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 (DQVA)19 Gal4 and G-Trace flies will provide negative control flies (see discussion). Fresh yeast paste serves as protein source to enhance egg production, so that increases number of progeny. Select virgin females and young male flies according to their phenotypes54.</li>
	<li>Incubate the flies at 18 degrees Celsius (oC) during the cross for 3 to 7 days, and then transfer the flies to a new vial to set up a new cross at 18 &#176;C. Continue to incubate the original vial at 18 &#176;C until progeny flies eclose. 
	NOTE: Transfer the parent flies to new vials to avoid overcrowding of progeny at the original vial. Parent flies can produce progeny with fresh food and yeast paste at the first 2 to 3 switches, and then the productivity will significantly decrease with time. Raising flies 18 &#176;C can reduce non-specific signal of CaspaseTracker biosensor (see discussion).</li>
	<li>Select progeny flies with correct phenotypes54 for following experiments. 
	NOTE: The transgenes of both caspase-sensitive Gal4 and G-Trace here are located at the second chromosome, balanced with CyO balancer. Select the non-curly wing progeny (without CyO), which has both transgenes of caspase-sensitive Gal4 and G-Trace.</li>
</ol>
2) Application of Transient Cell Death Induction to CaspaseTracker Biosensor Flies
<ol>
	<li>Transfer 10 to 20 newly eclosed female flies to a new vial with fresh fly food and fresh yeast paste for 1 day at 18 &#176;C to allow egg chamber production by oogenesis. 
	NOTE: Keeping the female with male flies might enhance egg chamber production.</li>
	<li>To induce egg chambers to undergo apoptosis by cold shock, transfer the female flies to new empty vial, which is then placed at -7 &#176;C for 1 h. 
	NOTE: Cold shock injures cells by inducing plasma membrane rupture55,56.</li>
	<li>To induce egg chambers to undergo apoptosis by protein starvation, transfer the female flies to a new vial with 8% sucrose and 1% agar food at 18 &#176;C for 3 days. 
	NOTE: Protein starvation (non-protein food) can trigger egg chambers to undergo apoptosis57,58,59 and autophagy60,61. Switch flies to a new vial with 8% sucrose and 1% agar food every day to keep optimal condition of the sucrose fly food.</li>
	<li>Transfer the stressed flies back to a new vial with fresh fly food and fresh yeast paste for 3 days at 18 &#176;C to allow them to recover. Dissect the starved and the starved-recovered flies to obtain egg chambers at ovaries as described62. 
	NOTE: To dissect Drosophila to obtain ovaries, anesthetize flies with CO2, and use 2 pairs of forceps to remove fly head, and use the forceps to pull the base of the abdomen to remove the ovaries of the flies.</li>
</ol>
3) Fixation and Staining of Dissected Egg Chambers for Imaging
<ol>
	<li>Transfer the dissected egg chambers together with around 0.5 mL phosphate buffered saline (PBS) to 1 mL centrifuge tubes. Allow the eggs to settle down. 
	NOTE: Coat the plastic pipette tips with 1% bovine serum albumin (BSA) dissolved in water or PBS to prevent the egg chambers to stick on the plastic surface of the tips. Perform the following procedures in dark to avoid photobleaching of red fluorescent protein (RFP, also known as DsRed) and green fluorescent protein (GFP) in the egg chambers.</li>
	<li>Remove the PBS by pipetting, and then apply 0.5 mL 4% paraformaldehyde in PBS to fix the egg chambers at room temperature in dark for 20 to 30 min. 
	NOTE: Apply gentle rotation in this and the following incubation steps.</li>
	<li>Remove the paraformaldehyde by pipetting, and then washed the egg chamber with 0.5 mL PBST (PBS + 0.1% Triton X-100) for 3 times. 
	NOTE: Prolonged fixation could reduce the RFP and GFP signals.</li>
	<li>Incubate the egg chambers with PBST for 1 to 2 h&#160;at room temperature or overnight at 4 &#176;C with gentle rotation to permeabilize the egg chambers. 
	NOTE: PBST can also avoid egg chambers to stick to the non-BSA coated plastic surface.</li>
	<li>Remove the PBST by pipetting, and then apply 0.5 mL of 10 &#956;g/mL of blue nuclear Hoechst dye in PBST to egg chambers for 1 to 2 h&#160;at room temperature to stain for nucleus. 
	NOTE1: Avoid prolonged incubation with nuclear dye as this will increase non-specific signal. 
	NOTE2: Alternative approach to stain nucleus without Hoechst is to add 200 &#956;L anti-bleaching mounting agent with DAPI (see materials) and incubate overnight63, before mounting the tissues on glass cover slip as described at protocol 3.8. 
	NOTE3: Perform the staining and the following procedures in dark to avoid photobleaching.</li>
	<li>Remove the nuclear dye by pipetting, and then apply 0.5 mL PBST to wash the egg chambers in the 1 mL centrifuge tubes for 3 times, with 10 to 20 min&#160;incubation with gentle rotation between each washing step.</li>
	<li>Remove all PBST with fine pipette, and then apply 200 &#956;L anti-bleaching mounting agent (see materials) to incubate the egg chambers at room temperature for 3 h&#160;or overnight at 4 &#176;C until the egg chambers sink to the bottom of the tube. 
	NOTE: Tissues that have fully absorbed the mounting agent sink to the bottom of the tube.</li>
	<li>Mount the stained egg chambers by transferring them with 200 &#956;L anti-bleaching mounting agent on a pre-cleaned glass slide for imaging by pipetting, cover the egg chambers with a 20 x 20 mm pre-cleaned glass cover slip, and seal the cover slip on glass slide by putting nail polish at the edge of the cover slip. 
	NOTE1: Pre-clean the glass slide and cover slip with water or 70% ethanol. 
	NOTE2: Apply petroleum jelly between the glass slide and the cover slip to avoid destroying the egg chambers by overcompression.</li>
	<li>Image the egg chambers using fluorescence or confocal microscope, using a 20x, NA 0.8 Plan-Apochromat objective, with excitation light wavelength 405 nm for nuclear staining (detect emission ~461 nm), 561 nm for RFP (ongoing or recent caspase activity) signal (detect emission ~590 nm), and 488 nm for GFP (past caspase activity) signal (detect emission ~518nm). 
	Note: See our published protocols for microscopy20,64.</li>
</ol>

