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In vivo CaspaseTracker biosensor system for detecting anastasis and non-apoptotic caspase activity.

Tang HL, Tang HM, Fung MC, Hardwick JM - Sci Rep (2015)

Bottom Line: Importantly, when stressed flies were returned to normal conditions, morphologically healthy egg chambers and new progeny flies were labeled by the biosensor, suggesting functional recovery from apoptotic caspase activation.In striking contrast to developing egg chambers, which lack basal caspase biosensor activation under normal conditions, many adult tissues of normal healthy flies exhibit robust caspase biosensor activity in a portion of cells, including neurons.The widespread persistence of CaspaseTracker-positivity implies that healthy cells utilize active caspases for non-apoptotic physiological functions during and after normal development.

View Article: PubMed Central - PubMed

Affiliation: 1] W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205 USA [2] School of Life Sciences and Center for Soybean Research of the State Key Laboratory of Agrobiotechnology, Chinese University of Hong Kong, Shatin, Hong Kong SAR, China.

ABSTRACT
The discovery that mammalian cells can survive late-stage apoptosis challenges the general assumption that active caspases are markers of impending death. However, tools have not been available to track healthy cells that have experienced caspase activity at any time in the past. Therefore, to determine if cells in whole animals can undergo reversal of apoptosis, known as anastasis, we developed a dual color CaspaseTracker system for Drosophila to identify cells with ongoing or past caspase activity. Transient exposure of healthy females to environmental stresses such as cold shock or starvation activated the CaspaseTracker coincident with caspase activity and apoptotic morphologies in multiple cell types of developing egg chambers. Importantly, when stressed flies were returned to normal conditions, morphologically healthy egg chambers and new progeny flies were labeled by the biosensor, suggesting functional recovery from apoptotic caspase activation. In striking contrast to developing egg chambers, which lack basal caspase biosensor activation under normal conditions, many adult tissues of normal healthy flies exhibit robust caspase biosensor activity in a portion of cells, including neurons. The widespread persistence of CaspaseTracker-positivity implies that healthy cells utilize active caspases for non-apoptotic physiological functions during and after normal development.

No MeSH data available.


Related in: MedlinePlus

The CaspaseTracker System for detection of apoptosis and anastasis in vivo.(a) Schematic of Drosophila ovary, and flow chart for cold shock-, and protein starvation-induced cell death in 1-day-old flies, followed by 3-days recovery at normal condition. Drosophila ovary drawing is provided by Polan Santos; Drosophila image is provided by Darren Obbard. Used with permission (b) Egg chambers from the ovary of 6-day female CaspaseTracker flies fed with normal fly food for 6 days (untreated). (c) Caspase biosensor activity in egg chambers of CaspaseTracker Drosophila at 1 day after cold shock (−7°C, 1 hour, followed by 25°C for 24 hours) to induce apoptosis in egg chambers. (d) Caspase biosensor activity in egg chambers of CaspaseTracker Drosophila fed 3 days with 8% sucrose in 1% agar (starved) to induce apoptosis in egg chambers. (e) Like panel c except flies were then switched to normal conditions for 3 days after cold shock (CS recovered). (f) Like panel d except flies were switched to normal yeast-based fly food for 3 days after starvation (refed). Panel at left most is merged confocal image of RFP, NucGFP, nuclei, cleaved-caspase immuno-staining and DIC for overview of egg chambers at the ovaries; middle left panel is enlarged view of the dotted box at the left most panel; middle, middle right, and right most panels display biosensor RFP and NucGFP, nucleus, and cleaved caspase, respectively. (g) Quantification of RFP and NucGFP expression in egg chambers of CaspaseTracker (DQVD) flies before and after apoptosis induction. Caspase insensitive CaspaseTracker (DQVA) files serve as controls. Data presented are from 3 different batches of flies (n = 20), counting 100 egg chambers from each batch per condition. Error bars denote SD. (h) Confocal image of egg chambers recovered 3 days after starvation. Nuclear GFP in nurse cells (black arrows), oocytes (white arrows) and follicle cells (yellow arrows) of egg chambers, and in the germarium (green arrow). (i) GFP and non-GFP expressing progeny from starved and refed CaspaseTracker (DQVD) female flies.
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f2: The CaspaseTracker System for detection of apoptosis and anastasis in vivo.(a) Schematic of Drosophila ovary, and flow chart for cold shock-, and protein starvation-induced cell death in 1-day-old flies, followed by 3-days recovery at normal condition. Drosophila ovary drawing is provided by Polan Santos; Drosophila image is provided by Darren Obbard. Used with permission (b) Egg chambers from the ovary of 6-day female CaspaseTracker flies fed with normal fly food for 6 days (untreated). (c) Caspase biosensor activity in egg chambers of CaspaseTracker Drosophila at 1 day after cold shock (−7°C, 1 hour, followed by 25°C for 24 hours) to induce apoptosis in egg chambers. (d) Caspase biosensor activity in egg chambers of CaspaseTracker Drosophila fed 3 days with 8% sucrose in 1% agar (starved) to induce apoptosis in egg chambers. (e) Like panel c except flies were then switched to normal conditions for 3 days after cold shock (CS recovered). (f) Like panel d except flies were switched to normal yeast-based fly food for 3 days after starvation (refed). Panel at left most is merged confocal image of RFP, NucGFP, nuclei, cleaved-caspase immuno-staining and DIC for overview of egg chambers at the ovaries; middle left panel is enlarged view of the dotted box at the left most panel; middle, middle right, and right most panels display biosensor RFP and NucGFP, nucleus, and cleaved caspase, respectively. (g) Quantification of RFP and NucGFP expression in egg chambers of CaspaseTracker (DQVD) flies before and after apoptosis induction. Caspase insensitive CaspaseTracker (DQVA) files serve as controls. Data presented are from 3 different batches of flies (n = 20), counting 100 egg chambers from each batch per condition. Error bars denote SD. (h) Confocal image of egg chambers recovered 3 days after starvation. Nuclear GFP in nurse cells (black arrows), oocytes (white arrows) and follicle cells (yellow arrows) of egg chambers, and in the germarium (green arrow). (i) GFP and non-GFP expressing progeny from starved and refed CaspaseTracker (DQVD) female flies.

