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.