The authors have nothing to disclose.

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 activity79,80, learning and memory81,82,83,84, suppression of necroptotic cell death85,86, spermatid individualization87,88, microRNA processing89, cell proliferation90, and cell fate patterning91. In addition to apoptosis and anastasis, the CaspaseTracker biosensor system can therefore detect non-apoptotic caspase activity, which is present in the brain and optic lobes, cardia, gut, Malpighian tubules, trachea, muscles, and other tissues of Drosophila19,46,64. This biosensor signal could also represent the current or past anastatic activity during embryo development or normal homeostasis in these tissues. To study anastasis after transient cell death-inducing environmental stress in live animals, it is critical to choose tissues with cells that exhibit no caspase biosensor activity under normal physiological conditions, but that can be induced to undergo caspase activation by transient cell death induction. Egg chambers are ideal for this purpose, because they typically have no caspase activity from germarium to stage 10 during oogenesis58,59,92.
Exposing female Drosophila to transient environmental stresses, such as protein starvation and cold shock, can efficiently trigger caspase activation and cell death in egg chambers19,57,58,78. Critical steps within the protocol include avoiding prolonged cell death induction to flies. The optimized conditions of protein starvation (8% sucrose in 1 % agar for 3-days) and cold shock (1 h&#160;at -7 oC) to female flies can trigger caspase-activated cell death process in egg chambers, and allow them to recover after the stressed flies are returned to normal condition19. Prolonged treatment with a cell death stimulus can trigger more egg chambers to undergo the cell death process, but the recovery rate is also reduced, presumably because the dying egg chambers experience massive damage beyond repair.
An additional critical step in this protocol is to reduce the CaspaseTracker background signal in egg chambers by crossing, raising and maintaining the CaspaseTracker flies at a lower temperature, such as 18 oC. While the majority of egg chambers from optimally reared flies do not display caspase activity in the germarium through stage 10 during oogenesis58, about 1% of egg chambers could exhibit caspase biosensor activity without cell death induction19. This may reflect the normal attrition rate due to innate errors or may be triggered unintentionally during oogenesis by standard laboratory conditions. As Gal4 displays less activity in flies at low temperature93, raising flies at 18 oC, rather than at room temperature, can reduce the endogenous signal that activates the CaspaseTracker system. Alternatively, switching flies to a higher temperature, such as 29 oC, can increase the sensitivity of CaspaseTracker system, due to increase in the Gal4 activity93, and potentially other endogenous temperature-dependent enzymatic activities.
It is important to distinguish the CaspaseTracker-positive cells that undergo cell death process or anastasis from cells that exhibit non-apoptotic caspase activity. Apoptotic cells express RFP (transient marker for on-going or recent caspase activity), and often GFP (permanent maker for past caspase activity) in treatment of apoptosis induction, as these dying cells have ongoing caspase activity that cleave-activated Gal4, which then activates the transient (Gal4 activity-dependent RFP) and permanent (Gal4 triggered FLPase-FRT mediated GFP) reporters of the G-Trace system. Apoptosis of these cells can be confirmed by morphological and biochemical hallmarks such us nuclear condensation detected by staining with nuclear dyes14,19,58, and caspase cleavage detected by immunostaining19,94. Cells that have already undergone anastasis display permanent GFP expression due to the FLPase-mediated recombination event of G-Trace system. These cells express no RFP as they have no on-going caspase activity, nor other hallmarks of apoptosis19. These cells also display normal nuclear morphology. Cells do have on-going non-apoptotic caspase activity often display both RFP and GFP expression, and also have a normal nuclear morphology19.
