10 May 2012 00:00 hrs.
Third generation genetically encoded FRET probes – Application to GPCR signaling

Prof. Dr. Theodorus W.J. Gadella Jr. and Dr. Ir. Joachim Goedhart, Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, Amsterdam


Dr. Ir. Merel Adjobo-Hermans, Department of Biochemistry, NCMLS Nijmegen

10-05-2012 00:00:00Europe/AmsterdamThird generation genetically encoded FRET probes – Application to GPCR signaling

Remarks / more information:

Genetically encoded probes and sensors have found a wide application for visualizing biological processes at different scales, ranging from single molecules to complete organisms. Sensors for specific small molecules (second messengers, metabolites), protein activity (kinase activity, activation of GTPases) and protein-protein interactions are often based on Förster Resonance Energy Transfer (FRET) between spectral variants of green fluorescent protein. Cyan Fluorescent Proteins (CFPs) are widely used as donors in these FRET sensors. The prototypal CFP has long been the modestly bright ECFP. Saturation mutagenesis, guided by structural information, combined with fluorescence lifetime-based screening generated a novel variant, mTurquoise2. This is a brighter variant with faster maturation, longer mono-exponential lifetime and the highest quantum yield (0.93) ever measured for a fluorescent protein. We discuss applications of bright CFP variants and other genetically encoded probes for visualizing cellular processes, with a focus on signal transduction cascades that are activated by G-protein coupled receptors (GPCRs).

Signaling cascades triggered by GPCRs control a variety of important physiological processes and misregulation is implicated in hypertension, cancer and neurological disorders. Upon receptor activation, the G-protein complex undergoes an activating conformational change. The classical view is that a linear pathway downstream of the G-proteins is activated. However, a more complex picture has emerged recently, involving multiple effectors, feedback circuitry and competition between pathways. We will discuss how we can increase our understanding of signaling through G-proteins, by acquiring high-quality quantitative kinetic data from single living cells using microscopic methods.


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