Name: detecting anastasis in vivo by caspasetracker biosensor
Uploaded: 2018-02-01T12:30:00
Description: Watch this Scientific Journal Video about Detecting Anastasis In Vivo by CaspaseTracker Biosensor at JoVE.com

We thank Darren Obbard for Drosophila image in Figure 3C and in video manuscript; J. Marie Hardwick, Wade Gibson, and Heather M. Lamb for valuable discussion of this manuscript. This work was supported by a Sir Edward Youde Memorial Fellowship (H.L.T.), Dr. Walter Szeto Memorial Scholarship (H.L.T.), Fulbright grant 007-2009 (H.L.T.), Life Science Research Foundation fellowship (H.L.T.), and NCI K22 grant CA204458 (H.L.T.). Ho Lam Tang was a Shurl and Kay Curci Foundation Fellow of the Life Sciences Research Foundation (2014-2017).

1) Preparation of CaspaseTracker Biosensor Flies
<ol>
	<li>Anesthetize flies with CO2, and use a paintbrush to transfer 7 to 10 caspase-sensitive Gal4 (DQVD)19 virgin females and 7 to 10 G-Trace53 Gal4 reporter young male flies (or vice versa) in the same vial with fly food and fresh yeast paste. 
	NOTE: Cross of Caspase-sensitive (DQVD) Gal4 and G-Trace flies will produce CaspaseTracker progeny flies. Cross of Caspase-insensitive...

The CaspaseTracker dual biosensor system is a novel and unique tool that allows detection of recent or ongoing caspase activity, and tracking of cells that have reversed cell death process and survive after experiencing caspase activity in vivo. While caspase activity has been traditionally assumed as a hallmark of apoptosis, growing studies reveal that non-apoptotic caspase activity plays potential roles in diverse normal cell functions, such as regulation of neuronal activity<s...

Programmed cell death, such as apoptosis, plays an essential role in embryonic development and normal homeostasis by eliminating unwanted, injured, or dangerous cells in multicellular organisms1,2,3. The loss of balance between cell death and survival can lead to fatal consequences such as cancer, heart failure, autoimmunity, and degeneration4,5,6,7,8. Activation of executioner caspases has traditionally been considered as the &#8220;point of no return&#8221; in apoptosis9,10,11, as it triggers rapid and massive cellular demolition12,13,14,15,16. Challenging this general dogma, we demonstrated that cultured dying primary cells and cancer cells can recover not only after caspase activation, but also following important cell death hallmarks including plasma membrane blebbing, cell shrinkage, mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, nuclear and chromatin condensation, DNA damage, nuclear fragmentation, cell surface exposure of phosphatidylserine (PS), and formation of apoptotic bodies17,18,19,20,21. We propose that anastasis is an intrinsic cell recovery phenomenon, as dying cells can recover after removal of cell death stimuli17,18,19,20,21. We coined the term &#8220;Anastasis&#8221; (&#913;&#957;&#945;&#963;&#964;&#940;&#963;&#951;&#962;)18, which means &#8220;rising to life&#8221; in Greek, to describe this unexpected cell recovery phenomenon. Our observation of anastasis is further supported by recent independent studies that also reveal recovery of cells after phosphatidylserine externalization22,23,24, limited mitochondrial outer membrane permeabilization25,&#160; activation of mixed lineage kinase-like (MLKL), and cell shrinkage26.
Characterizing the mechanisms regulating anastasis will have paradigm-shifting physiological, pathological, and therapeutic implications. Anastasis could represent a previously unknown cytoprotective mechanism to rescue or preserve important postmitotic cells and tissues that are difficult to replace, and possibly account for heart failure reversal by ventricular unloading with left ventricular assist devices (LVADs)27,28, recovery of photoreceptor cells after transient exposure of excessive light29,30,31, or repair of neurons after brain injury32. If so promoting anastasis could enhance cell and tissue recovery. Conversely, anastasis could be an unexpected escape tactic used by cancer cells to survive cell-death-inducing therapy, causing cancer recurrence17,18. Therefore, suppressing anastasis in dying cancer cells during and after cancer treatment could be a novel therapeutic strategy to cure cancers by preventing their relapse.
During the process of anastasis, we have found that some recovered cells acquired permanent genetic alterations and underwent oncogenic transformation, likely due to DNA damage incurred during apoptosis18,20,21. Reversing the death process of DNA-damaged cells could be a mechanism of tumorigenesis, potentially underlying the observation that repeated tissue injury increases the risk of cancer in a variety of tissues, such as chronic thermal injury in the esophagus induced by the consumption of very hot beverages33,34,35, liver damage due to alcoholism36,37, tumor evolution after genotoxic cancer therapy38,39,40, and development of new cancers from normal tissues that arise during the intervals between cycles of anti-cancer therapy41,42,43,44. If true, targeting anastasis could prevent or arrest cancer development and progression. We have found that starvation-induced dying germ cells undergo anastasis in re-fed Drosophila19.&#160; If anastasis occurs in germ cells with DNA damage, it could be account for the observation that prolonged environmental stress promotes development of genetic diseases. For example, famines contribute to the development of transgenerational inheritable diseases such as diabetes and coronary heart diseases45. Therefore, understanding anastasis could lead to strategies for the prevention of developing inheritable diseases caused by this potential mechanism.
To harness the discovery of anastasis and direct it to develop innovative therapies, it is essential to study the cause and consequence of anastasis in live animals. However, it is technically challenging to identify and track anastatic cells in vivo, because the cells that recovered from cell death process appear morphologically indistinguishable from normal healthy cells, and there is no biomarker of anastasis identified yet17,18,21. To address these problems, we recently developed a new in vivo caspase biosensor designated &#8220;CaspaseTracker&#8221;19, to identify and track cells that survive apoptosis after caspase activation19,46, the hallmark of apoptosis10,14. Distinguishing it from the &#8220;real-time&#8221; caspase biosensors such as SCAT12,47, Apoliner48, CA-GFP49, ApoAlert18,50, C3AIs51 and iCasper52 that detect on-going caspase activity, the CaspaseTracker biosensor additionally features the ability to permanently label cells that express caspase activity even transiently. Therefore, the CaspaseTracker biosensor enables long term tracking of anastasis after reversal of caspase-mediated cell death process in vivo.