It can be difficult to distinguish cells that experienced anastasis from those cells that had past non-apoptotic caspase activity, because both display only GFP and have no hallmark of cell death. Therefore, careful control experiments must be included19. For examples, to study anastasis in egg chambers, it is essential to examine GFP expression in both stressed-recovered flies and non-stressed flies (negative control). Recovered flies should show more GFP-expressing cells than unstressed flies, if anastasis has occurred after caspase activation. It is also important to distinguish CaspaseTracker fluorescent signals from nonspecific auto-fluorescence, as observed in cuticle, pigments, and fat bodies19. We generated negative control biosensor flies by mutating the caspase cleavage site of the biosensor (DQVD) to a non-cleavable sequence (DQVA) and thereby rendering the control biosensor non-responsive to caspase activity19. The signal detected in the caspase sensitive (DQVD) but not in the negative control (DQVA) biosensor flies is the signal of interest, triggered by caspase activity, rather than auto-fluorescence.
Our current Drosophila dual CaspaseTracker biosensor can identify cells with &#34;recent&#34; caspase activity by expression of RFP, and cells with &#34;past&#34; caspase activity by expression of GFP19. We note that RFP is not a &#34;real-time&#34; caspase activity indicator, because it takes a few hours of reaction time for Gal4 to drive the expression of RFP in response to caspase activity. To add a &#34;real time&#34; function, the Drosophila CaspaseTracker biosensor can be combined with the recently developed iCasper biosensor52, a &#34;real-time&#34; and &#34;dark to bright&#34; in vivo biosensor that only shows its far-red signal when it is cleaved by caspases.
Apart from apoptosis, CaspaseTracker could potentially also be activated during alternative types of cell death as cells can switch cell death pathways during their course that directly or indirectly involves caspase activation, such as autophagy after starvation95,96, necrosis by the cold shock-induced plasma membrane rupture19,55,56,78, and staurosporine-induced necroptosis97,98. Our present and previous studies demonstrated that the CaspaseTracker biosensors can label cells recovered from these cell death inductions in vitro or in vivo17,18,19,20,21. As these cell death inductions alone could simultaneously activate multiple cell death pathways in the same cells, the recovery of these dying cells suggests that anastasis is a general cell recovery phenomenon that can reverse different cell death processes including apoptosis, autophagy, necrosis and necroptosis, individually or simultaneously.
The in vivo CaspaseTracker biosensor will facilitate pursuit of the yet unknown functions, mechanisms, and therapeutic implications of anastasis (Figure 4)21. To reveal the molecular signature of anastasis, we performed time-course whole-genome gene expression microarray studies to analyze mouse primary liver cells during reversal of ethanol-induced apoptosis, and interestingly, found striking changes in transcription of genes involved in multiple pathways including pro-survival, anti-oxidation, DNA damage response, histone modification, angiogenesis, cell migration, and transformation18,21. This finding is supported by our validation study of the recovery of human liver cancer HepG2 cells from apoptosis18,21, and also a recent independent RNA-sequencing study of HeLa cells in recovery of apoptosis99. Interestingly, up-regulation of some pro-survival factors found in anastatic cells are also observed in heart failure reversal, tumor progression, and cancer recurrence100,101,102, suggesting the potential participation of anastasis. To study the physiological, pathological and therapeutic potentials of anastasis, it is important to identify the anastatic cells and track their fate in small animals, as this will enable mechanistic and therapeutic studies. Our Drosophila and mammalian CaspaseTracker biosensors will be useful tools for testing the potential contributions of anastasis in normal development, homeostasis, tissue recovery, tumor evolution, cancer recurrence, and metastasis. Finding from these studies will increase our understanding of the natural role of anastasis, and offer potential to identify revolutionary new therapeutic approaches for intractable diseases by mediating cell death and survival through controlling anastasis.

