The overall goal of this experiment is to detect and track non-apoptotic caspase activity in live animals using Drosophila Melanogaster as a model. Caspases are proteases that cleave many cellular components to cause apoptotic cell death. Therefore, the presence of active caspases in cells is often assumed to reflect impending cell death.
However, growing evidence indicates that the same caspases that cause cell death also have normal roles in healthy cells. And these day jobs of caspases include regulation of neuronal activity, mitochondrial functions, even the suppression of alternative cell death pathways. There are two major difficulties in studying the day jobs of caspases.
First, normal basal levels of caspase activity are typically below the limits of detection by available technologies. And secondly, even when basal caspase activity is detectable, it is difficult to distinguish from the caspase activity that will lead to cell death in the near future. To solve this problem, we developed a caspase tracker:a more sensitive caspase biosensor that can permanently label cells that have survived that caspase activation.
This biosensor will enable us to detect and track the cells with non-apoptotic caspase activities and to study its functions in live animals. The drosophila in vivo caspase tracker biosensor system consists of two components. The caspase activatable yeast transcriptor Gal4 and the Gal4 activated fluorescent reporter known as G-Trace.
Caspase biosensor flies are crossed with G-Trace flies to generate CaspaseTracker flies that report caspase activity as red and green fluorescence. Caspase activity is detected in progeny flies when the biosensor is cleaved by active caspases at the sequence DQVD, releasing Gal4 from the plasma membrane. Released Gal4 translocates to the nucleus and induces expression of red fluorescent protein, RFP, until caspases become inactive.
However, Gal4 also induces the expression of FLP recombinase, leading to continuous expression of nuclear-targeted GFP for the life of the cell. A control biosensor has a mutation, DQVA, in the caspase cleavage site. To begin, cross transgenic flies carrying the caspase-sensitive Gal4 biosensor with flies carrying the G-Trace fluorescence reporter.
Pair seven to 10 virgin biosensor females with seven to 10 newly-eclosed male G-Trace flies in a fresh food vial. An important negative control is the caspase insensitive version of this biosensor, which has a single point mutation in the aspartate residue required for caspase cleavage. Use the same mating procedure for these control flies.
After setting up the crosses, keep the vials at 18 degrees Celsius. When larvae are developing, and before new progeny flies eclose, transfer the parent flies to new vials to continue producing new progeny. When the progeny flies emerge, collect those with straight, non-curly wings.
This phenotype indicates that these flies carry transgenes for both the caspase biosensor and the fluorescent G-Trace reporter. Then, following established protocols, dissect flies to isolate intact internal soft tissues from both caspase tracker and control flies. To begin, prepare pipette tips and centrifuge tubes that will not stick to the dissected tissues.
To do this, coat the tips and tubes with one percent bovine serum albumin dissolved in water. To fix the dissected tissues, load the coated clear-walled tubes with point-five milliliters of fresh four percent paraformaldehyde in PBS and transfer the freshly dissected tissues into these tubes. Then, place tissues at room temperature in the dark to avoid photobleaching and incubate for 20 to 30 minutes with a gentle rotation to bathe tissues with fix while avoiding damage to the tissues.
Longer fixations can lead to suboptimal staining of tissues. To remove paraformaldehyde and to initiate membrane permeablization of the fixed tissues, gently remove the paraformaldehyde solution with a coated pipette tip and wash the tissues three times with point five milliliters of PBST. To complete tissue permeablization, and to block non-specific binding in subsequent staining procedures, incubate the tissues in point five milliliters of PBST containing one percent BSA at four degrees Celsius overnight in the dark with gentle rotation.
The next day, remove the PBST BSA solution by washing the permeablized tissues three times with point five milliliters of PBST. To help visualize different tissue structures, co-stain nuclear DNA and cytoplasmic F-actin by incubating the prepared tissues in point five milliliters of PBST containing hoaxed 33342 blue nuclear dye and Alexofluor 633 phalloidin F-actin stain for one hour at room temperature with gentle rotation. Remove excess stain by washing tissues three times with point three milliliters of PBST.
Incubating each wash for five minutes at room temperature with gentle rotation. Remove all solution from the tissues in the tube carefully by fine pipette tip. To preserve the fluorescent signal during imaging, add 200 microliters of anti-bleach mounting agent completely covering the tissues.
Incubate the tissues one to three hours at room temperature, or at four degrees celsius overnight. Optimally, tissues that have fully absorbed the mounting agent will sink to the bottom of the tube. Before mounting the tissues, clean the glass slide with water or 70%ethanol.
Apply petroleum jelly to the glass slide and to the cover slip to avoid destroying the tissues by overcompression. Then, transfer the tissues with mounting agent to the glass slide. Remove any extra mounting agent using tissue paper.
Finally, seal the cover slip by applying nail polish all along the edges. To begin, place the slide on the stage of an inverted microscope. And capture cell images using a 20x Plan-Apochromat objective with an aperture of point eight.
Take a few test images at low resolution to verify an image tiling plan that will cover the region of interest, which in this case is the complete internal organ system of the fly. Set up the image sequence starting with the longest excitation wavelength and ending with the shortest excitation wavelength. This will minimize photobleaching which increases at shorter wavelengths.
Then, take images at a suitably high resolution. Each image should have a 10%overlap with the next for successful tiling. Collect two to eight imaging scans per tile to reduce the background noise.
To eliminate the shadow between image tiles from the differential interference contrast, DIC, take a reference DIC image at the empty region. Then, perform shadow correction on the tile image with the reference DIC image. This is a dissected newly eclosed caspase tracker fly with mouthparts, foregut, crop, midgut, hindgut, anus, oviduct, and ovaries.
By confocal microscopy, caspase activity is visualized as RFP fluorescence which indicates recent caspase activity, and by GFP fluorescence which indicates past caspase activity. Enlarged images reveal RFP and GFP biosensor activity in the optic lobe, cardia, midgut, hindgut, and oviduct. Specific cells along Malpighian tubules prominently display both RFP and GFP, indicating persistent caspase activity.
In contrast, no RFP or GFP biosensor activity was expressed in egg chambers, though the biosensor was expressed in muscle cells between egg chambers based on co-staining with F-actin stain. However, the cells of egg chambers are capable of activating caspases if flies are treated with a cell death stimulus such as starvation or cold shock which causes nuclear destruction and massive caspase activation based on immunostaining. Caspase tracker likely appears early and is subsequently destroyed during cell death.
In the optic lobe of the healthy caspase tracker fly brain, biosensor activity is detected in healthy cells that appear to be neurons, based on costaining with a pan-neuronal marker anti-ELAVI antibody. This suggests a role for non-apoptotic caspase activity in neurons. Other strategies can be used to demonstrate long-term survival of cells with caspase activity, such as turning off biosensor expression using genetic tools.
For example, GalADTS that inhibits Gal4. Caspase tracker specifically reports caspase activity as the control uncleavable biosensor, DQVA is not activated. The only signal on controls is due to autofluorescence from the remaining cuticle on the mouthpart and anus, and ingested food, yeast, in the crop and cardiac.
After watching this video, you should have a good understanding of how to use the caspase tracker biosensor to follow non-apoptotic caspase activity in fruit flies. While attempting this procedure, it's important to include negative controls to distinguish caspase biosensor signals from autofluorescence. For example the cuticle and the fat bodies produce strong autofluorescence in flies with or without the biosensor.
The current version of the caspase tracker biosensor is designed to detect the active caspase DRICE. Other applications may include biosensors specific for different caspases by replacing the cleavage site recognized by DRICE with the cleavage site recognized by other caspases.
