The overall goal of this methodology is to visualize one of the first steps in the activation of an initiator caspases. This step is called induced proximity and occurs when caspase molecules come together to form active dimers during apoptotic cell death. This method can help answer key questions in the caspase field such as when, where, and how efficiently specific initiator caspases can get be activated.
The main advantage of this technique is that one can visualize the assembly of caspase activation complexes in living cells in real time. This biomolecular fluorescence complementation method uses fragments of the thyroxine protein, Venus, that have infused to a caspase. The fragments are not fluorescent on their own but when the caspase undergoes induced proximity, this brings the two Venus halves together and they light up.
To transfect the cells, first add 12 microliters of the transfection reagent to 750 microliters of reduced serum media in a steril tube. Incubate the mixture at room temperature for five minutes. Then add 10 nanograms of a reporter plasmid to a steryl 1.5 milliliter tube for each well to be transfected.
Add 20 nanograms of each caspase BiFC plasma to each tube. Bring each tube up to a total volume of 100 microliters by adding reduced serum media. Using a P200 pipette, add 100 microliters of transfection reagent solution to the plasmid mixture in a dropwise manner.
After incubating the plasmid transfection reagent mix for 20 minutes at room temperature, aspirate the medium from the cells using a pipette or with suction. Then using a P1000 pipette, pipette 800 microliters of serum-free media gently down the side of the well. Using a P200 pipette, add 200 microliters of the DNA lipid complex dropwise to each well.
Incubate the cells in a tissue culture incubator at 37 degrees Celsius for three hours. Following incubation, remove the serum-free media containing the DNA lipid complexes by aspiration using suction or with the pipette taking care not to disrupt the monolayer. Pipette two milliliters of pre-warmed complete growth media gently down the side of each well before incubating the cells as described in the text protocol.
Prior to data acquisition, perform induction of caspase activation as described in the text protocol. Turn on the microscope following the manufactures instructions and launch the acquisition software. Select the 60x or 63x oil objective and place a drop of oil onto the objective.
Then place the culture dish on the microscope stage using the correct plate holder. Turn on the heater and set the temperature to 37 degrees Celsius. Focus on the reporter fluorescence using the RFP laser or filter.
Input the percent laser power at 50%and the exposure time at 100 milliseconds for both Venus and RFP as a starting point to determine the optimal settings to use for the experiment. Turn on the live capture and inspect the resulting image and the displayed histograms for both channels. If the signal is low and the image is hard to make out increase the percentage laser power and or exposure time in increments until the signal and the image looks good.
Next, select or open the Z stack module in the microscope software. Adjust the focus in one direction until the cell is not longer visible. Select this as the top position.
Adjust the focus in the opposite direction until the cell is again no longer visible and select this as the bottom position. Choose the optimal step size and start the acquisition. If needed adjust the display histogram to improve the visual appearance of the fluorescent signal.
Finally, use three dimensional rendering software to reconstruct the 3D image. If a carbon dioxide source is available, place the carbon dioxide delivery device on top of the plate holder. Set the carbon dioxide level to 5%and turn on the carbon dioxide controller from within the software.
Navigate to a field of transfected cells. Visualize the live image of the cells as acquired by the camera and displayed by the acquisition software on the computer screen using the RFP laser. Next, input the settings for percentage laser power and exposure time for the RFP laser.
Then, input the setting for percentage laser power and exposure time for the YFP laser light. For each well of the plate, choose a number of different positions that contain one or more cells expressing the reporter. Input the time interval between each frame of the time laps and total number frames to be taken.
Revisit each position and correct and update the focus as needed. Begin the time lapse and save the data. Finally, analyze the data using available imaging software as described in the text protocol.
Shown here is an example of caspase-2 BiFC following DNA damage. Cells were treated with a topoisomerase one inhibitor:Camptothecin. The red fluorescent protein, mCherry, was used as a reporter to show that the cells express the BiFC probe and help visualize the total number of cells.
Venus fluorescence is shown in green and the large puncta represent caspase-2 induced proximity. To provide high resolution visualization of the subcellular localization of the caspase activation complex, single cells can be imaged in three dimensions. caspase-2 activation is shown here in green and the nucleus is shown in red.
The orthoganal slices view of the cell is shown where the xy plane, the yz plane and the xz plane can be visualized side-by-side. The three dimensional image can also be displayed in a movie where the cell is rotated around the y axis. To provide kinetic data of caspase BiFC time lapse imaging can be used.
This movie shows caspase-2 activation in green following treatment with the proteasome inhibitor:Bortezomib. The mitochondria are shown in red. After watching this video you should have a good understanding of how to use caspase BiFC to track the assembly of the caspase activation complexes in real time and to distinctly determine the size, shape and localization the complexes produced.
This protocol outlines a few microscope based approaches to collecting the results to the caspase BiFC experiment;however, it should be noted that a number of variations and combinations of these approaches can be used to creatively interrogate caspase activation pathways. While attempting this procedure, it is important to remember to include controls from microscopy induced artifacts such as photo toxicity and photo bleaching. The accompanying text protocol includes tips to accomplish this.
Deciding which acquisition and analysis method to use will be determined by the perimeters to be explored and the availability of instrumentation and imaging software. Following this procedure, imaging analysis approaches can be used to interpret the data. You can measure changes in fluorescence intensity over time or the percentage of fluorescent cells.
Number of foci or object size can also be calculated.
