Name: strategies for tracking anastasis, a cell survival phenomenon that reverses apoptosis
Uploaded: 2015-02-16T13:00:00.000+00:00
Duration: 12 min 55 s
Description: Watch this Scientific Journal Video about Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis at JoVE.com

We thank Rev. Dr. Ralph Bohlmann and Rev. Dr. James Voelz for suggesting the word &#8220;anastasis&#8221; to describe reversal of apoptosis; Douglas R. Green for HeLa cells stably expressing cytochrome c-GFP; Charles M. Rudin and Eric E. Gardner for H446 cells; Heather Lamb for assistance in cartoon drawing at the video; Yee Hui Yeo for valuable discussion of this manuscript. This work was supported by a Sir Edward Youde Memorial Fellowship (H.L.T.), the Dr. Walter Szeto Memorial Scholarship (H.L.T.), Fulbright grant 007-2009 (H.L.T.), Life Science Research Foundation fellowship (H.L.T.), NIH grants NS037402 (J.M.H.) and NS083373 (J.M.H.), and University Grants Committee of the Hong Kong AoE/B-07/99 (M.C.F.). Ho Lam Tang is a Shurl and Kay Curci Foundation Fellow of the Life Sciences Research Foundation.

The authors have nothing to disclose.

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 apoptosis monitored with several biomarkers for verification. Regarding technical issues, glass bottom dishes were used to culture cells for imaging12,13, because of the highly transparent thin glass between cells and the microscope objective, which allows long working distances for high quality confocal or epi-fluorescence microscopy in high magnification, facilitates observation of classic apoptosis including mitochondrial fragmentation, release of cytochrome c, and nuclear condensation and fragmentation. Glass also does not distort the polarity of light, to allow high quality DIC microscopy for monitoring of membrane blebbing, cell shrinkage of cells, and apoptotic body formation during apoptosis. DIC microscopy has advantages over phase contrast microscopy to observe these morphological hallmarks of apoptosis, as DIC enhances the contrast of unstained and transparent cell samples, improving detection of cell morphologies. If DIC and phase contrast microscopy are not available, CellTracker can be used to stain the cytosol to outline the morphology of live cells for confocal or epi-fluorescence microscopy and monitor the cell morphology.
The application of appropriate doses of death stimuli and the strategies of the removal of the stimuli are critical steps for detection of anastasis. We have chosen to use low concentrations of ethanol as a death stimulus for the experiments described here because a large percent of treated cells can uniformly undergo apoptosis and can recover from this, potentially milder, death stimulus. Nevertheless, these approaches can also be applied successfully with other death stimuli, as we have reported12,13. However, some death stimuli are inherently more challenging than others, such as staurosporine (STS), a commonly used apoptosis-inducing agent. Perhaps because it binds its substrates with high affinity46-48, repeated washes are essential but still may not efficiently remove STS from the cells. In this case, using lower drug concentrations and shorter drug incubation time that can still activate apoptotic dying process could be helpful to allow more cells to reverse apoptosis. Re-feeding cells with conditioned cell culture medium can also enhance reversal of apoptosis better than the fresh cell culture medium. Apoptotic cells are usually loosely attached to the culture dish surface particularly on glass surfaces, hence it is important to wash away the apoptosis&#160;inducers gently to avoid detaching cells. Coating glass dishes with poly-D-lysine, collagen and/or fibronectin could increase cell adherence for allowing certain types of cells, such as cardiomyocytes37, to attach properly on the glass surface of cell culture dishes, particularly after exposure of cells to a death stimulus.
The process of apoptosis, and likely the reversal of apoptosis, depends on enzymatic activities that can be influenced by temperature. Therefore, maintaining cells at 37&#160;&#176;C or at their normal culture condition is important throughout the live imaging process to ensure reproducibility of experimental results. Pre-warm microscope before starting live cell imaging is also a critical step that allows the microscope components to reach thermo-equilibrium. This can avoid the drift of focus and shift of x-y plane due to the thermal expansion and contraction of the components during live cell imaging. Applying or removing the death stimulus by changing the medium a culture dish with either a pipet or by perfusion in the pre-warmed microscopy system during time-lapse imaging can still cause drift of focus due to changes in temperature or volume of the medium. Z-stack acquisition can help to image cells at the correct focal plane, but will increase the risk of phototoxicity due to additional exposure of cells to fluorescent lasers. Alternatively, use of focus drift compensation systems, such as an infrared laser-based automatic focus correction system, can keep cells at the same focal plane during time-lapse live cell imaging by maintaining the distance between the cells/glass and the objective without additional exposure of the cells to short wavelength, phototoxic fluorescence.
Mitochondrial release of apoptogenic proteins such as cytochrome c, and activation of effector caspases, such as the downstream/effector caspase-3 and caspase-7, have been generally considered to be the &#8220;point of no return&#8221; in the apoptotic cell death process2,5,6. Western blots have been used to detect the cleavage (activation) of caspase-3 and its substrates such as poly(ADP)-ribose polymerase-1 (PARP) and DFF45/ICAD in the whole cell population during apoptotic induction and after removal of apoptotic stimuli, and revealed that cleaved caspase-3, ICAD, and PARP showed up in cells during exposure to death stimuli, but disappeared after the cells were re-feed with fresh culture medium13. However, these methods reveal the general condition in cell population, but do not distinguish the individual cells that will ultimately die from those that will reverse apoptosis and then survive. Biochemical fractionation to analyze release of mitochondrial cytochrome c has similar caveats. Therefore, to track the fate of individual cells after cytochrome c release and caspase-activation, two of the most recognized hallmarks of apoptosis2,5,6, GFP-tagged cytochrome c (CytoC-GFP) and a caspase biosensor, such as the one used here NES-DEVD-YFP-NLS, combined with live cell microscopy circumvent these problems by directly observing morphological recovery and the translocation of the CytoC-GFP biosensor in the same cells. These results indicate the reversibility of apoptosis after mitochondrial cytochrome c release and caspase activation.
The current challenge for studying anastasis is the lack of labeling techniques to detect cells that have undergone anastasis at any point throughout their lifespan, in vivo or in vitro. Stable expression of the caspase biosensor NES-DEVD-YFP-NLS also permits detection of caspase activity if caspases are activated again in the future, but does not permanently record caspase activity as the activated biosensor will be degraded without sustained caspase activity13. Other previously reported caspase biosensors, such as the FRET-based SCAT biosensors (e.g. ECFP-[DEVD]-Venus)26,49 as well as fluorescent reporters Apoliner (CD8-RFP-[DQVD]-GFP)50 and CPV (e.g. CD8-[DEVD]-Venus)51,52, can be used to detect caspase activity in whole animals and in cultured cells, but have similar limitations. Similarly, CytoC-GFP cannot be used to track cells that reversed apoptosis in long term, as it is released from mitochondria into the cytosol during apoptosis and is subsequently degraded. Annexin V labeling has been used to track cells that reversed apoptosis, but the signal intensity also declines with time, so is not useful for long term tracking of anastasis13,45. Continuous long-term in vivo imaging could be used to track the fate of the same cells after exposure of death stimuli. However, long-term in vivo imaging is still challenging to perform in most live animals. Therefore additional approaches are needed to extend anastasis research to whole animal studies, and for screening drugs that could promote or inhibit anastasis using tissue culture cells
In the future, newer protocols and techniques that track long-term cell fate are needed to develop and test the physiological, pathological and therapeutic implications of anastasis. We proposed that anastasis could be a general cell survival mechanism to rescue injured cells that are difficult to replace, such as mature neurons and cardiac cells13. However, anastasis of apoptotic cancer cells after chemotherapeutic treatments could result in recovery of dangerous cells leading to recurrence of resistant tumors12,13. Anastasis could also be an important mechanism of tumorigenesis, as some cells display oncogenic transformation after they reversed apoptosis13. Therefore, enhancing anastasis could be a new therapeutic strategy for inhibiting neurodegeneration and heart failure, while suppressing anastasis as a new way for preventing or treating cancers. Developing biosensors to track reversal of apoptosis in disease model systems will advance the understanding of the cell survival mechanisms of anastasis, and potential therapeutics to intractable diseases by modulating anastasis.

