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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

Anastasis (Greek for “rising to life”) 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.

Introduction

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 “point of no return” 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 “Anastasis” (Αναστάσης)18, which means “rising to life” 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,  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.  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 “CaspaseTracker”19, to identify and track cells that survive apoptosis after caspase activation19,46, the hallmark of apoptosis10,14. Distinguishing it from the “real-time” 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.

Protocol

1) Preparation of CaspaseTracker Biosensor Flies

  1. 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.
  2. 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 °C. Continue to incubate the original vial at 18 °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 °C can reduce non-specific signal of CaspaseTracker biosensor (see discussion).
  3. 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.

2) Application of Transient Cell Death Induction to CaspaseTracker Biosensor Flies

  1. 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 °C to allow egg chamber production by oogenesis.
    NOTE: Keeping the female with male flies might enhance egg chamber production.
  2. To induce egg chambers to undergo apoptosis by cold shock, transfer the female flies to new empty vial, which is then placed at -7 °C for 1 h.
    NOTE: Cold shock injures cells by inducing plasma membrane rupture55,56.
  3. 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 °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.
  4. Transfer the stressed flies back to a new vial with fresh fly food and fresh yeast paste for 3 days at 18 °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.

3) Fixation and Staining of Dissected Egg Chambers for Imaging

  1. 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.
  2. 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.
  3. 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.
  4. Incubate the egg chambers with PBST for 1 to 2 h at room temperature or overnight at 4 °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.
  5. Remove the PBST by pipetting, and then apply 0.5 mL of 10 μg/mL of blue nuclear Hoechst dye in PBST to egg chambers for 1 to 2 h 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 μL 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.
  6. 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 incubation with gentle rotation between each washing step.
  7. Remove all PBST with fine pipette, and then apply 200 μL anti-bleaching mounting agent (see materials) to incubate the egg chambers at room temperature for 3 h or overnight at 4 °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.
  8. Mount the stained egg chambers by transferring them with 200 μL 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.
  9. 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.

Results

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...

Discussion

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

Disclosures

The authors have nothing to disclose.

Acknowledgements

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).

Materials

NameCompanyCatalog NumberComments
CONSUMABLES AND REAGENTS
Vectashield mounting mediumVector ProductsH-1000Antifade mounting medium
Vectashield mounting medium (with DAPI)Vector ProductsH-1200Antifade mounting medium with DAPI
ForcepsTed Pella#505 (110mm, #5)Dumont tweezer biology grade, stainless steel
Hanging Drop SlidesFisher Scientific12-565BGlass slides
Hoechst 33342Molecular ProbesH1399DNA stain
Mitotracker Red CMXRos Molecular ProbesM-7512Mitochondria stain
Cleaved caspase-3 (Asp175) antibodyCell Signaling Technology#9661Stain for active fragment of caspase-3
Bovine Serum Albumin (BAS)Sigma-AldrichA8806Blocking agent for immunostaining
Phosphate Buffered Saline VWR114-056-101Medium for washing and immunostaining
Triton™ X-100Sigma-AldrichT8787Detergent for cell permeabilization
NameCompanyCatalog NumberComments
EQUIPMENT
LSM780 confocal microscopeCarl ZeissN/AImaging
Carl Zeiss Stereomicroscope Stemi 2000 Carl ZeissN/ADrosophila dissection
AmScope Fiber Optic Dual Gooseneck Microscope Illuminator, 150WAmScopeWBM99316 Light source

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