Detecting Anastasis In Vivo by CaspaseTracker Biosensor

The overall goal of this procedure is to detect and track reversal of the cell death process after caspase activation in live animals using Drosophila melanogaster as a model. In response to death stimulus, cells undergo programmed cell death such as apoptosis which has traditionally been considered as an irreversible cascade leading to cell death. Interestingly, however, after removal of the death stimulus, some dying cells can reverse the cell death process even at the late stage.

Through a newly discovered cell recovery phenomenon called anastasis. Live cell microscopy shows that apoptotic dying cells display unique morphological hallmarks, such as plasmid membrane blebbing, nuclear condensation, and cell shrinkage. In general, these cells are expected to die.

However, after washing and incubating the dying cells with fresh medium, they undergo anastasis, recover, and can later divide. It is difficult to detect and track anastasis in live animals because the recovered cells can be morphologically indistinguishable from the surrounding healthy cells. To solve this problem, the mammalian CaspaseTracker biosensor system was developed to identify and track the cells that reverse the cell death process after execution or caspase activation, the hallmark of apoptosis.

This biosensor system consists of two components:the caspase activatable transactivator, rtTA, and the Cre LoxP based rtTA activity reporter. In healthy cells, without caspase activity, rtTA is tethered to a plasma membrane anchor through caspase-cleavable DEVD linker. As tethered rtTA cannot translocate from cytosol to nucleus, the rtTA reporter remains inactive.

However, upon activation in response to cell death induction, caspases cleave the DEVD linker, freeing the rtTA to translocate to the nucleus to activate the rtTA reporter. Once in the nucleus, the rtTA binds to the tet response element, or TRE, and triggers transient expression of Cre recombinase, leading to an irreversible recombination event that removes the stop codon cassette between the CAG promoter and the coding sequences for the red fluorescent protein DsRed. This results in permanent expression of DsRed which serves as the permanent fluorescent marker for cells that can remain alive after they have experienced caspase activity as well as their progeny.

Live cell confocal microscopy shows that after transient cell death induction, the CaspaseTracker biosensor cells express DsRed during and after their recovery, and therefore, distinguish them from the non-recovered cells, indicated by white arrows, as well as the control biosensor cells that were not exposed to the cell death stimulus. Programmed cell death, such as apoptosis, is generally assumed to be irreversible because it is executed by a rampant and massive cellular destruction. However, we find that that dying cell can actually recover even at the late stage that has been considered as the point of no return, such as after cytochrome c release, caspase-3 activation, DNA damage, plasma membrane blebbing, cell shrinkage, and formation of apoptotic bodies.

We named these reversal of cell death process anastasis, which means rising to life in Greek. By using live cells microscopy, we can observe anastasis in cultured cells. However, it is technically challenging to observe anastasis in live animals because the recovered death cell can look like normal healthy cells that did not attempt cell death at all.

Therefore, we developed a CaspaseTracker biosensor system to detect and track anastasis in live animals. The Drosophila version of the CaspaseTracker system is a dual biosensor that consists of two components:the caspase-activatable use transcription factor, GAL4, and the GAL4 activity reporter, known as G-TRACE. In the cells without caspase activity, the yeast transcription factor, GAL4, is tethered to a plasma membrane anchor domain through a caspase-cleavable DQVD linker.

Upon caspase activation, activated caspases cleave the DQVD linker to release GAL4, which then translocates to the nucleus to activate the G-TRACE reporter. The nuclear GAL4 binds to the specific upstream activating sequences, abbreviated UAS, to trigger transient expression of RFP, which serves as the reporter of recent or current caspase activity until the caspase and the GAL4 activity stop and RFP protein is degraded. GAL4 also triggers the expression of the FLP recombinase, which leads to a recombination event that removes the stop cassette between the ubiquitin, abbreviated Ubi, promoter and the coating sequences for the nuclear-targeted GFP.

This results in permanent expression of nuclear GFP, which serves as a permanent marker for cells that have experienced caspase activity but remain alive as well as their progeny. Therefore, this system can identify cells with ongoing or recent caspase activity by the transient RFP signal and past caspase activity with the permanent GFP signal. A control biosensor has the mutation DQVA at the caspase cleavage site, which is insensitive to caspase activity.

