This method can be used to detect the activation of Aurora kinase A, in a fast and robust manner using fluorescent biosensors, relying on the principles of FRET. By measuring FRET efficiency in a rapid and online manner, we can instantaneously determine whether or not Aurora kinase A, is activating. The FRET-FLIM device is relatively easy to mount and can be used on any white film microscope that has a free side port.
After selecting a donor accept FRET pair in the fluorescent protein database, in the tools menu, select spectra viewer, click close to bypass the welcome window and enter the name of the floral force pair to be visualized into the search box. To simulate the properties of the donor, acceptor pair with a specific light source, select a given laser and click normalize emission to this. To adjust the flora force spectra to the desired wavelength.
The day after synchronization, replace the no cortisol supplemented Supernat with sterile prewarmed PBS and wash the cells with gentle rocking two times at room temperature. After the second wash, feed the culture with sterile prewarmed imaging medium and quickly place the cells in the thermostatic chamber of the microscope. For FLET-FLIM acquisition, turn on the laser, camera, microscope set up and imaging software.
Select the appropriate excitation and emission wavelengths for the donor floral four and set the exposure time in the imaging software. Before launching FLET-FLIM acquisitions, check for the formation of the bipolar spindle in the cells under the microscope using an external light source. When the spindle has appeared, select at least a 63 times objective and locate a cell in metaphase.
Adjust the X, Y Z coordinates, to place the cell at the center of the field of view and select a single Z plane in which the mitotic spindle is more visible or intense. Then start the recording. The majority of available commercial setups will generate both a fluorescence micrograph, and a pixel by pixel lifetime map.
Save both images. To calculate the Delta lifetime values for the donor accepter pair, select the region of interest, corresponding to specific sub regions before extracting the lifetime values, from the whole pixel by pixel lifetime map. Then calculate the mean lifetime of the cells expressing the donor only vector, to obtain the mean donor lifetime.
For FLIM value comparison, subtract each calculated independent lifetime value, from the mean donor lifetime for each cell in each of the experimental conditions analyzed, to obtain the Delta lifetime value for each condition. The Delta lifetime values for the donor only, biosensor kinase-dead and pharmacological inhibitor treated constructs for example, can then be compared. After synchronizing cells in G2M and releasing them into mitosis, for lifetime of all the transfected constructs at the mitotic spindle can be measured.
As expected, in this analysis, the Lifetime values of GFP-AURAKA, and AURAKA-mTurquoise2, were close to zero. Indicating that the values measured for these constructs fluctuated around the mean value. Conversely, that the Delta lifetime values for GFP AURAKA-mCherry, was statistically different from the donor only condition.
With the Delta lifetime value increasing approximately 130 picoseconds. Similar observations were made for shadowG-AURAKA-mTurquoise2, and for super YFP-AURAKA-mTurquoise2. With the Delta lifetime values increasing approximately 150 and 220 picoseconds from the donor only condition respectively.
In this analysis, the values of Delta Lifetime in cells, expressing the donor only constructs around zero, appear to be pseudo colored yellow. While more significant differences are observed as a pseudo colored red purple signal in cells expressing either biosensor. This effect was also observed when the GFP-AURAKA-mCherry biosensor, was treated with a pharmacological inhibitor.
In constructs with kinase-dead biosensors, Delta lifetime values were significantly higher, compared to the donor only condition. But are also significantly lower than their normal counterparts. Make sure that the variability of the donor only condition fluctuates around its main value, and that the value is significantly different from the lifetime of the biosensor constructs.
Since the AURORA-kinase A biosensors works well with dark acceptors, we can consider coupling it to a second biosensor to follow its enzymatic activity toward a substrate.
