Name: in vivo biosensor tracks non-apoptotic caspase activity in drosophila
Uploaded: 2016-11-27T13:00:00
Description: Watch this Scientific Journal Video about In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila at JoVE.com

We thank Polan Santos and Darren Obbard for Drosophila illustrations in Fig. 2a, Marcelo Jacobs-Lorena for use of the JHMRI insectary. This work was supported by the Life Science Research Foundation fellowship (H.L.T.), University Grants Committee of the Hong Kong AoE/B-07/99 (M.C.F.), and NIH grants NS096677,&#160;NS037402 and NS083373 (J.M.H.). Ho Lam Tang is a Shurl and Kay Curci Foundation Fellow of the Life Sciences Research Foundation.

1. Preparation of CaspaseTracker Flies
<ol>
	<li>To prepare CaspaseTracker (DQVD) flies for experiments, perform this cross: UBI-CaspaseTracker x G-TRACE (UAS-RFP; UAS-FLP; Ubi&#62;Stop&#62;GFP-nls), by transferring 7-10 virgin female (or male) flies carrying the caspase biosensor substrate mCD8-DIAP1-Gal4 driven by the ubiquitin promoter35 together with the same number of male (or female) G-TRACE flies, which have the second chromosome CyO balancer to avoid lethality of the homozygous comb...

Here we illustrate the construction and inner workings of CaspaseTracker that facilitate detection of widespread basal caspase activity in healthy tissues. The critical steps for detecting non-apoptotic caspase activity in vivo are: 1) generating flies with the biosensor transgene, 2) verifying caspase-specific reporter function with appropriate controls, 3) practicing dissection techniques to observe all internal organ systems of adult Drosophila, and 4) distinguishing biosensor activity from autofluor...

Caspases are cysteine proteases that mediate apoptotic cell death by cleaving many intracellular proteins after key aspartate residues. For example, initiator caspases activate effector caspases, derepress DNA nucleases, cleave cytoskeletal components and alter the lipid composition of cell membranes to rapidly dismantle cells and stimulate their recognition and engulfment by neighboring cells that dispose of the cell corpses.1-4 It is estimated that billions of cells die per day in the human body, and apoptosis is an important mechanism of chemotherapy-induced tumor cell death.5 A different set of caspases can cause cell death by distinct non-apoptotic processes to stimulate innate immunity.6 Therefore, most research on caspases has focused on their pro-death functions.
Interestingly, early evidence in the field revealed that the same caspases responsible for promoting cell death also have non-death functions. Pioneering studies have demonstrated that caspases are involved in diverse cellular functions in healthy cells, including the regulation of cell proliferation and migration during embryogenesis.7-9 Caspases are required for spermatid individualization in Drosophila10,11, for blocking an alternative necroptotic cell death pathway in mice12,13, and for microRNA processing in C. elegans.14,15 In perhaps the longest-lived cells, neurons, caspases and other apoptotic machinery are implicated in the regulation of neuronal activity by pruning synaptic endings, a process believed to be essential to strengthen other synapses for learning and memory.16-18 It is possible that caspases facilitate synaptic pruning by a type of mini-apoptosis of tiny neuronal projections without whole cell death.19 However, caspases may have alternative functions unrelated to apoptosis-like events.20,21 Dual roles in life and death are not unique to caspases; BCL-2 family proteins and cytochrome c have roles in cellular energetics in healthy cells but are also part of the core apoptotic pathway that is activated by many types of cell stress.22-25 Although not proven, it seems logical that evolution has linked day-jobs to death-jobs within the same molecules to ensure timely elimination of unfit or undesirable cells.
At present, the molecular mechanisms of non-apoptotic caspase activity are not understood, and the extent of non-apoptotic caspase activity during embryonic development and in adult tissues is also not known. A major challenge is the difficulty in distinguishing day-jobs from death-jobs of caspases. In contrast to apoptosis and pyroptosis, when caspase activity is amplified by a proteolytic cascade, the day-jobs of caspases are expected to occur at much lower levels of enzymatic activity, likely below detection by many available technologies.
Prior to the work presented here, others developed a variety of caspase biosensors for different purposes. The SCAT biosensors (e.g., ECFP-DEVD-Venus) rapidly detect real-time caspase activity in cultured cells and animal tissues using FRET.26,27 Upon caspase cleavage, the nuclear-targeted GFP moiety of Apoliner (mCD8-RFP-DQVD-nucGFP) undergoes subcellular relocalization within minutes when its plasma membrane-tether is cleaved by caspases.28 Similarly, ApoAlert-pCaspase3-Sensor (NES-DEVD-YFP-NLS) relocalizes from the cytosol to the nucleus upon caspase cleavage.29,30 More recently, the chromophore in iCasper was cleverly engineered to fluoresce when cleaved by caspases, permitting detection of biosensor activity in real time in neurons of Drosophila embryos, but primarily in association with developmental cell death.31 Caspase-dependent death of olfactory neurons during aging was demonstrated by immuno-detection of the caspase-cleaved form of CPV biosensors (e.g., mCD8-PARP-Venus).32,33 Importantly, the activated form of caspase-3 was detected in the absence of cell death by sensitive immunostain in spines of cultured neurons, and in the soma using the caspase-dependent fluorescence of the nuclear CellEvent reporter dye, but difficulties were encountered due to photo-toxicity, although cell death was delayed until after spine elimination.19 Thus, new caspase biosensors are needed to detect and track cells with basal caspase activity in vivo.
To overcome these difficulties, we generated a novel dual color caspase biosensor, designated CaspaseTracker. This strategy combines a modified version of the Drosophila caspase-sensitive Apoliner biosensor28 with the Drosophila G-TRACE FRT recombinase system34 to permanently label and track cells in vivo.35 The Gal4-activated G-TRACE system allows very low levels of caspases to activate CaspaseTracker, resulting in RFP expression in the cytoplasm and permanent nuclear-targeted GFP expression in any cell that has ever experienced caspase activity.35 This system can label cells throughout life in whole animals using Drosophila melanogaster, a tractable and widely used model system for the study of caspases and cell death.36-38