Mentions: Environmental insults such as cold shock and protein starvation cause physiological stress that triggers cell death including death by apoptosis in Drosophila353637. Cold shock can cause massive cellular damage such as loss of cell membrane selective permeability3839. Cell death induced by starvation during Drosophila oogenesis is due in part to caspases, apparently to match female fertility to environmental resources4041. CaspaseTracker (DQVD) biosensor flies were analyzed the day after a cold shock (1 hour at −7°C) and after 3 days of protein starvation (8% sucrose in 1% agar). Egg chambers in the ovaries of only the treated flies developed both cytoplasmic red (recent/ongoing) and nuclear green (permanent) biosensor activity (Fig. 2a–d). Biosensor activation was accompanied by evidence of apoptosis, including nuclear condensation and immunoreactivity for cleaved effector caspases, a marker of activated caspases in mammals and Drosophila364243 (Fig. 2c and d, and Supplementary Figs. 2 and 3). In contrast, mutation of the caspase cleavage site in the control caspase-insensitive (DQVA) biosensor abolished CaspaseTracker activity, indicating caspase-specific cleavage after the known Asp cleavage site in the DQVD biosensor. This difference was not explained by the lack of cell death in DQVA flies, as egg chambers from both DQVD and DQVA biosensor flies exhibited similar morphological changes and cleaved/active caspase immunoreactivity (Supplementary Figs. 2 and 3).


In vivo CaspaseTracker biosensor system for detecting anastasis and non-apoptotic caspase activity.

Tang HL, Tang HM, Fung MC, Hardwick JM - Sci Rep (2015)

The CaspaseTracker System for detection of apoptosis and anastasis in vivo.(a) Schematic of Drosophila ovary, and flow chart for cold shock-, and protein starvation-induced cell death in 1-day-old flies, followed by 3-days recovery at normal condition. Drosophila ovary drawing is provided by Polan Santos; Drosophila image is provided by Darren Obbard. Used with permission (b) Egg chambers from the ovary of 6-day female CaspaseTracker flies fed with normal fly food for 6 days (untreated). (c) Caspase biosensor activity in egg chambers of CaspaseTracker Drosophila at 1 day after cold shock (−7°C, 1 hour, followed by 25°C for 24 hours) to induce apoptosis in egg chambers. (d) Caspase biosensor activity in egg chambers of CaspaseTracker Drosophila fed 3 days with 8% sucrose in 1% agar (starved) to induce apoptosis in egg chambers. (e) Like panel c except flies were then switched to normal conditions for 3 days after cold shock (CS recovered). (f) Like panel d except flies were switched to normal yeast-based fly food for 3 days after starvation (refed). Panel at left most is merged confocal image of RFP, NucGFP, nuclei, cleaved-caspase immuno-staining and DIC for overview of egg chambers at the ovaries; middle left panel is enlarged view of the dotted box at the left most panel; middle, middle right, and right most panels display biosensor RFP and NucGFP, nucleus, and cleaved caspase, respectively. (g) Quantification of RFP and NucGFP expression in egg chambers of CaspaseTracker (DQVD) flies before and after apoptosis induction. Caspase insensitive CaspaseTracker (DQVA) files serve as controls. Data presented are from 3 different batches of flies (n = 20), counting 100 egg chambers from each batch per condition. Error bars denote SD. (h) Confocal image of egg chambers recovered 3 days after starvation. Nuclear GFP in nurse cells (black arrows), oocytes (white arrows) and follicle cells (yellow arrows) of egg chambers, and in the germarium (green arrow). (i) GFP and non-GFP expressing progeny from starved and refed CaspaseTracker (DQVD) female flies.
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Related In: Results  -  Collection