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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;width:273px;">Vectashield mounting medium</td>
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			<td style="width:225px;">H-1000</td>
			<td>Antifade mounting medium</td>
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			<td height="21" style="height:21px;">Vectashield mounting medium (with DAPI)</td>
			<td style="width:229px;">Vector Products</td>
			<td style="width:225px;">H-1200</td>
			<td>Antifade mounting medium with DAPI</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>
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			<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 height="21" style="height:21px;">Mitotracker Red CMXRos&#160;</td>
			<td style="width:229px;">Molecular Probes</td>
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			<td>Mitochondria stain</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;width:273px;">Bovine Serum Albumin (BAS)</td>
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			<td style="width:225px;">A8806</td>
			<td>Blocking agent for immunostaining</td>
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			<td height="21" style="height:21px;width:273px;">Phosphate Buffered Saline&#160;</td>
			<td style="width:229px;">VWR</td>
			<td style="width:225px;">114-056-101</td>
			<td>Medium for washing and immunostaining</td>
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			<td height="21" style="height:21px;">Triton&#8482; X-100</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:225px;">T8787</td>
			<td>Detergent for cell permeabilization</td>
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			<td height="21" style="height:21px;width:273px;">Name</td>
			<td style="width:229px;">Company</td>
			<td style="width:225px;">Catalog Number</td>
			<td>Comments</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>
		</tr>
		<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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		<tr height="42">
			<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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detecting anastasis in vivo by caspasetracker biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

While time-lapse live cell microscopy is a reliable method to tract anastasis in cultured cells20, it is challenging to identify which cells have undergone anastasis in animals, because the recovered cells appear morphologically indistinguishable from normal healthy cells that have not attempted cell death. For example, human cervical cancer HeLa cells display morphological hallmarks of apoptosis1,2,14, s...

Watch this Scientific Journal Video about Detecting Anastasis In Vivo by CaspaseTracker Biosensor at JoVE.com

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

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