scholarly journals Real-Time Monitoring of Somatostatin Receptor-cAMP Signaling in Live Pituitary

Endocrinology ◽  
2010 ◽  
Vol 151 (9) ◽  
pp. 4560-4565 ◽  
Author(s):  
Stefan Jacobs ◽  
Davide Calebiro ◽  
Viacheslav O. Nikolaev ◽  
Martin J. Lohse ◽  
Stefan Schulz
Keyword(s):  
Real Time ◽  
Somatostatin Receptor ◽  
Somatostatin Receptors ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Pituitary Cells ◽  
Gi Protein ◽  
Camp Levels ◽  
Pituitary Receptors

Fluorescence resonance energy transfer using genetically encoded biosensors has proven to be a powerful technique to monitor the spatiotemporal dynamics of cAMP signals stimulated by Gs-coupled receptors in living cells. In contrast, real-time imaging of Gi-mediated cAMP signals under native conditions remains challenging. Here, we describe the use of transgenic mice for cAMP imaging in living pituitary slices and primary pituitary cells. This technique can be widely used to assess the contribution of various pituitary receptors, including individual Gi protein-coupled somatostatin receptors, to the regulation of cAMP levels under physiologically relevant settings.

Development and use of fluorescent nanosensors for metabolite imaging in living cells

2005 ◽  
Vol 33 (1) ◽  
pp. 287-290 ◽  
Author(s):  
M. Fehr ◽  
S. Okumoto ◽  
K. Deuschle ◽  
I. Lager ◽  
L.L. Looger ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Real Time ◽  
Conformational Change ◽  
Binding Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Periplasmic Binding Proteins ◽  
Living Organisms ◽  
Subcellular Resolution

To understand metabolic networks, fluxes and regulation, it is crucial to be able to determine the cellular and subcellular levels of metabolites. Methods such as PET and NMR imaging have provided us with the possibility of studying metabolic processes in living organisms. However, at present these technologies do not permit measuring at the subcellular level. The cameleon, a fluorescence resonance energy transfer (FRET)-based nanosensor uses the ability of the calcium-bound form of calmodulin to interact with calmodulin binding polypeptides to turn the corresponding dramatic conformational change into a change in resonance energy transfer between two fluorescent proteins attached to the fusion protein. The cameleon and its derivatives were successfully used to follow calcium changes in real time not only in isolated cells, but also in living organisms. To provide a set of tools for real-time measurements of metabolite levels with subcellular resolution, protein-based nanosensors for various metabolites were developed. The metabolite nanosensors consist of two variants of the green fluorescent protein fused to bacterial periplasmic binding proteins. Different from the cameleon, a conformational change in the binding protein is directly detected as a change in FRET efficiency. The prototypes are able to detect various carbohydrates such as ribose, glucose and maltose as purified proteins in vitro. The nanosensors can be expressed in yeast and in mammalian cell cultures and were used to determine carbohydrate homeostasis in living cells with subcellular resolution. One future goal is to expand the set of sensors to cover a wider spectrum of metabolites by using the natural spectrum of bacterial periplasmic binding proteins and by computational design of the binding pockets of the prototype sensors.

scholarly journals A Fluorescence Resonance Energy Transfer-Based Analytical Tool for Nitrate Quantification in Living Cells

ACS Omega ◽  
2020 ◽  
Vol 5 (46) ◽  
pp. 30306-30314
Author(s):  
Urooj Fatima ◽  
Fuad Ameen ◽  
Neha Soleja ◽  
Parvez Khan ◽  
Abobakr Almansob ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Analytical Tool ◽  
Fluorescence Resonance

scholarly journals Time-resolved luminescence resonance energy transfer imaging of protein-protein interactions in living cells

2010 ◽  
Vol 107 (31) ◽  
pp. 13582-13587 ◽  
Author(s):  
H. E. Rajapakse ◽  
N. Gahlaut ◽  
S. Mohandessi ◽  
D. Yu ◽  
J. R. Turner ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Protein Protein Interactions ◽  
Time Resolved

scholarly journals AIE-Driven Fluorescent Polysaccharide Polymersome as Enzyme-responsive FRET Nanoprobe to Study the Real-time Delivery Aspects in Live-Cells

2021 ◽  
Author(s):  
Nilesh Umakant Deshpande ◽  
Mishika Virmani ◽  
Manickam Jayakannan
Keyword(s):  
Energy Transfer ◽  
Confocal Microscopy ◽  
Real Time ◽  
Fluorescence Resonance Energy Transfer ◽  
Imaging Techniques ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Intracellular Enzyme ◽  
Bio Imaging

We report aggregation induced emission (AIE) driven polysaccharide polymersome as fluorescence resonance energy transfer (FRET) nanoprobes to study their intracellular enzyme-responsive delivery by real-time live-cell confocal microscopy bio-imaging techniques. AIE...

scholarly journals Visualization of growth signal transduction cascades in living cells with genetically encoded probes based on Förster resonance energy transfer

2008 ◽  
Vol 363 (1500) ◽  
pp. 2143-2151 ◽  
Author(s):  
Kazuhiro Aoki ◽  
Etsuko Kiyokawa ◽  
Takeshi Nakamura ◽  
Michiyuki Matsuda
Keyword(s):  
Signal Transduction ◽  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Small Gtpases ◽  
Forster Resonance Energy Transfer ◽  
Fluorescence Probes ◽  
Förster Resonance Energy Transfer ◽  
Förster Resonance

Fluorescence probes based on the principle of Förster resonance energy transfer (FRET) have shed new light on our understanding of signal transduction cascades. Among them, unimolecular FRET probes containing fluorescence proteins are rapidly increasing in number because these genetically encoded probes can be easily loaded into living cells and allow simple acquisition of FRET images. We have developed probes for small GTPases, tyrosine kinases, serine–threonine kinases and phosphoinositides. Images obtained with these probes have revealed that membrane protrusions such as nascent lamellipodia or neurites provide an active signalling platform in the growth factor-stimulated cells.

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