Caspase-sensitive GAL4 virgin female flies were crossed with G-TRACE young male flies, or vice versa, to generate the CaspaseTracker flies. To test reversibility of the cell death process after caspase activation, the flies are first exposed to transient, cell death-inducing environmental stress, such as cold shock or protein starvation. Then the stressed flies are transferred back to normal culture conditions for recovery.

Tissues were obtained from the egg chambers in the ovaries from the control, stressed, or recovered flies. The samples were subjected to confocal microscopy to detect the fluorescent signals of the CaspaseTracker biosensor. To begin, anesthetized, recently eclosed caspase-sensitive DQVD GAL4 flies using carbon dioxide and, with a paintbrush, transfer 7 to 10 virgin females to a fresh food vial with a small dollop of yeast paste.

Mate the females with 7 to 10 young, G-TRACE GAL4 reporter males. Newly eclosed virgin flies show the dark green meconium, indicated by arrows, which are the remnants of larval food. The newly eclosed flies tend to be bigger than the mature flies.

Males have a darker abdomen, indicated by circles, and tend to be smaller than females. Setup the cross at 18 degrees Celsius to reduce non-specific signals from the CaspaseTracker biosensor in the progeny because GAL4 activity increases with temperature. Every three to seven days, move the adults into a new vial with yeast paste until their productivity diminishes.

When progeny eclosed, connect the flies with both transgenes of caspase-sensitive GAL4 and G-TRACE, which will function as the CaspaseTracker progeny flies. Both insertions are balanced by Curly-O. So the non-Curly winged flies will have both transgenes.

For a negative control, collect dual transgene progeny from a cross between caspase-insensitive DQVA GAL4 females and G-TRACE GAL4 reporter males or vice versa. From the experimental crosses, transfer cohorts of 10 to 20 newly eclosed female flies to new vials with yeast paste. Adding some males will help females for egg production.

Transfer the well-fed flies to 18 degrees Celsius for a day so their ovaries produce egg chambers through OO-genesis. Then to induce their egg chambers to undergo apoptosis use a cold shock. Transfer the flies to a new vial without food and freeze them at 7 degrees Celsius for an hour.

An alternative method to induce egg chamber apoptosis is to use protein starvation by housing the flies on 8 percent sucrose, 1 percent agar food at 18 degrees Celsius for three days. Change the no-protein food vials daily when using this method. After stressing the flies by either method return them to the normal housing condition and food with yeast paste for three days to allow them to recover.

Later dissect these flies to isolate their egg chambers and ovaries. After anesthetizing them, use two pairs of forceps to remove their heads. Then pull the flies at the base of the abdomen to remove their ovaries.

Prepare to collect the ovaries by coating plastic pipette tips with 1 percent BSA dissolved in water or PBS so the egg chambers will not stick to the pipette tips. After dissecting the egg chambers, collect them in prepared tips with about one half milliliter of PBS. Then transfer them to a one milliliter centrifuge tube and allow the eggs to settle.

Continue the procedure under low light or completely dark conditions to prevent photo bleaching of the fluorescent tags. Aspirate off the PBS and load 500 microliters of four percent paraformaldehyde in PBS into the centrifuge tube. Let the eggs fix for 20 to 30 minutes at room temperature with gentle rotation.

Avoid prolonged fixation, as it will deplete the signal of the fluorescent proteins in the egg chambers. Once fixed, remove the PFA and wash the egg chambers with 500 microliters of PBS-T three times. Then incubate the egg chambers with PBS-T overnight at 4 degrees Celsius with gentle rotation to permeabilize the egg chambers.

The next morning, add 500 microliters of PBS-T with 5 micrograms of blue nuclear dye, such as Hoechst, to the egg chambers. Let them incubate with gentle rotation at room temperature for one to two hours, but no longer, or the signals will become non-specific. Next, wash the egg chamber with PBS-T three times for 10 minutes per wash.

Then, using a fine pipette, aspirate off all the PBS-T and apply 200 microliters of anti-bleaching mounting agent. The tissues float on the top before they have fully absorbed the mounting agent. Incubate the egg chamber in the agent at 4 degrees Celsius for three hours to overnight.