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.

<table><tbody><tr><td>&lt;strong&gt;CONSUMABLES AND REAGENTS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Vectashield mounting medium</td><td>Vector Products</td><td>H-1000</td><td>Antifade mounting medium</td></tr><tr><td>Vectashield mounting medium (with DAPI)</td><td>Vector Products</td><td>H-1200</td><td>Antifade mounting medium with DAPI</td></tr><tr><td>Forceps</td><td>Ted Pella</td><td>#505 (110mm, #5)</td><td>Dumont tweezer biology grade, stainless steel</td></tr><tr><td>Hanging Drop Slides</td><td>Fisher Scientific</td><td>12-565B</td><td>Glass slides</td></tr><tr><td>Hoechst 33342</td><td>Molecular Probes</td><td>H1399</td><td>DNA stain</td></tr><tr><td>Mitotracker Red CMXRos&amp;nbsp;</td><td>Molecular Probes</td><td>M-7512</td><td>Mitochondria stain</td></tr><tr><td>Cleaved caspase-3 (Asp175) antibody</td><td>Cell Signaling Technology</td><td>#9661</td><td>Stain for active fragment of caspase-3</td></tr><tr><td>Bovine Serum Albumin (BAS)</td><td>Sigma-Aldrich</td><td>A8806</td><td>Blocking agent for immunostaining</td></tr><tr><td>Phosphate Buffered Saline&amp;nbsp;</td><td>VWR</td><td>114-056-101</td><td>Medium for washing and immunostaining</td></tr><tr><td>Triton&amp;trade; X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>Detergent for cell permeabilization</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>LSM780 confocal microscope</td><td>Carl Zeiss</td><td>N/A</td><td>Imaging</td></tr><tr><td>Carl Zeiss Stereomicroscope Stemi 2000&amp;nbsp;</td><td>Carl Zeiss</td><td>N/A</td><td>Drosophila dissection</td></tr><tr><td>AmScope Fiber Optic Dual Gooseneck Microscope Illuminator, 150W</td><td>AmScope</td><td>WBM99316&amp;nbsp;</td><td>Light source</td></tr></tbody></table>

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.

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 ...

detecting-anastasis-in-vivo-by-caspasetracker-biosensor

Universidad San Sebastian

Research

JoVE Journal

Medicine

9.2K Views.  Johns Hopkins University School of Medicine. 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.

Video: Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first to be activated in these pathways. This group includes caspase-2, -8, and -9, as well as the inflammatory caspases, caspase-1, -4, and -5. The initiator caspases are all activated by dimerization following recruitment to specific multiprotein complexes called activation platforms. Caspase Bimolecular Fluorescence Complementation (BiFC) is an imaging-based approach where split fluorescent proteins fused to initiator caspases are used to visualize the recruitment of initiator caspases to their activation platforms and the resulting induced proximity. This fluorescence provides a readout of one of the earliest steps required for initiator caspase activation. Using a number of different microscopy-based approaches, this technique can provide quantitative data on the efficiency of caspase activation on a population level as well as the kinetics of caspase activation and the size and number of caspase activating complexes on a per cell basis.

This protocol describes caspase Bimolecular Fluorescence Complementation (BiFC); an imaging-based method that can be used to visualize induced proximity of initiator caspases, which is the first step in their activation.

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first ...