Apoptosis (Greek for &#8220;falling to death&#8221;) is generally assumed to be a one-way process ending in cell suicide1-7. 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 pathway8,9. Similarly, genetic manipulations&#160;allow&#160;healthy mammalian cells that artificially display &#8220;eat me&#8221; signals or that lose adhesiveness to their extracellular matrix to escape death by whole cell phagocytosis or entosis, respectively10,11. However,&#160;we and others have shown that without genetic manipulation normal healthy mammalian cells and cell lines can also recover from the early stages of apoptosis12-15. Using tools to track individual cells, we have further demonstrated recovery from late stages of apoptosis12,13, after cells have passed important checkpoints that typically mark the &#8220;point of no return&#8221;2-6. These checkpoints of late stage apoptosis&#160;include mitochondrial release of cytochrome c, activation of caspases, nuclear fragmentation, and formation of apoptotic bodies. We adopted a Greek compound word &#8220;anastasis&#8221;, which means &#8220;rising to life&#8221;, to describe this reversal of apoptosis at the brink of cell death2-6.
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 events16-19. These events promote self-destruction of cells to regulate developmental and homoeostatic processes in unicellular and multicellular organisms by eliminating damaged or dangerous cells16-19. While apoptotic cells can be readily distinguished by standardized morphological, biochemical and molecular manifestations of apoptosis1,5,6,16,20, currently there is no known marker specific to anastasis12,13. Importantly, cells that have undergone anastasis appear to be normal healthy cells, and cells that just start reversing apoptosis appear as apoptotic dying cells12,13. 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 minutes23,24, followed by cytoplasmic and nuclear condensation within 10 min25-27, and cell death shortly thereafter25-27. Activated caspases&#160;orchestrate apoptosis by cleaving and inactivating key structural and functional components for the purpose of cellular demolition2,28, such as the endonuclease inhibitor DFF45/ICAD29,30. Caspases also activate pro-apoptotic factors, such as BCL-2 family member BID, which translocates to mitochondria to promote mitochondrial release of cytochrome c31,32. Caspase activity also results in cell surface exposure of phosphatidylserine as an &#8220;eat me&#8221; signal for promoting engulfment of dying cells by macrophages or neighbor cells through phagocytosis33. Furthermore, apoptotic events render mitochondria dysfunctional, disrupting cellular bioenergetics and metabolism34,35,36. 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 lines12,13. 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&#160;methods and a caspase biosensor for&#160;detecting cells that have previously reversed late stage apoptosis and then survived.

<table><tbody><tr><td>LSM780 confocal microscopy</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Glass bottom culture dish</td><td>MatTek Corporation</td><td>P35G-0-14-C</td><td></td></tr><tr><td>Transparent CultFoi</td><td>Carl Zeiss</td><td>000000-1116-084</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; independent medium</td><td>Life Technologies</td><td>18045-088</td><td></td></tr><tr><td>CellTracker</td><td>Life Technologies</td><td>C34552</td><td></td></tr><tr><td>Mitotracker Red CMXRos</td><td>Life Technologies</td><td>M-7512</td><td></td></tr><tr><td>Hoechst 33342</td><td>Life Technologies</td><td>H1399</td><td></td></tr><tr><td>Fluorescently labeled annexin V</td><td>Biovision</td><td>K201</td><td></td></tr></tbody></table>

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.

