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;width:273px;">CONSUMABLES AND REAGENTS</td>
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			<td height="21" style="height:21px;">Vectashield</td>
			<td style="width:229px;">Vector Products</td>
			<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>
			<td style="width:229px;">Molecular Probes</td>
			<td style="width:225px;">H1399</td>
			<td>DNA stain</td>
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			<td height="21" style="height:21px;">Alexa Fluor 633 Phalloidin</td>
			<td style="width:229px;">Molecular Probes</td>
			<td style="width:225px;">A22284</td>
			<td>Actin stain</td>
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			<td height="42" style="height:42px;width:273px;">Rat-Elav-7E8A10 anti-elav antibody</td>
			<td style="width:229px;">&#160;Developmental Studies Hybridoma Bank (DSHB)</td>
			<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>
			<td style="width:229px;">Cell Signaling Technology</td>
			<td style="width:225px;">#9661</td>
			<td>Stain for active fragment of caspase-3</td>
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			<td height="21" style="height:21px;">ProLong Gold antifade reagent</td>
			<td style="width:229px;">Life Technologies</td>
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			<td>to preserve fluorophores&#160;</td>
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			<td height="21" style="height:21px;width:273px;">ProLong Diamond Antifade Mountant</td>
			<td style="width:229px;">Life Technologies</td>
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			<td>to preserve fluorophores&#160;</td>
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			<td height="21" style="height:21px;width:273px;">SylGard 182 Silicone Elastomer Kit</td>
			<td style="width:229px;">Dow Corning&#160;</td>
			<td style="width:225px;">Product code: 0001023934</td>
			<td>for dissection plates</td>
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			<td height="21" style="height:21px;width:273px;">EQUIPMENT</td>
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			<td></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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		<tr height="21">
			<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 ...

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