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Medicine

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Published: February 1st, 2018

DOI:

10.3791/54107

1Institute for Basic Biomedical Sciences, Johns Hopkins University School of Medicine, 2School of Life Sciences, Chinese University of Hong Kong, 3Department of Neurosurgery, Johns Hopkins University School of Medicine

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 “rising to life”) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Programmed cell death, such as apoptosis, plays an essential role in embryonic development and normal homeostasis by eliminating unwanted, injured, or dangerous cells in multicellular organisms1,2,3. The loss of balance between cell death and survival can lead to fatal consequences such as cancer, heart failure, autoimmunity, and degeneration4,5,6,7,8. Activation of executioner caspases has traditionally been consi....

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1) Preparation of CaspaseTracker Biosensor Flies

  1. Anesthetize flies with CO2, and use a paintbrush to transfer 7 to 10 caspase-sensitive Gal4 (DQVD)19 virgin females and 7 to 10 G-Trace53 Gal4 reporter young male flies (or vice versa) in the same vial with fly food and fresh yeast paste.
    NOTE: Cross of Caspase-sensitive (DQVD) Gal4 and G-Trace flies will produce CaspaseTracker progeny flies. Cross of Caspase-insensitive.......

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While time-lapse live cell microscopy is a reliable method to tract anastasis in cultured cells20, it is challenging to identify which cells have undergone anastasis in animals, because the recovered cells appear morphologically indistinguishable from normal healthy cells that have not attempted cell death. For example, human cervical cancer HeLa cells display morphological hallmarks of apoptosis1,2,14, s.......

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The CaspaseTracker dual biosensor system is a novel and unique tool that allows detection of recent or ongoing caspase activity, and tracking of cells that have reversed cell death process and survive after experiencing caspase activity in vivo. While caspase activity has been traditionally assumed as a hallmark of apoptosis, growing studies reveal that non-apoptotic caspase activity plays potential roles in diverse normal cell functions, such as regulation of neuronal activity

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We thank Darren Obbard for Drosophila image in Figure 3C and in video manuscript; J. Marie Hardwick, Wade Gibson, and Heather M. Lamb for valuable discussion of this manuscript. This work was supported by a Sir Edward Youde Memorial Fellowship (H.L.T.), Dr. Walter Szeto Memorial Scholarship (H.L.T.), Fulbright grant 007-2009 (H.L.T.), Life Science Research Foundation fellowship (H.L.T.), and NCI K22 grant CA204458 (H.L.T.). Ho Lam Tang was a Shurl and Kay Curci Foundation Fellow of the Life Sciences Research Foundation (2014-2017).

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Name Company Catalog Number Comments
CONSUMABLES AND REAGENTS
Vectashield mounting medium Vector Products H-1000 Antifade mounting medium
Vectashield mounting medium (with DAPI) Vector Products H-1200 Antifade mounting medium with DAPI
Forceps Ted Pella #505 (110mm, #5) Dumont tweezer biology grade, stainless steel
Hanging Drop Slides Fisher Scientific 12-565B Glass slides
Hoechst 33342 Molecular Probes H1399 DNA stain
Mitotracker Red CMXRos  Molecular Probes M-7512 Mitochondria stain
Cleaved caspase-3 (Asp175) antibody Cell Signaling Technology #9661 Stain for active fragment of caspase-3
Bovine Serum Albumin (BAS) Sigma-Aldrich A8806 Blocking agent for immunostaining
Phosphate Buffered Saline  VWR 114-056-101 Medium for washing and immunostaining
Triton™ X-100 Sigma-Aldrich T8787 Detergent for cell permeabilization
Name Company Catalog Number Comments
EQUIPMENT
LSM780 confocal microscope Carl Zeiss N/A Imaging
Carl Zeiss Stereomicroscope Stemi 2000  Carl Zeiss N/A Drosophila dissection
AmScope Fiber Optic Dual Gooseneck Microscope Illuminator, 150W AmScope WBM99316  Light source

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