In this video, kinase and phosphatase activities are monitored throughout the cell cycle using Forrester Resonance Energy transfer or fret. Here a polo like kinase one or LK one activity probe has cyan fluorescent protein CFP placed on one end and yellow fluorescent protein YFP placed on the other. This probe is transfected into U2 OS cells.
Then to examine the phosphorylation status of LK one targets during the G two two M transition, the TRANSFECTED U2 OS cells are subjected to time lapse fluorescence imaging. When CFP and YFP are in close proximity and CFP is excited, energy is transferred to YFP and YFP admission can be detected. However, when the probe becomes phosphorylated, a confirmational change in the protein separates CFP and YFP.
Now when CFP is excited, a strong CFP emission and only a weak YFP emission are detected quantification of the ratio of YFP emission after both CFP and YFP excitation demonstrates that PLK one activity increases in G two and peaks during mitosis. So the main advantage of this technique over existing methods like standard geometric filming, is that specific attention is put to that the cell cycle is not perturbed either by filming conditions or by expression of a probe. This method can help answer key questions in the cell cycle field, such as where and when specific kinases or phosphatases are activated.
Although this method can provide insight into cell cycle regulated enzymes, it can also be applied to other systems where unres conditions are required, such as cell proliferation assays. To monitor the human cell cycle, U2 OS cells are transfected with a fret based PK one activity probe. Using a standard calcium phosphate transfection method, the cells are selected in pur mycin for at least seven days.
This enriches the population of cells expressing the probe and limits the number of cells with toxic expression levels or expression levels that severely affect the cell cycle. Stable clones are then selected by limited dilution expression of CFP and YFP is then examined to verify that they are present in all cells at approximately the same ratio. If some cells contain only CFP or YFP recombination has likely occurred in this case, repeat the plasmid preparation using bacteria that are less prone to homologous recombination, such as sure two cells then linearize the plasmid and repeat the transfection.
Once the cells stably express the probe, seed them on number 1.5 glass bottom dishes after they have attached and are 30 to 50%confluent. Change the medium to one that is CO2 independent and does not contain phenol red mount the cells on a motorized epi fluorescence microscope With temperature control set to 37 degrees centigrade. The microscope should be equipped with CFP and YFP excitation and emission filters.
A ROIC mirror suitable for monitoring CFP and YFP and an objective suitable for the required resolution. Here, a 20 x NA 0.75 air objective is used. Using transmitted light, bring the cells into focus.
Do not use fluorescent light at this point since it will induce a stress response that may perturb the cell cycle. To avoid phototoxicity, set the bending to the maximum available that permits the required subcellular resolution. For this experiment, it should be possible to distinguish the nucleus and the cytoplasm.
Set a short exposure time and insert a high neutral density filter. Then acquire a 12 or 16 bit test image Using YFP excitation and emission, adjust the exposure time and neutral density filters so that the average intensity in a cell is approximately equal to the background intensity plus five times the difference between minimum and maximum background intensities. To verify that the images are not saturated, examine the fluorescence intensity for the sample image.
The maximum intensity in the image should be less than half of the dynamic range to allow for intensity differences when cells enter mitosis. Next, repeat this process using CFP excitation and YFP emission filters. Select multiple regions with transfected cells, excluding the regions used for focusing and optimizing exposure conditions.
Since these procedures may have invoked a stress response, ensure that the maximum intensity is less than half of the dynamic range in all of the cells. Instruct the software to acquire two images every 45 minutes using YFP excitation, YFP emission and CFP excitation. YFP emission filters.
Also set the total length of the experiment to exceed the duration of the cell cycle. Once everything is set, begin acquiring images at after the acquisition is complete, open the acquired images and monitor the cell cycle time from mitosis to mitosis. For cells that express the transfected probe here, the cell cycle time was roughly 20 to 25 hours.
Compare this to control data for the cell type used. If cell cycle time corresponds to reference timings fret can be analyzed. Once the timing has been verified, the images can be analyzed.
The analysis can be performed by most software dedicated to microscopy. Here, image j freeware is used with the ratio plus plugin. Begin the analysis by opening the images in Image J.If the images are in a multicolor stack format, separate the individual channels by converting them to a hyper stack By selecting image.
Then hyper stack then stack to hyper stack. The convert to hyper stack window opens with the settings displayed click okay. The image will now be displayed with two slider bars below it.
Next to split the images to single channel stacks, click on image and select hyper stack reduce dimensionality. This will open the reduce window, ensure that frames and keep source are selected. Then click Okay.
A new image containing only one channel will appear. Repeat this process to bring up a new image with the second channel. Next, to increase the visual clarity of the final images, select the YFP excitation YFP emission stack.
Click on process, then click on math. Then multiply When the multiply window opens, enter three to multiply the YFP excitation YFP emission stack by three. The step is optional and ensures that ratios are not in the range between zero and one.
Next, on the image J toolbar, select the freehand tool. Then in the YFP excitation, YFP emission stack, draw a region of interest or ROI in an area that is devoid of cells, but that is close to a cell that will be measured to add the ROI to the ROI manager, click on analyze tools. Then ROI manager.
The ROI manager window will appear. Click on add to save the ROI coordinates to measure the average intensity of the ROI In the YFP excitation YFP emission stack. Click on analyze, then select measure.
The results window will open. Select the CFP excitation YFP emission stack. Then double click on the ROI in the ROI manager and repeat the measurement.
The measurement is added to the results window. Scroll to other places in the stack to repeat this process at multiple time points. Verify that background intensities close to the cell of interest are similar throughout the film by comparing measurements in the results window.
To set measurements to include minimal intensity, click on analyze, then click on set measurements and indicate the minimum and maximum gray value. A window showing measurement alternatives will appear next. After adjusting brightness and contrast as necessary, draw an ROI covering most of a cell and measure the minimal intensity in both stacks.
Then take measurements as before this time using a cell instead of the background. Using the values from the results sections, calculate the difference between the measured minimal intensity and the background intensity and divide it by two. This provides a starting estimate for a clipping value to open ratio plus select plugins.
Then ratio plus. Then for fret ratio, select the CFP excitation YFP emission S stack one or for inverted fret ratio visualizing probes where fret efficiency decreases upon phosphorylation. Select the YFP excitation YFP emission S stack one to insert the measured background intensities and clipping values.
Use the values from the result window and paste them in the ratio plus window. Set the scaling of the resulting ratio stack and apply a suitable lookup table to visualize ratio changes. U2 OS cells expressing a fret based probe monitoring PLK one activity were filmed for 60 hours as described in this video.
This image shows cumulative mitotic entry of 50 fret probe expressing cells, including divisions of daughter cells. The majority of the cells expressing the probe proliferate with a cell cycle time of between 20 and 25 hours, indicating that the imaging conditions and expression levels of the probe do not affect cell cycle timing. A false color representation of inverted fret ratio following one cell through four divisions demonstrates that although there is considerable noise, a trend is seen in which PK one activity increases in G two and peaks during mitosis.
When the raw data is mean filtered and presented as a graph, the time between activity peaks is easy to estimate. This figure shows quantification of the inverted fret ratio of the cell shown in the previous images from the graph. The timing of activation can be estimated and as can be seen here, phosphorylation of the probe becomes visible approximately four hours before the phosphorylation is peaking in mitosis.
While attempting this procedure, it's important to keep the cells in an unstress condition, so this is done by minimizing exposure to light as well as to minimize changes in temperature or pH. It is also important to remember that this method monitors the balance between kinase and phosphatase activity in a cell. This procedure can be combined with RNA interference or inhibitor screens to identify the upstream regulators of these kinases and phosphotases.