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&#160;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 biomarkers to track cell fates using the methods described below.
We first describe our strategies to detect reversal of apoptosis in tissue culture cells by using time-lapse live cell microscopy, which is required for tracking and recording the response of individual cells before, during, and after exposure to an apoptotic stimulus12,13. Healthy adherent cells spread on their attached substrate (Figure 1Bi)12,13, and displayed filamentous mitochondria and round nuclei prior to treatment (Figures 1Ci, Di, Ei, Fi, Gi)12,13. After exposure to 3.8-4.5% ethanol in cell culture medium (vol/vol), DIC or phase contrast microscopy revealed that the cells exhibited morphological hallmarks of apoptosis, including cytoplasmic condensation, plasma membrane blebbing, and cell shrinkage (Figures 1Bii, 1Cii-iii, and 1Eii-vi)12,13. Apoptotic body formation by the same cells was&#160;readily observed by DIC (Figures 1Eii-vi). Fragmentation of MitoTracker-labeled mitochondria (Figures 1Dii-iii, 1Fii-vi)12,13 as well as the condensation of Hoechst-stained nuclear DNA (Figures 1Dii-iii, Fii-iii, Gii)12,13, and fragmentation of nuclei&#160;(Figures 1Evi and Fvi, white arrows), were detected simultaneously by confocal or epi-fluorescence microscopy. The cells that successfully reversed apoptosis were&#160;subsequently observed to regain normal morphology, apparently repairing their damage (Figures 1Biii, C and Div-vi, E and Fvii-xii, and Giii)12,13. Cells under these conditions also had&#160;been shown to regain organelle motility, cell migration, and functional endocytosis based on uptake of fluorescence-emitting Quantum Dots and cell division13. Interestingly, some cells that revered apoptosis displayed irregular nuclear morphology (Figure 1Fxii), and also abnormal cell division and formation of micronuclei (Figures 1Giv-vii)13, which is a biomarker of DNA damage, chromosome breakage and whole chromosome loss in dividing cells39,40. Displaced chromosomes or chromosome fragments outside fail to be included in the daughter cell nuclei are enclosed by nuclear membrane39,40. Thus, a consequence of anastasis could be the harboring of genomes that were damaged during aborted apoptosis, leading to tumorigenesis and cancer progression. The presence of micronuclei is also common in cancer cells40,41.
Release of mitochondrial cytochrome c is a critical step to mediate or amplify caspase-dependent apoptosis20-22. Using HeLa cells that stably express&#160;GFP-tagged cytochrome c, we monitored the subcellular relocation of cytochrome c during apoptosis and anastasis by confocal microscopy. As reported23,24, cytochrome c-GFP localized&#160;to mitochondria prior to treatment (Figures 2Ai and B), but then was&#160;released into the cytosol after a death stimulus had&#160;been applied (Figures 2Aii, 2Aiii and B). Other characteristic morphologies such as mitochondrial fragmentation and plasma membrane blebbing, were&#160;also observed in cells that had&#160;released cytochrome c-GFP (Figure 2Aiii). These cells were&#160;reported to undergo cell death shortly after release of cytochrome c23-27. However, after removal of the death stimulus, we observed that cytosolic cytochrome c-GFP levels declined, mitochondria regained filamentous structures, and the plasma membrane recovered a normal appearance (Figures 2Aiv-vii, and B). Therefore, apoptotic cells can undergo anastasis after mitochondrial release of cytochrome c.
Amplification of downstream DEVD-cleaving caspases is generally assumed to be the &#8220;point of no return&#8221; in apoptosis2,5. Therefore, we used a caspase biosensor NES-DEVD-YFP-NLS expressed from a plasmid13, making it possible to detect surviving cells that have previously experienced caspase activity (Figure 3A). This caspase biosensor is a polypeptide with an N-terminal nuclear exclusion signal (NES), a linker with the caspase-3/7 consensus cleavage site (DEVD)26,42,43, followed by a yellow fluorescent protein (YFP) and a C-terminal nuclear localization signal (NLS). In healthy cells, this biosensor predominantly localizes to the cytosol as a function of the NES (Figures 3Bi, Ci-iv, and D)13. During apoptosis, activated caspases cleave the DEVD motif, releasing YFP-NLS, which efficiently translocates from the cytosol to the nucleus in cells that simultaneously, or subsequently, display