean intensity in this ROI to the results window. To subtract this value from the stack, choose process math and subtract. Then enter the background mean intensity and choose preview.Okay and yes, Thresholding is the most difficult aspect of the procedure. To ensure success, define thresholding parameters such that the resulting image maintains the actual size of the fluorescent object in the original image. Using exactly the same exposure conditions for each experiment will help produce consistent thresholding throughout image processing.A threshold that is too low will include background pixels. A threshold that is too high will eliminate parts of the mitochondria from the analysis using the subtracted Zack, find the middle slice and select image adjust and threshold and then threshold the mitochondria after finding an appropriate threshold value. Click apply to apply this threshold to all slices in the stack.Then check set background pixels to NAN or not a number and click okay and yes, repeat this process for the other image. Create the ratio Z stack by clicking on process and then image calculator. Divide the reduced stack by the oxidized C stack for MIT O-R-G-F-P one analysis.The same protocol is used for migo AAM two analysis. In that case, divide the A TP bound 560 nanometer image by the A TP unbound 510 nanometer image. Draw an ROI around the area of interest.Choose analyze tools. ROI manager and click add to record the ROI then choose more multi measure and okay. Finally copy the slice number area and mean and export the data to a spreadsheet for analysis, use the spreadsheet to calculate the area times mean the weighted sum and the weighted average cells expressing mitochondria targeted GFP and row GFP one grow at normal rates.The maximum growth rate is similar in cells expressing mito GFP and MIT row GFP one. Furthermore, mitochondria and yeast expressing MIT RO GF P one are tubular align along the mother bud axis and accumulate at the tips of mother and daughter cells. This normal morphology indicates that MIT RO GF P one does not perturb mitochondrial function.To assess the dynamic range of mitg FP one midlock phase wild type yeast cells were treated with hydrogen peroxide and dihi three etol and the mean mitochondrial reduced to oxidized ratio was measured to assess mitochondrial redox state titration with hydrogen peroxide or DTT from zero to 10 millimolar results in a dose dependent change. In mean mitochondrial reduced to oxidized ratio with a measured range from 0.6 under oxidizing conditions to 1.23 under reducing conditions. Thus mitg FP one is an effective biosensor for analysis of mitochondrial redate in living cells.Mito RO GFP one also offers subcellular resolution of mitochondrial redox state. This subcellular resolution reveals that mitochondria within individual yeast cells differ in relative redox state light can induce changes in the GFP Chromo four upon exposure to high intensity 400 nanometer light row GFP one undergoes photo conversion to a species with a different emission spectrum using low intensity excitation. There is no significant photo conversion during the period analyzed.The preferred way to reduce photo conversion is to excite the oxidized form of row GFP at 365 nanometers MIGO AAM two co localizes with DAPI stained mitochondrial DNA in yeast and is found in tubular structures typical of wild type yeast mitochondria. Additionally, the growth rate of cells expressing MIGO AAM two is similar to that of cells expressing mitochondria targeted GFP in both glucose and glycerol media. Thus expression of migo A two does not appear deleterious to the cell or mitochondria to test whether migo A two responds to changes in mitochondrial A TP levels cells were treated with antimycin A, an agent that inhibits mitochondrial A TP production.The median fret ratio decreases in cells treated with antimycin. A interestingly, the preliminary data indicates that there may be differences in a TP levels in different mitochondria within the same cell. Once mastered, this technique can be performed in several hours.Furthermore, while attempting this procedure, it is important to keep imaging parameters constant while monitoring cells for mitochondrial fragmentation and other signs of phototoxicity. Avoid photobleaching and develop consistent criteria for thresholding. These biosensors can be used to monitor mitochondrial changes in live plant fungi and mammalian cells.They can also be used to monitor mitochondrial changes in neurodegeneration, metabolic disease, and other diseases associated with defects in mitochondrial functions. After watching this video, you should be able to use mitochondria targeted ratio metric fluorescent dyes to monitor mitochondrial redox state and a TP in living cells.

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Research

JoVE Journal

Bioengineering

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Learn ratiometric biosensor techniques to measure mitochondrial redox state and ATP levels in living yeast cells using GFP variants. This method enables real-time subcellular analysis of organelle function.

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

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