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.

1. Preparation of Cells for Live Cell Imaging
<ol>
	<li>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 cells12,13, including primary mouse liver cells, primary mouse macrophages, primary rat cardiomyocytes, and also cell lines such as human embryoni...

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...

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.

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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 wi...

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 ...

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17.9K visualizações.  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. 