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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

Post developmental caspase activation, and persistence in the adult of cells that experienced developmental caspase activity at Drosophila anterior midgut.Biosensor fluorescence of anterior midgut of (a) Newly eclosed Gal80ts–temperature sensitive(ts) CaspaseTracker flies raised at 18°C. The thermosensitive Gal80 (Gal80ts) conditionally represses Gal4 at 18°C; biosensor is functional “Off” during development. (b) Newly eclosed 18°C raised (ts) adult CaspaseTracker flies were then shifted to 30°C for 10 days. Gal80ts cannot repress Gal4 at 30°C; biosensor is functional “On” at the 10-day period. (c) Newly eclosed tsCaspaseTracker flies raised at 30°C; biosensor is functional “On” at the development. (d) Newly eclosed 30°C raised (ts) adult CaspaseTracker flies were shifted to 18°C for 10 days. Gal80ts represses Gal4 at 18°C; biosensor is functional “Off” at the 10-day period.
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f4: Post developmental caspase activation, and persistence in the adult of cells that experienced developmental caspase activity at Drosophila anterior midgut.Biosensor fluorescence of anterior midgut of (a) Newly eclosed Gal80ts–temperature sensitive(ts) CaspaseTracker flies raised at 18°C. The thermosensitive Gal80 (Gal80ts) conditionally represses Gal4 at 18°C; biosensor is functional “Off” during development. (b) Newly eclosed 18°C raised (ts) adult CaspaseTracker flies were then shifted to 30°C for 10 days. Gal80ts cannot repress Gal4 at 30°C; biosensor is functional “On” at the 10-day period. (c) Newly eclosed tsCaspaseTracker flies raised at 30°C; biosensor is functional “On” at the development. (d) Newly eclosed 30°C raised (ts) adult CaspaseTracker flies were shifted to 18°C for 10 days. Gal80ts represses Gal4 at 18°C; biosensor is functional “Off” at the 10-day period.

Mentions: Like human gut tissue, the Drosophila gut also undergoes considerable cell turnover47, potentially consistent with the RFP (ongoing/recent) caspase biosensor activity observed throughout the gut, but particularly in regions of the hindgut (Fig. 3a). To determine if cell turnover in the Drosophila midgut also occurs in adults, we generated new biosensor flies with a Gal80ts temperature-sensitive (ts) biosensor (Supplemental Fig. 9). Gal80ts binds and suppresses Gal4 activity at 18°C48, turning off the biosensor, consistent with only rare biosensor-positive cells in tsCaspaseTracker flies raised at 18°C (Fig. 4a). However, the inhibitory effect of Gal80ts was abolished at 30°C48, which turns on the biosensor. Therefore, shifting newly eclosed adult flies from 18°C to 30°C resulted in both GFP and RFP biosensor-positive cells 10 days later in midguts (Fig. 4b) and elsewhere. The presence of RFP biosensor activity at 10 days strongly indicates that caspases were activated in adults.


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

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

Post developmental caspase activation, and persistence in the adult of cells that experienced developmental caspase activity at Drosophila anterior midgut.Biosensor fluorescence of anterior midgut of (a) Newly eclosed Gal80ts–temperature sensitive(ts) CaspaseTracker flies raised at 18°C. The thermosensitive Gal80 (Gal80ts) conditionally represses Gal4 at 18°C; biosensor is functional “Off” during development. (b) Newly eclosed 18°C raised (ts) adult CaspaseTracker flies were then shifted to 30°C for 10 days. Gal80ts cannot repress Gal4 at 30°C; biosensor is functional “On” at the 10-day period. (c) Newly eclosed tsCaspaseTracker flies raised at 30°C; biosensor is functional “On” at the development. (d) Newly eclosed 30°C raised (ts) adult CaspaseTracker flies were shifted to 18°C for 10 days. Gal80ts represses Gal4 at 18°C; biosensor is functional “Off” at the 10-day period.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4355673&req=5

f4: Post developmental caspase activation, and persistence in the adult of cells that experienced developmental caspase activity at Drosophila anterior midgut.Biosensor fluorescence of anterior midgut of (a) Newly eclosed Gal80ts–temperature sensitive(ts) CaspaseTracker flies raised at 18°C. The thermosensitive Gal80 (Gal80ts) conditionally represses Gal4 at 18°C; biosensor is functional “Off” during development. (b) Newly eclosed 18°C raised (ts) adult CaspaseTracker flies were then shifted to 30°C for 10 days. Gal80ts cannot repress Gal4 at 30°C; biosensor is functional “On” at the 10-day period. (c) Newly eclosed tsCaspaseTracker flies raised at 30°C; biosensor is functional “On” at the development. (d) Newly eclosed 30°C raised (ts) adult CaspaseTracker flies were shifted to 18°C for 10 days. Gal80ts represses Gal4 at 18°C; biosensor is functional “Off” at the 10-day period.
Mentions: Like human gut tissue, the Drosophila gut also undergoes considerable cell turnover47, potentially consistent with the RFP (ongoing/recent) caspase biosensor activity observed throughout the gut, but particularly in regions of the hindgut (Fig. 3a). To determine if cell turnover in the Drosophila midgut also occurs in adults, we generated new biosensor flies with a Gal80ts temperature-sensitive (ts) biosensor (Supplemental Fig. 9). Gal80ts binds and suppresses Gal4 activity at 18°C48, turning off the biosensor, consistent with only rare biosensor-positive cells in tsCaspaseTracker flies raised at 18°C (Fig. 4a). However, the inhibitory effect of Gal80ts was abolished at 30°C48, which turns on the biosensor. Therefore, shifting newly eclosed adult flies from 18°C to 30°C resulted in both GFP and RFP biosensor-positive cells 10 days later in midguts (Fig. 4b) and elsewhere. The presence of RFP biosensor activity at 10 days strongly indicates that caspases were activated in adults.

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