After fully absorbing the mounting agent, the tissues sink to the bottom of the tubes and are ready to be mounted. To begin, apply petroleum jelly on the glass slide or the cover slip to avoid destroying the egg chambers by over-compression. To mount the stained egg chambers, transfer them with 200 microliters of mounting agent to a glass slide and cover them with a 20 square millimeter glass cover slip.

Seal the cover slip with nail polish and proceed with using confocal microscopy. In the egg chambers of the ovary in the healthy CaspaseTracker flies there was no CaspaseTracker biosensor activity, indicated by a lack of green and red fluorescence. However, after subjecting the flies to cell death-inducing environmental stress, such as protein starvation for three days, the egg chambers underwent the cell death process with caspase activation, which induced RFP and GFP biosensor signals.

In contrast, mutation of the caspase cleavage site in the control caspase-insensitive DQVA flies abolished biosensor sensitivity, indicating that the biosensor signals depend on caspase activity. Then the starved, caspase-sensitive biosensor flies were given protein food for three days. The egg chambers of the recovered flies did not express the RFP transient caspase reporter, indicating no recent or ongoing caspase activity.

Importantly, the recovered egg chambers only expressed GFP, indicating that they had reversed the cell death process after caspase activation. Anastasis is also observed in vivo after transient cell death-inducing temperature stress. In response to cold shock the dying egg chambers express the RFP and GFP biosensor markers, indicating recent or ongoing caspase activity, represented by the RFP signal, and past caspase activity, represented by the GFP signal.

However, after relieving stress by returning to normal culture condition for three days, the egg chambers in the recovered flies displayed only the GFP caspase reporter, indicating that the cells in these egg chambers underwent anastasis at a point after caspase activation. Notably, GFP CaspaseTracker signal reveals that multiple cell types within the egg chambers are capable of reversing the cell death process and repairing the damage from caspases, including germ line cells, such as oocytes and nurse cells, and somatic follicle cells. Interestingly, GFP also labelled cells in the germarium, which contain stem cells, along with its associated egg chambers in the same ovarial chain.

Therefore, these GFP-positive egg chambers may have been derived from stem cells that underwent anastasis. Here are images showing the tissues that display the CaspaseTracker biosensor activity without exposure to environmental stress. These are the mouth parts, foregut, crop, midgut, hindgut, Malpighian tubules, anus, oviduct, and ovaries from a dissected, newly eclosed, Day 0 female fly.

Confocal microscopy reveals recent caspase activity, as indicated by RFP, and the past caspase activity, indicated by GFP. Enlarged images show the recent or ongoing and past caspase activity at the optic lobe, cardia, Malpighian tubules, hindgut, and the muscle walls on egg chambers that co-localized with F-actin stain. Some tissues, such as the oviduct, only demonstrate past caspase activity.

These biosensor activities suggest potential anesthetic activity during embryonic development or normal homeostasis. It could also indicate current or past non-apoptotic caspase activity. Please see the manuscript for more detail.

The in vivo CaspaseTracker biosensor specifically reports caspase activity as the insensitive CaspaseTracker with the non-caspase-cleavable sequence DQVA has no biosensor activity. The only signal here is the non-specific signal of auto fluorescence from pigments, cuticle, and fat bodies. After watching this video, you should have a general understanding of how we can use the CaspaseTracker biosensor system to detect and track anastasis in live animals.

These biosensors can facilitate the studies of the currency and known function of anastasis. Their identification of reach has a wide-range of physiological, pathological, and therapeutic applications. While these biosensors can detect reversal of apoptosis after caspase activation, it can also detect the recovery of the cells after they attempt other forms of cell death that directly or indirectly involve caspase activities.

When designing an experiment, it is very important to include reciprocal controls to distinguish the fluorescent biosensor signal that tracks anesthetic cells from the dying cells with ongoing caspase activities. The healthy cells with the current or past known apoptotic caspase activities and the non-specific auto fluorescence signal from the pigment, fat, and critical. Please find the detailed discussion in the PDF version of this manuscript.