A fluorescent reporter of caspase activity for live imaging.
There is a growing interest in the mechanisms that control the apoptosis cascade during development and adult life. To investigate the regulatory events that trigger apoptosis in whole tissues, we have devised a genetically encoded caspase sensor that can be detected in live and fixed tissue by standard confocal microscopy. The sensor comprises two fluorophores, mRFP, monomeric red fluorescent protein (mRFP) and enhanced green fluorescent protein (eGFP), that are linked by an efficient and specific caspase-sensitive site. Upon caspase activation, the sensor is cleaved and eGFP translocates to the nucleus, leaving mRFP at membranes. This is detected before other markers of apoptosis, including anti-cleaved caspase 3 immunoreactivity. Moreover, the sensor does not perturb normal developmental apoptosis and is specific, as cleavage does not occur in Drosophila embryos that are unable to activate the apoptotic cascade. Importantly, dying cells can be recognized in live embryos, thus opening the way for in vivo imaging. As expected from the high conservation of caspases, it is also cleaved in dying cells of chick embryos. It is therefore likely to be generally useful to track the spatiotemporal pattern of caspase activity in a variety of species.
Bardet
Pierre-Luc
PL
Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.
Kolahgar
Golnar
G
Mynett
Anita
A
Miguel-Aliaga
Irene
I
Briscoe
James
J
Meier
Pascal
P
Vincent
Jean-Paul
JP
D000818:Animals; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival; D002645:Chickens; D004331:Drosophila melanogaster / Q000196:embryology / Q000201:enzymology; D004625:Embryo, Nonmammalian / Q000196:embryology / Q000201:enzymology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D013131:Spine / Q000201:enzymology; D013379:Substrate Specificity; D000097573:Red Fluorescent Protein
D020169:Caspases; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein; C485184:enhanced green fluorescent protein
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