other features of apoptosis such as DNA/chromatin condensation and cell shrinkage (Figures 3Bii-iv, and D) 13. Thus, these cells retain nuclear import function following caspase activation. After removal of the apoptosis inducer, cells that undergo anastasis display morphological recovery even after caspase activation has occurred (Figures 3Bv-x, and D)13, apparently reversing late stage apoptosis. During recovery of normal morphology, the nuclear YFP signal decreases in intensity in some cells when they start to reverse apoptosis after removal of death stimulus&#160;(Figures 3Bv-x, Cell 1)13, suggesting that cells degrade the caspase-cleaved biosensor, possibly by the same mechanism used to remove other caspase-cleaved products during and after anastasis.
Anastasis can also be detected without live cell microscopy. This can be achieved by using fluorescently labeled annexin V38,44, which binds and marks externalized phosphatidylserine (PS) at an early step in apoptosis (Figure 4A). For example, as expected, healthy and untreated neonatal rat primary cardiac ventricular myocytes cannot be labeled with annexin V (Figure 4B)13, as phosphatidylserine is restricted to the inner leaflet of the plasma membrane of healthy cells38. In contrast, ethanol-induced apoptotic dying cells expose phosphatidylserine on the cell surface(Figure 4B)13, and therefore, can be labeled with the fluorescent annexin V38,44. Noticeably, annexin V-labeled cells can regain normal morphology after removal of the death stimuli, suggesting that primary cardiomyocytes have reversed apoptosis (Figure 4B)13. Others have previously applied a similar strategy to suggest that recovery from apoptosis could occur in vivo. In an in vivo model of transient ischemic injury, caspase-dependent annexin V labeling of cardiomyocytes in rabbits and mice apparently resulted in the internalization of annexin V by surviving cells after transient ischemia45, suggesting that anastasis could occur in live animals. In addition, two other studies used cell sorting to identify a fraction of annexin V-labeled mouse BCL1.3B3 B lymphoma cells (exposed to anti-immunoglobulin antibodies that induce apoptosis in BCL1.3B3) and mouse mammary carcinoma MOD cells expressing temperature-sensitive p53 (that causes apoptosis after incubated below permissive temperature) continued to proliferate after they were returned to normal culture conditions14,15. Collectively, these studies suggest that annexin V is useful for determining the fate of cells that have reversed apoptosis without live-cell microscopy imaging. However, there are caveats with this strategy, as annexin V fluorescence is not permanent (Figure 4B)13, remaining detectable for only a few hr after removal of the death stimulus, so that these&#160;methods cannot provide long term tracking of cells that reversed apoptosis.
<img alt="Figure 1" src="/files/ftp_upload/51964/51964fig1highres.jpg" /> 
Figure 1: Tracking reversal of apoptosis by live cell imaging. (Reprinted with permission from Tang et al., MBoC 23, 2240-225213, for Figures 1A-D, and G). (A) Approach to induce apoptosis and subsequently allow cultured cells to recover after washing away the apoptosis inducer. (B) Time-lapse live cell phase contrast microscopy of healthy primary mouse liver cells (untreated), the same group of cells that were treated with 4.5% ethanol in culture medium (vol/vol) for 5 hr (treated), and then washed and further incubated with fresh culture medium for 24 hr (washed). Scale bar, 100 &#956;m. (C) Continuous time-lapse live cell epi-fluorescence microscopy of the same primary mouse liver cell before ethanol treatment (i), at the indicated times after treatment with 4.5% ethanol in cell culture medium for 2.5 hr (ii and iii), and after washing and incubating with fresh medium to allow recovery for 1 hr (iv - vi). Merged images of MitoTracker-stained mitochondria (red) and the Hoechst-stained nucleus (blue) were visualized by epi-fluorescence microscopy, and cell morphology by DIC microscopy. Scale bar, 10 &#956;m. (D) Merged fluorescence images only (without DIC) from panel C reveal organelle morphologies. (E) Continuous time-lapse live cell confocal microscopy of the human small cell lung carcinoma H446 cells before ethanol treatment (i), after treatment with 3.8% ethanol in cell culture medium for 2 hr 28 min (ii-vi), and after washing and incubating with fresh medium to allow recovery (vii-xii). Merged images of MitoTracker-stained mitochondria (red) and the Hoechst-stained nuclei (blue) were visualized by confocal microscopy, and cell morphology by DIC microscopy. White arrows indicate a fragmented nucleus in apoptotic bodies (vi). Scale bar, 10 &#956;m. (F) Merged fluorescence images only (without DIC) from panel E reveal organelle morphologies. (G) Continuous time-lapse live cell epi-fluorescence microscopy for monochrome Hoechst-stained nuclear imaging of a single HeLa cell that undergoes imprecise cell division. Before ethanol treatment&#160;(i), treatment with 4.3% ethanol in cell culture medium for 5 hr (ii) and after ethanol removal (washed, iii-vi). Panels iv reveals abnormal cell divisions. Red arrows indicate the major nuclei in the divided cells (iv- vi). Scale bar, 30 &#956;m. Times are indicated as hr:min. <a href="https://www.jove.com/files/ftp_upload/51964/51964fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/51964/51964fig2highres.jpg" /> 
Figure 2: Detecting reversal of apoptosis after mitochondrial release of cytochrome c (A) Continuous time-lapse live cell confocal microscopy of HeLa cells stably expressing cytochrome c-GFP (CytoC-GFP) before (i), during (ii-iii) and after (iii-vii) exposure to 3.9% ethanol in cell culture medium. Merged confocal images of CytoC-GFP (green), MitoTracker-stained mitochondria (red), and Hoechst-stained nuclei (blue) are combined with DIC images (left panel). Unmerged versions are shown separately for CytoC-GFP, presented as a heat map of the signal intensity (left middle panel), monochrome image of CytoC-GFP (middle panel), and monochrome image of mitochondria&#160;(right middle panel). Merged images of CytoC-GFP and mitochondria (right panel). (B) The change of the cytosolic cytochrome c-GFP signal intensity of the cells, Cell 1 and Cell 2, as indicated in the monochrome CytoC-GFP image at Panel Ai. Times are indicated as hr:min. <a href="https://www.jove.com/files/ftp_upload/51964/51964fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/51964/51964fig3highres.jpg" /> 
Figure 3: Detecting reversal of apoptosis after caspase activation (Reprinted with permission from Tang et al., MBoC 23, 2240-225213, for Figures 3A-D). (A) Diagram of the caspase biosensor fusion protein composed of a nuclear export signal (NES), the DEVD caspase cleavage site, yellow fluorescent protein (YFP), and the nuclear localization signal (NLS). (B) Continuous time-lapse live cell confocal microscopy of HeLa cells expressing the caspase biosensor NES-DEVD-YFP-NLS before (i), during (ii-iv) and after (v-x) exposure to 4.3% ethanol in cell culture medium. Merged confocal images of the caspase biosensor (green), Hoechst-stained nuclei (blue) and DIC images (left panel), a monochrome images of YFP signal (middle panel), and a heat map indicating the YFP signal intensity (right panel). (C) Untreated controls analyzed as in panel B. (D) Quantified fluorescence intensities for the activated nuclear caspase biosensor in individual cells (numbered 1 and 2 in panel Bi) before, during, and after exposure of ethanol, and in untreated controls (numbered 3 and 4 in panel Ci) were calculated as the percent of YFP present in the nucleus (nuclear/total cell YFP signal intensity x 100%). Times are indicated as hr:min. Scale bar, 10 &#956;m. <a href="https://www.jove.com/files/ftp_upload/51964/51964fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/51964/51964fig4highres.jpg" /> 
Figure 4: Detecting reversal of apoptosis by fluorescent-labeled annexin V (Reprinted with permission from Tang et al., MBoC 23, 2240-225213, for Figures 4A-B). (A) Diagram of the annexin V-FITC anastasis assay used in panel B. (B) Neonatal rat primary cardiac ventricular myocytes were exposed to 4.5% ethanol in cell culture medium for 5 hr, and then incubated with annexin V-FITC for 10 min before removal of the ethanol medium. Cells were either fixed immediately (treated), or fixed after refeeding with fresh medium for an additional 2 hr recovery (washed). Untreated cells that were exposed annexin V-FITC serve as control (Untreated). MitoTracker-stained mitochondria (red), Hoechst-stained nuclei (blue) and annexin V-FITC (green) were visualized by confocal microscopy, and cell morphology was visualized by DIC microscopy. Scale bar, 10 &#956;m. <a href="https://www.jove.com/files/ftp_upload/51964/51964fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis at JoVE.com