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f2: The CaspaseTracker System for detection of apoptosis and anastasis in vivo.(a) Schematic of Drosophila ovary, and flow chart for cold shock-, and protein starvation-induced cell death in 1-day-old flies, followed by 3-days recovery at normal condition. Drosophila ovary drawing is provided by Polan Santos; Drosophila image is provided by Darren Obbard. Used with permission (b) Egg chambers from the ovary of 6-day female CaspaseTracker flies fed with normal fly food for 6 days (untreated). (c) Caspase biosensor activity in egg chambers of CaspaseTracker Drosophila at 1 day after cold shock (−7°C, 1 hour, followed by 25°C for 24 hours) to induce apoptosis in egg chambers. (d) Caspase biosensor activity in egg chambers of CaspaseTracker Drosophila fed 3 days with 8% sucrose in 1% agar (starved) to induce apoptosis in egg chambers. (e) Like panel c except flies were then switched to normal conditions for 3 days after cold shock (CS recovered). (f) Like panel d except flies were switched to normal yeast-based fly food for 3 days after starvation (refed). Panel at left most is merged confocal image of RFP, NucGFP, nuclei, cleaved-caspase immuno-staining and DIC for overview of egg chambers at the ovaries; middle left panel is enlarged view of the dotted box at the left most panel; middle, middle right, and right most panels display biosensor RFP and NucGFP, nucleus, and cleaved caspase, respectively. (g) Quantification of RFP and NucGFP expression in egg chambers of CaspaseTracker (DQVD) flies before and after apoptosis induction. Caspase insensitive CaspaseTracker (DQVA) files serve as controls. Data presented are from 3 different batches of flies (n = 20), counting 100 egg chambers from each batch per condition. Error bars denote SD. (h) Confocal image of egg chambers recovered 3 days after starvation. Nuclear GFP in nurse cells (black arrows), oocytes (white arrows) and follicle cells (yellow arrows) of egg chambers, and in the germarium (green arrow). (i) GFP and non-GFP expressing progeny from starved and refed CaspaseTracker (DQVD) female flies.
Mentions: Environmental insults such as cold shock and protein starvation cause physiological stress that triggers cell death including death by apoptosis in Drosophila353637. Cold shock can cause massive cellular damage such as loss of cell membrane selective permeability3839. Cell death induced by starvation during Drosophila oogenesis is due in part to caspases, apparently to match female fertility to environmental resources4041. CaspaseTracker (DQVD) biosensor flies were analyzed the day after a cold shock (1 hour at −7°C) and after 3 days of protein starvation (8% sucrose in 1% agar). Egg chambers in the ovaries of only the treated flies developed both cytoplasmic red (recent/ongoing) and nuclear green (permanent) biosensor activity (Fig. 2a–d). Biosensor activation was accompanied by evidence of apoptosis, including nuclear condensation and immunoreactivity for cleaved effector caspases, a marker of activated caspases in mammals and Drosophila364243 (Fig. 2c and d, and Supplementary Figs. 2 and 3). In contrast, mutation of the caspase cleavage site in the control caspase-insensitive (DQVA) biosensor abolished CaspaseTracker activity, indicating caspase-specific cleavage after the known Asp cleavage site in the DQVD biosensor. This difference was not explained by the lack of cell death in DQVA flies, as egg chambers from both DQVD and DQVA biosensor flies exhibited similar morphological changes and cleaved/active caspase immunoreactivity (Supplementary Figs. 2 and 3).

Bottom Line: Importantly, when stressed flies were returned to normal conditions, morphologically healthy egg chambers and new progeny flies were labeled by the biosensor, suggesting functional recovery from apoptotic caspase activation.In striking contrast to developing egg chambers, which lack basal caspase biosensor activation under normal conditions, many adult tissues of normal healthy flies exhibit robust caspase biosensor activity in a portion of cells, including neurons.The widespread persistence of CaspaseTracker-positivity implies that healthy cells utilize active caspases for non-apoptotic physiological functions during and after normal development.

View Article: PubMed Central - PubMed

Affiliation: 1] W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205 USA [2] School of Life Sciences and Center for Soybean Research of the State Key Laboratory of Agrobiotechnology, Chinese University of Hong Kong, Shatin, Hong Kong SAR, China.

ABSTRACT
The discovery that mammalian cells can survive late-stage apoptosis challenges the general assumption that active caspases are markers of impending death. However, tools have not been available to track healthy cells that have experienced caspase activity at any time in the past. Therefore, to determine if cells in whole animals can undergo reversal of apoptosis, known as anastasis, we developed a dual color CaspaseTracker system for Drosophila to identify cells with ongoing or past caspase activity. Transient exposure of healthy females to environmental stresses such as cold shock or starvation activated the CaspaseTracker coincident with caspase activity and apoptotic morphologies in multiple cell types of developing egg chambers. Importantly, when stressed flies were returned to normal conditions, morphologically healthy egg chambers and new progeny flies were labeled by the biosensor, suggesting functional recovery from apoptotic caspase activation. In striking contrast to developing egg chambers, which lack basal caspase biosensor activation under normal conditions, many adult tissues of normal healthy flies exhibit robust caspase biosensor activity in a portion of cells, including neurons. The widespread persistence of CaspaseTracker-positivity implies that healthy cells utilize active caspases for non-apoptotic physiological functions during and after normal development.

No MeSH data available.


Related in: MedlinePlus