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 manipulati....

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 (Mu....

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