Transgenic fruit-flies expressing a FRET-based sensor for in vivo imaging of cAMP dynamics.
3'-5'-cyclic adenosine monophosphate (cAMP) is a ubiquitous intracellular second messenger that mediates the action of various hormones and neurotransmitters and influences a plethora of cellular functions. In particular, multiple neuronal processes such as synaptic plasticity underlying learning and memory are dependent on cAMP signalling cascades. It is now well recognized that the specificity and fidelity of cAMP downstream effects are achieved through a tight temporal as well as spatial control of the cAMP signals. Approaches relying on real-time imaging and Fluorescence Resonance Energy Transfer (FRET)-based biosensors for direct visualization of cAMP changes as they happen in intact living cells have recently started to uncover the fine details of cAMP spatio-temporal signalling patterns. Here we report the generation of transgenic fruit-flies expressing a FRET-based, GFP-PKA sensor and their use in real-time optical recordings of cAMP signalling both ex vivo and in vivo in adult and developing organisms. These transgenic animals represent a novel tool for understanding the physiology of the cAMP signalling pathway in the context of a functioning body.
Lissandron
Valentina
V
Dulbecco Telethon Institute at the Venetian Institute of Molecular Medicine, via Orus 2, Padova, Italy.
Rossetto
Maria Giovanna
MG
Erbguth
Karen
K
Fiala
André
A
Daga
Andrea
A
Zaccolo
Manuela
M
D000818:Animals; D030801:Animals, Genetically Modified / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods*; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics; D029721:Drosophila Proteins / Q000235:genetics; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics*; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000378:metabolism; D005123:Eye / Q000166:cytology / Q000201:enzymology; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism; D021621:Imaging, Three-Dimensional / Q000379:methods*; D007814:Larva / Q000166:cytology; D018613:Microscopy, Confocal; D009420:Nervous System / Q000196:embryology; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins; D012469:Salivary Glands / Q000166:cytology
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D011993:Recombinant Fusion Proteins; C113278:ey protein, Drosophila; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases