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

Summary

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.

Abstract

Anastasis (Greek for “rising to life”) 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 for identifying and tracking anastasis in mammalian cells.

Introduction

Apoptosis (Greek for “falling to death”) is generally assumed to be a one-way process ending in cell suicide^1-7. Genetic disruption of pro-death genes results in the survival of extra cells that would otherwise die in whole animals, including cells that have already initiated the apoptosis pathway^8,9. Similarly, genetic manipulations allow healthy mammalian cells that artificially display “eat me” signals or that lose adhesiveness to their extracellular matrix to escape death by whole cell phagocytosis or entosis, respectively^10,11. However, we and others have shown that without genetic manipulation normal healthy mammalian cells and cell lines can also recover from the early stages of apoptosis^12-15. Using tools to track individual cells, we have further demonstrated recovery from late stages of apoptosis^12,13, after cells have passed important checkpoints that typically mark the “point of no return”^2-6. These checkpoints of late stage apoptosis include mitochondrial release of cytochrome c, activation of caspases, nuclear fragmentation, and formation of apoptotic bodies. We adopted a Greek compound word “anastasis”, which means “rising to life”, to describe this reversal of apoptosis at the brink of cell death^2-6.

Unless the entire dying-recovery process is observed by live cell imaging, it is challenging to distinguish cells that have undergone anastasis from cells that never experienced apoptotic events. Decades of work have revealed that the morphological features of cell suicide by apoptosis are driven by evolutionarily conserved biochemical and molecular events^16-19. These events promote self-destruction of cells to regulate developmental and homoeostatic processes in unicellular and multicellular organisms by eliminating damaged or dangerous cells^16-19. While apoptotic cells can be readily distinguished by standardized morphological, biochemical and molecular manifestations of apoptosis^1,5,6,16,20, currently there is no known marker specific to anastasis^12,13. Importantly, cells that have undergone anastasis appear to be normal healthy cells, and cells that just start reversing apoptosis appear as apoptotic dying cells^12,13. Thus, new tools are needed to conclude with certainty that a given surviving cell had previously experienced active apoptotic processes.

Apoptosis is generally assumed as an irreversible cascade because it is a rapid and massive destruction process. While it could take minutes to days for some cells to initiate apoptosis, once mitochondria have released apoptogenic factors such as cytochrome c into the cytosol ^21,22, caspases can be activated within 5 minutes^23,24, followed by cytoplasmic and nuclear condensation within 10 min^25-27, and cell death shortly thereafter^25-27. Activated caspases orchestrate apoptosis by cleaving and inactivating key structural and functional components for the purpose of cellular demolition^2,28, such as the endonuclease inhibitor DFF45/ICAD^29,30. Caspases also activate pro-apoptotic factors, such as BCL-2 family member BID, which translocates to mitochondria to promote mitochondrial release of cytochrome c^31,32. Caspase activity also results in cell surface exposure of phosphatidylserine as an “eat me” signal for promoting engulfment of dying cells by macrophages or neighbor cells through phagocytosis³³. Furthermore, apoptotic events render mitochondria dysfunctional, disrupting cellular bioenergetics and metabolism^34,35,36. Thus, recovery from such destruction seems intuitively unlikely.

Contrary to original expectations, cells can reverse the apoptotic cell death process even at a late stage. By continuously monitoring the fate of dying cells in culture, we observed the reversibility of late stage apoptosis in a range of primary cells and cell lines^12,13. Removal of the death stimulus allowed recovery from the overt features of apoptosis, such as mitochondrial fragmentation, chromatin condensation, DNA damage, plasma membrane blebbing, cell surface exposure of phosphatidylserine, release of mitochondrial cytochrome c, caspase activation, nuclear fragmentation, cell shrinkage, and formation of apoptotic bodies. These observations raise unanswered questions regarding the functions, consequences, and mechanisms of anastasis. To address these questions, a prerequisite is to reliably identify cells that have undergone anastasis. Here, we describe live microscopy methods and a caspase biosensor for detecting cells that have previously reversed late stage apoptosis and then survived.

Protocol

1. Preparation of Cells for Live Cell Imaging

1. To facilitate the detection of morphological changes, choose adherent cells such as HeLa (human cervical carcinoma) cells that are flat on the substrate to better visualize alteration of the plasma membrane and intracellular organelles.
   Note: Reversal of apoptosis has been observed in various mammalian cells^12,13, including primary mouse liver cells, primary mouse macrophages, primary rat cardiomyocytes, and also cell lines such as human embryonic kidney HEK-293T cells, African green monkey renal epithelial COS-7 cells, mouse cardiac muscle HL-1 cells, mouse NIH 3T3 fibroblasts, ferret (Mustela putoris furo) brain CRL1656 cells, human skin cancer A375 cells, human testicular cancer CRL1973 cells, human small cell lung carcinoma H446 cells, human liver cancer HepG2 cells, human breast cancer MCF7 cells, human prostate cancer PC3 cells, and human neuroblastoma SH-SY5Y cells.
2. Pre-wash glass coverslips of glass bottom cell culture dishes with absolute ethanol for cleaning and sterilization.
   Note 1: Use of glass bottom dishes is recommended for high quality differential interference contrast (DIC) microscopy, and high magnification confocal or epi-fluorescence microscopy (See Discussion).
   Note 2: For high magnifications (40X, 60/63X or 100X objectives and high numerical apertures 1.3 or 1.4), use of cell culture dishes with thinner glass bottom (thickness 0.085-0.16 mm) provides longer working distances for imaging (See Protocol 3).
3. Depending on the cell type, glass bottom culture dishes may require coating with poly-d-lysine (0.1 mg/ml), collagen (0.01% solution in 0.01 M acetic acid) and/or fibronectin (5 μg/ml) prior to seeding cells.
   Note: Optimization of the type and concentration of coating agent is required for different cell lines. HeLa cells can adhere directly on glass without coating. However, some cells, such as mouse cardiac muscle HL-1 cells, cannot adhere directly to glass surfaces, but require specific culture and coating protocols³⁷.
4. Seed ~2-3 x 10⁵ cells on 35 mm pre-coated glass bottom cell culture dishes and incubate at 37 degree Celsius (°C) with 5% CO₂ for a day to achieve 80% confluency.
   Note 1: Optimal conditions of cell density, incubation time, and culture condition can vary, depending on the cell type. Cell confluency can affect the apoptotic response of cells (See Protocol 2). At high confluency, cells could be less sensitive to the same concentrations of apoptotic stimuli.
   Note 2: Avoid over confluent cells, as high cell density can obscure morphological changes of individual cells and their organelles.

2. Application and Removal of Apoptotic Cell Stimuli

1. Shortly before use, pre-mix the apoptosis-inducing agent with 37 °C cell culture medium. Ethanol (3.6-4.5% vol/vol) served as apoptosis inducer in the present demonstration.
   Note 1: Our published and unpublished data demonstrate that cells can also reverse apoptosis that is triggered by other apoptotic stimuli^12,13, such as dimethyl sulfoxide (DMSO, 8% vol/vol for 24 hr), staurosporine (STS, 0.5 μM for 1 hr), and taxol (1 μM for 12 hr). Optimization of dosage for triggering apoptosis is required for different kinds of cells to achieve the minimum dosage that can trigger the majority of cells in the population to undergo apoptosis.
   Note 2: Pre-mixing apoptosis-inducing agents with cell culture medium is important to avoid uneven exposure of the cells to apoptotic stimuli.
2. Image a group of health cells in the cell culture dish before applying the apoptosis-inducing agent to establish baseline morphologies.
3. Remove the original medium from the cell culture dish, and then apply 2 ml of premixed apoptosis-inducing agent to the dish to trigger cells to undergo apoptosis. Incubate the cells at 37 °C with 5% CO₂ (or other normal culture conditions).
4. Following incubation, observe the cells by light, epi-fluorescence or confocal microscopy to monitor the progression of apoptotic features.
   Note 1: Cells undergoing apoptosis will display morphological, biochemical and molecular hallmarks of apoptosis that can be detected by microscopy and fluorescence-based biosensors (See Protocols 3 and 4).
   Note 2: Incubation time of cells with different apoptosis inducers will vary, depending on cell type, cell conditions (such as confluency and nutrient status), and the dose of death stimulus applied. Careful titration may be required.
5. When cells display hallmarks of apoptosis, remove apoptosis-inducing agent by washing the cells once with warm (37 °C, or other normal culture temperature) fresh cell culture medium.
   Note 1: As apoptotic cells are more loosely attached on the culture plate, it is important to apply and remove medium gently so that cells will not be washed away.
   Note 2: Live suspended cells can be recovered from the supernatant by gentle centrifugation (160 x g for 1 min) and added back to the culture dish.
6. Incubate cells at 37 °C with 2 ml/35 mm dish of fresh cell culture medium in 5% CO₂. Alternatively, use conditioned medium (cell-free culture medium collected from healthy cells) to further enhance survival of apoptotic cells.
   Note: Washing and incubating cells with fresh medium allow the majority of cells to reverse ethanol-induced apoptosis after ethanol exposure^12,13. However, repeating this washing step may be required when cells are exposed to other apoptotic stimuli (See Discussion).

3. Live Cell Microscopy

1. We recommend using an inverted confocal or epi-fluorescence microscope with environmental control (37 °C, 5% CO₂) for live cells microscopy.
   Note: It is also possible to image the cells using upright microscopes with a water dipping objective. Dip the objective directly into the culture medium to image cells. However, cells can be crushed by the dipping objective during focusing.
2. Pre-warm the microscope (such as environmental control chamber, stage incubator, and objective heater) at least 2 hr before imaging.
   Note: This allows microscope components to reach thermo-equilibrium, so that it can avoid the drift of focus and shift of x-y plane due to the thermal expansion and contraction of the components.
3. Place a 35 mm glass bottom dish of cultured cells (See Protocol 1) on the stage of an inverted microscope and capture cell images using a 40X or 63X plan-Apochromat objective with a numerical aperture (N.A.) 1.3 or 1.4 for imaging cells from the bottom of the dish through glass coverslips.
   Note: Avoid applying over 2 ml of medium to 35 mm glass bottom dish, as the weight of the medium could cause curvature of the glass bottom, making it difficult to image a field of cells at the same focal plane.
4. Maintain cells at 37 °C or the corresponding normal temperature during the entire imaging process.
   Note: The process of apoptosis, and likely the reversal of apoptosis, depends on temperature sensitive enzymatic activities. Therefore, maintaining cells at 37 °C (e.g. with a microscope stage top incubator) is important throughout the experiment. Decreased temperature could slow down the apoptotic response and the recovery response after removal of apoptotic stimulus.
5. Use a humidity device or lay a transparent foil (See Materials) on the culture dish to reduce water loss by evaporation from the medium.
   Note: the foil could disrupt the polarity of light for DIC microscopy. Restore polarity by adjusting the polarizer at the light path.
6. Maintain pH in the cell culture medium (pH 6.8 -7.3) by incubating in 5% CO₂ with an environmental control chamber on the microscope.
   Note: Maintenance the pH in culture medium can be also achieved by adding HEPES buffer, or by using commercial CO₂-independent medium (See Materials). Optimal conditions can vary, depending on the cell type.
7. Minimize fluorescence/laser (excitation light intensity) exposure to cells during the imaging process to avoid phototoxicity by reducing the fluorescence intensity to the lowest required to obtain high quality images of cell/subcellular structures or expressed biosensors (Details in Protocol 4).

4. Strategies for Detecting and Tracking Anastasis during and After Apoptotic Events

1. Plasma membrane blebbing, cytoplasmic condensation, cell shrinkage and apoptotic body formation (See Figures 1A-C, E).
   1. Perform time-lapse live cell differential interference contrast (DIC) or phase contrast microscopy to track a group of health cells and to observe their cell morphology (See Protocol 3 for live cell microscopy, and See Discussion).
      Note 1: Reduce intensity of light source for DIC/ phase contract imaging to avoid phototoxicity to the cells.
      Note 2: If DIC and phase contrast microscopy are not available, use CellTracker to stain the cytosol to outline the morphology of live cells for confocal or epi-fluorescence microscopy and monitor the cell morphology.
   2. Apply cell death stimulus to trigger cells to undergo apoptosis (See Protocol 2 for application and removal of apoptotic stimuli).
   3. Observe treated cells for morphological hallmarks of apoptosis such as plasma membrane blebbing, cytoplasmic condensation, cell shrinkage and apoptotic body formation (Figures 1A, 1B, 1C, 1E).
   4. Wash away death stimuli, and re-supply cells with fresh medium when the cells display morphological hallmarks of apoptosis.
      Note 1: Apply cell death stimulus or changing the cell culture medium on the microscope stage during time-lapse live cell imaging can be achieved by using a perfusion cell culture chamber, or can be performed directly on the cell culture dish by carefully pipetting without touching the dish, during the intervals between imaging.
      Note 2: Use focus drift compensation systems to avoid out of focus of the cells due to the loss of thermo-equilibrium of the microscope system after changing the cell culture medium (See Discussion).
   5. Continuous time-lapse imaging to track the fate of cells that display hallmarks of apoptosis. Cells that reverse apoptosis can repair damage and regain normal flat morphology (Figures 1A, 1B, 1C, 1E).
2. Mitochondrial fragmentation, DNA/chromatin condensation, and nuclear fragmentation (See Figures 1D, 1F, 1G).
   1. To visualize mitochondria, stain cells with 50 nM MitoTracker red/deep red/green-fluorescent dye, and simultaneously stain the nucleus with 10 μg/ml of Hoechst 33342 blue nuclear dye in culture medium for 20 min at 37 °C with 5% CO₂.
      Note 1: Reduce the concentration of dyes and duration of incubation to avoid cytotoxicity and reduce background fluorescence when need. Optimize the staining conditions to obtain a good signal to noise ratio with the minimal amount of dye and incubation time for staining.
      Note 2: Use fluorescent stains for mitochondria such as MitoTracker Red CMXRos, MitoTracker Deep Red FM, or MitoTracker Green FM, that does not leak out from mitochondria during apoptosis. This allows visualizing mitochondrial morphology during apoptosis and anastasis. See the materials and equipment table for details about these stains.
   2. Keep stained cells in the dark to avoid photobleaching.
   3. Remove excess stain by washing the cells 3 times with 37 °C phosphate-buffer saline (PBS) solution or fresh cell culture medium, with 1 min incubation in the fresh medium between each wash, and then further incubate in the fresh medium for 20 min before the final wash to allow excessive stains to exit the cells to reduce non-specific signal background for imaging.
      Note: Shorten incubation times with cell medium after staining may result in high background for imaging.
   4. Perform real-time live cell confocal or epi-fluorescence microscopy to image healthy cells before apoptotic induction is applied (See Protocol 3 for live cell microscopy).
      Note: Healthy cells display tubular mitochondria and round nuclei in most cell types (Figures 1D, 1F, 1G).
   5. Trigger cells to undergo apoptosis (See Protocol 2 for application and removal of apoptotic stimuli to the cells).
   6. Continue time-lapse live cell microscopy to observe alterations in mitochondrial and nuclear morphologies immediately after the death stimulus is applied to the cells. Observe cells to display hallmarks of apoptosis such as mitochondrial fragmentation and swelling, chromatin condensation and nuclear fragmentation, in contrast to healthy cells displaying tubular mitochondria and round nuclei.
   7. When cells display hallmarks of apoptosis, wash and supply cells with fresh medium, and continue time-lapse imaging to track the fate of cells. Cells that reverse apoptosis regain normal mitochondrial and nuclear morphology (Figures 1D, 1F, 1G).
      Note: Perform DIC microscopy in parallel when possible to obtain additional information for accessing the stages of apoptosis by observing the alterations of cell morphology in the same group of cells (See Protocol 4.1 for DIC microscopy).
3. Detection of mitochondrial release of cytochrome c using cells stably expressing a cytochrome c-GFP fusion protein (See Figure 2).
   1. Stain cells stably expressing cytochrome c-GFP ^23,24 with MitoTracker Red to label polarized cell mitochondria (See Steps 4.2.1 to 4.2.3 for live cell mitochondria staining).
   2. Perform real-time live cell confocal or epi-fluorescence microscopy to track healthy cells (Details in Protocol 3 for live cell imaging).
      Note: The cytochrome c-GFP signal primarily localizes in the mitochondria of healthy cells (Figures 2Ai, 2B).
   3. Trigger cells to undergo apoptosis (See Protocol 2 for application and removal of apoptotic stimuli to the cells). Continue time-lapse microscopy to observe translocation of cytochrome c-GFP from mitochondria to the cytosol (Figures 2Aii-iii, 2B).
      Note: Mitochondrial release of cytochrome c into the cytosol is a marker of mitochondrial outer membrane permeabilization (MOMP)⁴, a critical step in mitochondria-dependent apoptosis^21,22
   4. When cells display the cytochrome c-GFP signal in the cytoplasm, remove the cell death stimulus, and supply cells with fresh medium (See Protocol 2). Continue time-lapse imaging to track the fate of cells with cytosolic GFP.
      Note: Cells that reverse apoptosis regain normal cell morphology, and reduce their cytosolic cytochrome c-GFP signal presumably through degradation or translocation back to mitochondria (Figures 2Aiv-vii, 2B).
   5. Capture DIC images in parallel to obtain additional information for accessing the stages of apoptosis by observing alterations in cell morphology in single cells or group of cells (See Protocol 4.1 for DIC microscopy).
4. Detection of caspase activity using a caspase biosensor (See Figure 3)
   1. Transfect cells with the caspase biosensor NES-DEVD-YFP-NLS for 16 to 24 hr before subjecting them to an apoptotic stimulus¹³.
   2. Stain the transfected cultures with Hoechst 33342 to label cell nuclei (See Steps 4.2.1 to 4.2.3 for live cell nuclear staining).
   3. Perform real-time live cell confocal or epi-fluorescence microscopy to track healthy cells (Details in Protocol 3 for live cell imaging).
      Note: The NES-DEVD-YFP-NLS biosensor signal primarily localizes in the cytosol of healthy cells due to its nuclear export signal (NES) (Figures 3A, 3Bi and 3C, see Discussion).
   4. Trigger cells to undergo apoptosis (See Protocol 2 for application and removal of apoptotic stimuli to the cells). Continue time-lapse microscopy to observe nuclear translocation of YFP.
      Note: Activation of caspases cleaves the DEVD motif in the caspase biosensor NES-DEVD-YFP-NLS, resulting in YFP-NLS translocation from the cytosol to the nucleus (Figures 3A, 3Bii-iv, see Discussion).
   5. When cells display nuclear YFP, remove the apoptotic stimulus, and supply cells with fresh medium (See Protocol 2).
      Note: Cells that reverse apoptosis regain normal cell morphology, but retain the nuclear YFP signal (Figures 3Bv-x, Cells 1 and 2, and 3D).
   6. Continue time-lapse imaging to track the fate of cells that display nuclear YFP.
      Note: The nuclear YFP signal will become degraded several hours after cells have reversed apoptosis (Figures 3B, vi-x, Cell 1, See Results and Discussion) presumably through normal cell clearance mechanisms such as proteasome degradation.
   7. Capture DIC images in parallel to obtain additional information for accessing the stages of apoptosis by observing alterations in cell morphology in single cells or a group of cells. (See Protocol 4.1 for DIC microscopy).
5. Cell surface exposure of phosphatidylserine (See Figure 4)
   1. Seed cells on glass coverslips to achieve 80% cell confluency (See Protocol 1).
   2. Co-stain the cells with red-fluorescent MitoTracker and blue Hoechst 33342 stain for mitochondria and nuclei, respectively (See Steps 4.2.1 to 4.2.3 for live cell mitochondrial and nuclear staining).
   3. Trigger cells to undergo apoptosis by using an apoptosis inducer (See Protocol 2).
   4. Apply fluorescent-labeled annexin V (See Materials) to stain apoptotic cells 10 min at 37 °C before removal of the apoptosis inducer to detect exposure of phosphatidylserine on the surface of apoptotic cells.
      Note 1: Healthy cells are not stained with annexin V because phosphatidylserine is normally sequestered on the inner-leaflet of the cell plasma membrane³⁸. Apoptotic cells are stained with annexin V, which binds to phosphatidylserine on cell surface (Figures 4A and 4B).
      Note 2: Fluorescent-labeled annexin V can also be applied to stain apoptotic cells immediately after removal of the apoptosis inducer, with 10 min incubation.
   5. Remove cell death stimulus, wash the stained cells with warm PBS or fresh medium once, and then supply cells with fresh medium for 2 hr at 37 °C with 5% CO ₂ (See Protocol 2).
      Note 1: Cells that reversed apoptosis regain normal morphology and retain fluorescently labeled annexin V signal after regaining normal morphology (Figures 4A, and 4B).
      Note 2: The annexin V signal decreases with time on recovered cells (See Discussion).
   6. Fix cells at chosen time points with 4% paraformaldehyde containing 8% sucrose in 1x PBS for 20 min in the dark at room temperature, and wash the fixed cells with PBS for 3 times to remove paraformaldehyde before mounting cells on glass slides coverslips with antifade mountant (See Materials) for confocal or epi-fluorescence microscopy and observe annexin V on cells after removal of apoptosis inducer.
      Note 1: Sucrose in fixative preserves cell membrane structure during fixation.
      Note 2: Store the paraformaldehyde solution in dark at 4°C to prevent degradation. 
      Note 3: Warm up paraformaldehyde solution to room temperature before applying it to cells for fixation to avoid cold shock to the cells.

Results

To study the reversal of apoptosis, tissue culture cells are first exposed to a death stimulus to trigger apoptosis. When the cells display hallmarks of apoptosis, fresh culture medium is then applied to wash away the stimulus and then incubate the dying cells to allow recovery (Figure 1A). Here, the key question being addressed is how far individual dying cultured cells can progress towards apoptosis and still undergo anastasis. This question can be definitively answered by continuous monitoring with bi...

Discussion

Anastasis refers to the phenomenon where cells that have activated cell death pathway subsequently reverse the dying process and survive. Here, we have demonstrated that live cell imaging can be used to confirm that the same individual cells in fact can reverse apoptotic cell death process at a late stage, and then continue surviving and reproducing. Our protocols describe several optimized cell type-specific treatment conditions to induce apoptosis and allow a large proportion of the cells to undergo the reversal of apo...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
LSM780 confocal microscopy	Carl Zeiss
Glass bottom culture dish	MatTek Corporation	P35G-0-14-C
Transparent CultFoi	Carl Zeiss	000000-1116-084
CO2 independent medium	Life Technologies	18045-088
CellTracker	Life Technologies	C34552
Mitotracker Red CMXRos	Life Technologies	M-7512
Hoechst 33342	Life Technologies	H1399
Fluorescently labeled annexin V	Biovision	K201