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			<td height="21" style="height:21px;">Vectashield</td>
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			<td style="width:225px;">H-1000</td>
			<td>Mounting medium</td>
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			<td height="21" style="height:21px;">Forceps</td>
			<td style="width:229px;">Ted Pella</td>
			<td style="width:225px;">#505 (110mm, #5)</td>
			<td>Dumont tweezer biology grade, stainless steel</td>
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			<td height="21" style="height:21px;">Hanging Drop Slides</td>
			<td style="width:229px;">Fisher Scientific</td>
			<td style="width:225px;">12-565B</td>
			<td>Glass slides</td>
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			<td height="21" style="height:21px;">Hoechst 33342</td>
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			<td>DNA stain</td>
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			<td>Actin stain</td>
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			<td height="42" style="height:42px;width:273px;">Rat-Elav-7E8A10 anti-elav antibody</td>
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			<td style="width:225px;">Antibody Registry ID:&#160; AB_528218&#160;</td>
			<td>Stain for Drosophla pan-neuronal ELAV</td>
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			<td height="21" style="height:21px;width:273px;">Cleaved caspase-3 (Asp175) antibody</td>
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			<td>Stain for active fragment of caspase-3</td>
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			<td height="21" style="height:21px;">ProLong Gold antifade reagent</td>
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			<td>to preserve fluorophores&#160;</td>
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			<td>for dissection plates</td>
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			<td height="21" style="height:21px;width:273px;">EQUIPMENT</td>
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			<td height="21" style="height:21px;">LSM780 confocal microscope</td>
			<td style="width:229px;">Carl Zeiss</td>
			<td style="width:225px;">N/A</td>
			<td>Imaging</td>
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			<td height="21" style="height:21px;width:273px;">Carl Zeiss Stereomicroscope Stemi 2000&#160;</td>
			<td style="width:229px;">Carl Zeiss</td>
			<td style="width:225px;">N/A</td>
			<td>Drosophila dissection</td>
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			<td height="42" style="height:42px;width:273px;">AmScope Fiber Optic Dual Gooseneck Microscope Illuminator, 150W</td>
			<td style="width:229px;">AmScope</td>
			<td style="width:225px;">WBM99316&#160;</td>
			<td>Light source</td>
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in vivo biosensor tracks non-apoptotic caspase activity in drosophila

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by available technologies, apoptosis is generally assumed to occur shortly thereafter. Caspases can cleave many functional and structural components to cause rapid and complete cell destruction within a few minutes. However, accumulating evidence indicates that in normal healthy cells the same caspases have other functions, presumably at lower enzymatic levels. Studies of non-apoptotic caspase activity have been hampered by difficulties with detecting low levels of caspase activity and with tracking ultimate cell fate in vivo. Here, we illustrate the use of an ultrasensitive caspase reporter, CaspaseTracker, which permanently labels cells that have experienced caspase activity in whole animals. This in vivo dual color CaspaseTracker biosensor for Drosophila melanogaster transiently expresses red fluorescent protein (RFP) to indicate recent or on-going caspase activity, and permanently expresses green fluorescent protein (GFP) in cells that have experienced caspase activity at any time in the past yet did not die. Importantly, this caspase-dependent in vivo biosensor readily reveals the presence of non-apoptotic caspase activity in the tissues of organ systems throughout the adult fly. This is demonstrated using whole mount dissections of individual flies to detect biosensor activity in healthy cells throughout the brain, gut, malpighian tubules, cardia, ovary ducts and other tissues. CaspaseTracker detects non-apoptotic caspase activity in long-lived cells, as biosensor activity is detected in adult neurons and in other tissues at least 10 days after caspase activation. This biosensor serves as an important tool to uncover the roles and molecular mechanisms of non-apoptotic caspase activity in live animals.

There are two key components that allow CaspaseTracker to detect caspase activity in normal healthy cells (Figure 1a). The first of these is a 146 amino acid caspase-cleavable polypeptide modeled after the caspase biosensor Apoliner (Figure 1b).28 This polypeptide is derived from DIAP1 (Drosophila inhibitor of apoptosis) containing a single naturally occurring caspase site that is cleaved during apoptosis typically by the caspase DrICE...

Watch this Scientific Journal Video about In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila at JoVE.com

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

To detect healthy cells in whole animals that contain low levels of caspase activity, the highly sensitive biosensor designated CaspaseTracker was generated for Drosophila. Caspase-dependent biosensor activity is detected in long-lived healthy cells throughout the internal organs of adult animals reared under optimized conditions in the absence of death stimuli.

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by ...

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.

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