Limits...
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

CaspaseTracker biosensor system.(a) Schematic of the CaspaseTracker biosensor system, which is composed of a plasma membrane anchor (mCD8), a caspase-sensitive natural substrate (derived from DIAP1) fused to the Gal4 transcription factor with a C-terminal 3x-myc tag that translocates to the nucleus upon caspase activation to induce the G-Trace system. (b) Schematic of caspase-sensitive (DQVD) and caspase-insensitive control (DQVA) biosensors with the indicated modifications (NN/GV and D135R)3334 to prevent biosensor degradation and to prevent caspase inhibition by the biosensor.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4355673&req=5

f1: CaspaseTracker biosensor system.(a) Schematic of the CaspaseTracker biosensor system, which is composed of a plasma membrane anchor (mCD8), a caspase-sensitive natural substrate (derived from DIAP1) fused to the Gal4 transcription factor with a C-terminal 3x-myc tag that translocates to the nucleus upon caspase activation to induce the G-Trace system. (b) Schematic of caspase-sensitive (DQVD) and caspase-insensitive control (DQVA) biosensors with the indicated modifications (NN/GV and D135R)3334 to prevent biosensor degradation and to prevent caspase inhibition by the biosensor.

Mentions: The CaspaseTracker biosensor system is composed of two genetically encoded components, a caspase-activatable yeast transcription factor Gal4, and the G-TRACE fluorescent protein system32 (Fig. 1a). The caspase-activatable Gal4 is sequestered in the cytoplasm by a fragment of mammalian CD8 (mCD8) fused to a caspase-cleavable linker28. This 146-amino acid linker contains the natural caspase cleavage site DQVD near the BIR1 domain of Drosophila DIAP1 (Fig. 1b). Upon caspase activation, Gal4 is released from its plasma membrane tether, translocates to the nucleus and drives the expression of cytosolic red fluorescent protein (RFP) to indicate recent or on-going caspase activity, and simultaneously drives the expression of FLP recombinase leading to ubiquitous and permanent expression of nuclear-targeted GFP (NucGFP) for the life of the cell and its progeny (Fig. 1a). The DIAP1 linker contains two modifications to improve biosensor function, a 21NN/GV22 mutation to prevent degradation of Gal4 by the N-end rule upon caspase cleavage after Asp202833, and a D135R mutation known to abolish the drICE caspase inhibitory function of the sequence DICG in the BIR1 domain34 (Fig. 1b). Control biosensor flies were generated using the same biosensor with an additional engineered mutation of the obligatory Asp required for cleavage by caspases (DQVD to DQVA)33. For ubiquitous expression, the ubiquitin promoter was used to drive expression of the mCD8-DQVD/A-Gal4-myc CaspaseTracker biosensors. Both the caspase-activatable (DQVD) and control uncleavable (DQVA) biosensors appear to be expressed similarly based on immunostaining of whole mount transgenic Drosophila with anti-myc (Supplementary Fig. 1).


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

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

CaspaseTracker biosensor system.(a) Schematic of the CaspaseTracker biosensor system, which is composed of a plasma membrane anchor (mCD8), a caspase-sensitive natural substrate (derived from DIAP1) fused to the Gal4 transcription factor with a C-terminal 3x-myc tag that translocates to the nucleus upon caspase activation to induce the G-Trace system. (b) Schematic of caspase-sensitive (DQVD) and caspase-insensitive control (DQVA) biosensors with the indicated modifications (NN/GV and D135R)3334 to prevent biosensor degradation and to prevent caspase inhibition by the biosensor.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: CaspaseTracker biosensor system.(a) Schematic of the CaspaseTracker biosensor system, which is composed of a plasma membrane anchor (mCD8), a caspase-sensitive natural substrate (derived from DIAP1) fused to the Gal4 transcription factor with a C-terminal 3x-myc tag that translocates to the nucleus upon caspase activation to induce the G-Trace system. (b) Schematic of caspase-sensitive (DQVD) and caspase-insensitive control (DQVA) biosensors with the indicated modifications (NN/GV and D135R)3334 to prevent biosensor degradation and to prevent caspase inhibition by the biosensor.
Mentions: The CaspaseTracker biosensor system is composed of two genetically encoded components, a caspase-activatable yeast transcription factor Gal4, and the G-TRACE fluorescent protein system32 (Fig. 1a). The caspase-activatable Gal4 is sequestered in the cytoplasm by a fragment of mammalian CD8 (mCD8) fused to a caspase-cleavable linker28. This 146-amino acid linker contains the natural caspase cleavage site DQVD near the BIR1 domain of Drosophila DIAP1 (Fig. 1b). Upon caspase activation, Gal4 is released from its plasma membrane tether, translocates to the nucleus and drives the expression of cytosolic red fluorescent protein (RFP) to indicate recent or on-going caspase activity, and simultaneously drives the expression of FLP recombinase leading to ubiquitous and permanent expression of nuclear-targeted GFP (NucGFP) for the life of the cell and its progeny (Fig. 1a). The DIAP1 linker contains two modifications to improve biosensor function, a 21NN/GV22 mutation to prevent degradation of Gal4 by the N-end rule upon caspase cleavage after Asp202833, and a D135R mutation known to abolish the drICE caspase inhibitory function of the sequence DICG in the BIR1 domain34 (Fig. 1b). Control biosensor flies were generated using the same biosensor with an additional engineered mutation of the obligatory Asp required for cleavage by caspases (DQVD to DQVA)33. For ubiquitous expression, the ubiquitin promoter was used to drive expression of the mCD8-DQVD/A-Gal4-myc CaspaseTracker biosensors. Both the caspase-activatable (DQVD) and control uncleavable (DQVA) biosensors appear to be expressed similarly based on immunostaining of whole mount transgenic Drosophila with anti-myc (Supplementary Fig. 1).

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