Biochemistry

cAMP Analysis with cADDis: Single-Cell to High-Throughput Screening

Real-Time cAMP Dynamics in Live Cells Using the Fluorescent cAMP Difference Detector In Situ

cAMP Difference Detector In Situ (cADDis) is a novel biosensor that allows for the continuous measurement of cAMP levels in living cells. The biosensor is created from a circularly permuted fluorescent protein linked to the hinge region of Epac2. This creates a single fluorophore biosensor that displays either increased or decreased fluorescence upon binding of cAMP. The biosensor exists in red and green upward versions, as well as green downward versions, and several red and green versions targeted to subcellular locations. To illustrate the effectiveness of the biosensor, the green downward version, which decreases in fluorescence upon cAMP binding, was used. Two protocols using this sensor are demonstrated: one utilizing a 96-well plate reading spectrophotometer&#160;compatible with high-throughput screening and another utilizing single-cell imaging on a fluorescent microscope. On the plate reader, HEK-293 cells cultured in 96-well plates were stimulated with 10 &#181;M forskolin or 10 nM isoproterenol, which induced rapid and large decreases in fluorescence in the green downward version. The biosensor was used to measure cAMP levels in individual human airway smooth muscle (HASM) cells monitored under a fluorescent microscope. The green downward biosensor displayed similar responses to populations of cells when stimulated with forskolin or isoproterenol. This single-cell assay allows visualization of the biosensor location at 20x and 40x magnification. Thus, this cAMP biosensor is sensitive and flexible, allowing real-time measurement of cAMP in both immortalized and primary cells, and with single cells or populations of cells. These attributes make cADDis a valuable tool for studying cAMP signaling dynamics in living cells.

Author Spotlight: Advancing Real-Time cAMP Detection in Cells Using cADDis Biosensor

The protocol presented in this study illustrates the effectiveness of the cAMP Difference Detector In Situ in measuring cAMP through two methods. One method utilizes a 96-well plate reading spectrophotometer with HEK-293 cells. The other method demonstrates individual HASM cells under a fluorescent microscope.

cAMP Difference Detector In Situ (cADDis) is a novel biosensor that allows for the continuous measurement of cAMP levels in living cells. The biosensor is ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Bioengineering

CRISPR-Cas Synthetic Urine Biomarker Test for Cancer Diagnostics

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for traditional biofluid measurements. Synthetic biomarkers generally make use of reporters that provide readable signals in the biofluid to reflect the biochemical alterations in the local disease microenvironment during disease incidence and progression. The pharmacokinetic concentration of the reporters and biochemical amplification of the disease signal are paramount to achieving high sensitivity and specificity in a diagnostic test. Here, a cancer diagnostic platform is built using one format of synthetic biomarkers: activity-based nanosensors carrying chemically stabilized DNA reporters that can be liberated by aberrant proteolytic signatures in the tumor microenvironment. Synthetic DNA as a disease reporter affords multiplexing capability through its use as a barcode, allowing for the readout of multiple proteolytic signatures at once. DNA reporters released into the urine are detected using CRISPR nucleases via hybridization with CRISPR RNAs, which in turn produce a fluorescent or colorimetric signal upon enzyme activation. In this protocol, DNA-barcoded, activity-based nanosensors are constructed and their application is exemplified in a preclinical mouse model of metastatic colorectal cancer. This system is highly modifiable according to disease biology and generates multiple disease signals simultaneously, affording a comprehensive understanding of the disease characteristics through a minimally invasive process requiring only nanosensor administration, urine collection, and a paper test which enables point-of-care diagnostics.

Author Spotlight: Engineering Molecular Tools for Disease Detection and Imaging

This protocol describes a CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test that enables point-of-care cancer diagnostics through the ex vivo analysis of tumor-associated protease activities.

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for ...

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1&#946; and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1&#946; release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...