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

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 ...

strategies-for-tracking-anastasis-cell-survival-phenomenon-that

Nanyang Technological University

Research

JoVE Journal

Biology

18.0K Views.  Johns Hopkins University Bloomberg School of Public Health. 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. 

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

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

Cancer Research

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Detection and Isolation of Apoptotic Bodies to High Purity

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. It has been proposed that ApoBDs can aid cell clearance as well as intercellular communication through trafficking biomolecules. Conventional approaches used for the identification and isolation of ApoBDs are often limited by the lack of accurate quantification and low sample purity. Here, we describe a workflow to confirm the induction of apoptosis, validate ApoBD formation, and isolate ApoBDs to high purity. We will also outline and compare fluorescence-activated cell sorting (FACS) and differential centrifugation based approaches to isolate ApoBDs. Furthermore, the purity of isolated ApoBDs will be confirmed using a previously establish flow cytometry-based staining and analytical method. Taken together, using the described approach, THP-1 monocyte apoptosis and apoptotic cell disassembly was induced and validated, and ApoBD generated from THP-1 monocytes were isolated to a purity of 97-99%.

A workflow using flow cytometry or differential centrifugation is developed to detect, quantify and isolate apoptotic bodies from an apoptotic sample to high purity.

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

Biochemistry

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer treatments as used in clinical oncology. It enables a quantitative and longitudinal analysis of the DNA damage response after genotoxic stress, as induced by radiotherapy and a multitude of anti-cancer drugs. The method covers all stages of the DNA damage response, providing endpoints for induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and cell death by apoptosis in case of repair failure. Combining these measurements provides information about cell cycle-dependent treatment effects and thus allows an in-depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. As the effect of many cancer therapeutics including chemotherapeutic agents and ionizing radiation is limited to or strongly varies according to specific cell cycle phases, correlative analyses rely on a robust and feasible method to assess the treatment effects on the DNA in a cell cycle-specific manner. This is not possible with single-endpoint assays and an important advantage of the presented method. The method is not restricted to any particular cell line and has been thoroughly tested in a multitude of tumor and normal tissue cell lines. It can be widely applied as a comprehensive genotoxicity assay in many fields of oncology besides radio-oncology, including environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells.

The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

Medicine

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.

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.

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

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9.
The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

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

Summary

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Chapters in this video

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Detection and Isolation of Apoptotic Bodies to High Purity

Cell Cycle-specific Measurement of γH2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death