The overall goal of this procedure is to visualize the movement of the actin binding protein in yeast that express a mutant actin isoform. This is accomplished by first making a plasmid and coating the actin interacting protein one or a IP one P gene tagged with a three x green fluorescent protein Fluor four. The second step is to generate a yeast strain expressing only wild type or mutant acton.
Next, the GFP tagged A IP one P plasmid is transformed into the wild type or mutant yeast strain. The final step is to image and analyze the movement of the tagged A IP one P in the wild type and mutant yeast cells. Ultimately total internal reflection.
Fluorescence or turf microscopy is used to show that the A IP one P moves slower in yeast expressing the R 2 56 H mutant acton than wild type acton. This method can help answer key questions in the cytoskeletal field, such as the relative temporal movements of binding partners. We have first had the idea for this method when I discovered that unlike the wild type strain, deleting A IP one P from the mutant yeast strain was lethal.
The main advantage of this technique over existing methods such as confocal microscopy, is the enhanced contrast and imaging speed. Generally, individuals new to this method may struggle to optimize the microscope settings for their individual experiment because of the numerous adjustments that can be made and the subjective nature of the image quality. Begin this protocol with generation of the A IP one PGFP plasmid and mutant yeast strain as instructed in the text protocol, digest the plasmid with a restriction enzyme to allow integration into the yeast chromosome culture.
The prepared S-C-V-C-A cells that are unable to synthesize uracil overnight in five milliliters of YPD media on a wheel at 30 degrees Celsius. Spin down one milliliter of the cells for five minutes at 1000 times g. Next, make the plate mixture as instructed in the text protocol and filter sterilize decant the supernatant from the cells and resuspend the cells in the remaining liquid to the suspended cells.
Add two microliters of 10 milligrams per milliliter salmon testes carrier DNA. Then add 10 microliters of the digested plasmid, DNA and vortex before adding 0.5 milliliters of plate and vortexing again. Finally, add 20 microliters of one molar DI three etol and vortex.
After incubating the mixture for six to eight hours at 25 degrees Celsius. Heat shock the cells in a water bath at 42 degrees Celsius for 10 minutes. Plate 100 microliters of the cells from the bottom of the micro centrifuge tube where they have settled out onto a minus U plate.
Incubate at 30 degrees Celsius for two to three days or until colonies form. Select individual colonies and streak out onto a minus.ATE. To prepare for microscopy, these cells contain the plasmid with the A IP one GFP and UroGen integrated into the chromosome.
Grow a culture of the yeast cells with the incorporated A IP one PGFP in YPD overnight on a wheel at 30 degrees Celsius subculture. One milliliter of the overnight culture into nine milliliters of minus Euro media. Grow the cells for an additional three to four hours on a shaker at 30 degrees Celsius to ensure that they are in log growth phase.
Next, add three microliters of cells to a glass microscope slide and add a cover slip. Allow the cells to settle on the slide for five minutes. Before placing the slide in the bracket of the stage plate, observe the A IP one PGFP protein with a total internal reflection fluorescence or turf microscope using an oil immersion 100 x turf objective and a digital CCD camera.
To focus on the cells, open slide book 5.0 software and click the focus window button in the toolbar. To access the focus controls, select the scope tab and select the 100 x turf objective. Turn the lamp on and slide the lamp bar to 15%Change the bin setting to two by two and change filter to brightfield on the microscope.
Use the fine focus style to bring the cells into focus as seen on the computer screen. Using the controls within the focus window, turn the lamp off, change the filter to live and click the T-I-R-F-M button on the T-I-R-F-M illuminator attached to the right side port of the microscope. Turn the laser emission to on select the stream tab within the focus window.
Check the box next to start recording, and then click the brightfield button on the TIR FM illuminator. Turn the micrometer to adjust the incident angle of the laser. This is used to optimize the fluorescent emission from the A IP one GFP foci and minimize the background flu fluorescence from the cells and slide when the desired image is displayed.
Press start. Obtain images for 20 seconds at a rate of five frames per second. After 20 seconds, press stop.
Save the images under the file tab on the main menu bar. Select save Slide as and enter the file, location, and name. Next, use Image J software.
To analyze the images, use the macro plugin manual tracker to track the movement and rate of the fluorescent A IP one P foci. Change the time interval parameter to 0.2 seconds. Using the add track selection, select and track an individual fluorescent protein through four to eight connective frames.
Use the N track command and the velocity of protein movement between each frame will display in a results window. Determine the mean rate between each frame. Using a Microsoft Excel spreadsheet.
Use the values to compute the average velocity of a IP one P movement, or each cell strain of interest to quantify A IP one P movement. More than 50 fluorescent A IP one P foci were tracked in more than 10 cells. Representative results of a IP one P movement in wild type cells are shown as expected.
The fluorescently tagged a IP one P moves quickly across the cell and then disappears in cells expressing the R 2 56 H mutant Acton. A IP one P has less movement compared to wild type cells. A IP one P is tracked using Image J to quantitatively map the movement of the protein.
A IP one P ranges across the cell in the wild type strain, but moves only in a restricted region. In the mutant acton strain, the average velocity was calculated for each fluorescent A IP one P area. In wild type cells, the average speed of the movement of a IP one P is 1.60 plus or minus 0.42 microns per second cells expressing the R 2 56 H mutant actin known to have abnormal morphology of the actin cytoskeleton have an altered A IP one P phenotype in the mutant strain.
A IP one P movement is restricted and slower. The average speed of A IP one P movement is 0.88, plus or minus 0.30 microns per second. After watching this video, you should have a good understanding of how to image cytoskeletal proteins using turf microscopy.
While using this procedure, it is important to remember to not let the cells dry out or become photo bleached.