Immunology and Infection

Evaluation of Caspase Activation to Assess Innate Immune Cell Death

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is the initiation of innate immune programmed cell death (PCD) to eliminate infected or damaged cells and propagate immune responses. However, excess PCD is associated with inflammation and pathology. Therefore, understanding the activation and regulation of PCD is a central aspect of characterizing innate immune responses and identifying new therapeutic targets across the disease spectrum.
This protocol provides methods for characterizing innate immune PCD activation by monitoring caspases, a family of cysteine-dependent proteases that are often associated with diverse PCD pathways, including&#160;apoptosis,&#160;pyroptosis, necroptosis, and PANoptosis. Initial reports characterized caspase-2, caspase-8, caspase-9, and caspase-10 as initiator caspases and caspase-3, caspase-6, and caspase-7 as effector caspases in apoptosis, while later studies found the inflammatory caspases, caspase-1, caspase-4, caspase-5, and caspase-11, drive pyroptosis. It is now known that there is extensive crosstalk between the caspases and other innate immune and&#160;cell death molecules across the previously defined PCD pathways, identifying a key knowledge gap in the mechanistic understanding of innate immunity and PCD and&#160;leading to the characterization of PANoptosis. PANoptosis is a unique innate immune inflammatory PCD pathway regulated by PANoptosome complexes, which integrate components, including caspases,&#160;from other cell death pathways.
Here, methods for assessing the activation of caspases in response to various stimuli are provided. These methods allow for the characterization of PCD pathways both in vitro and in vivo, as activated caspases undergo proteolytic cleavage that can be visualized by western blotting using optimal antibodies and blotting conditions. A protocol and western blotting workflow have been established that allow for the assessment of the activation of multiple caspases from the same cellular population, providing a comprehensive characterization of the PCD processes. This method can be applied across research areas in development, homeostasis, infection, inflammation, and cancer to evaluate PCD pathways throughout cellular processes in health and disease.

This protocol describes a comprehensive method for assessing caspase activation (caspase-1, caspase-3, caspase-7, caspase-8, caspase-9, and caspase-11) in response to both in vitro and in vivo (in mice) models of infection, sterile insults, and cancer to determine the initiation of cell death pathways, such as pyroptosis, apoptosis, necroptosis, and PANoptosis.

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is ...

Optimized CRISPR-Based Molecular Diagnostics Protocol for Point-of-Care Use

Point-of-care CRISPR-based Diagnostics with Premixed and Freeze-dried Reagents

Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes that are suitable for use at point-of-care settings such as fast turnaround times for results, convenient simple readouts, and no requirement of complicated instruments. However, the reactions can be cumbersome to perform at the point of care due to their many components and manual handling steps. Herein, we provide a step-by-step, optimized protocol for the robust detection of disease pathogens and genetic markers with recombinase-based isothermal amplification and CRISPR-based reagents, which are premixed and then freeze-dried in easily stored and ready-to-use formats. Premixed, freeze-dried reagents can be rehydrated for immediate use and retain high amplification and detection efficiencies. We also provide a troubleshooting guide for commonly found problems upon preparing and using premixed, freeze-dried reagents for CRISPR-based diagnostics, to make the detection platform more accessible to the wider diagnostic/genetic testing communities.

Author Spotlight: Development of Simplified CRISPR-Based Tests for Rapid Detection of Infectious Diseases

Here, we present the step-by-step preparation of premixed, lyophilized recombinase-based isothermal amplification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based reactions, which can be used for the detection of nucleic acid biomarkers of infectious disease pathogens or other genetic markers of interest.

Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

Biology

Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System

Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of A. baumannii is crucial for prompt treatment, effective infection control, and curbing antibiotic resistance. However, there is no suitable method for rapid and easy on-site detection of A. baumannii. The DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) system offers a rapid, precise, and sensitive approach to A. baumannii detection by integrating the target-specific recognition capabilities of Cas12a with the isothermal amplification efficiency of Recombinase Polymerase Amplification (RPA). This protocol details the detection of A. baumannii using RPA combined with LbaCas12a endonuclease. The following steps are described in this article: extraction of DNA, selection of a specific DNA sequence, design of primer and CRISPR RNA (crRNA), construction of positive recombinant plasmid, setup of Cas12a-RPA assay, optimization of the RPA amplification system, visualization of the RPA-CRISPR/Cas12a assay using a fluorescence detection tool such as a real-time PCR instrument, and evaluation of sensitivity and specificity evaluation.

To facilitate the rapid and precise detection of Acinetobacter baumannii, we present a protocol that employs Recombinase Polymerase Amplification (RPA) in conjunction with LbaCas12a endonuclease for identifying A. baumannii infections.

Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of ...

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.

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.

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

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Summary

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Chapters in this video

Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation

Real-Time cAMP Dynamics in Live Cells Using the Fluorescent cAMP Difference Detector In Situ

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Evaluation of Caspase Activation to Assess Innate Immune Cell Death

Point-of-care CRISPR-based Diagnostics with Premixed and Freeze-dried Reagents

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila