FRET as a biomolecular research tool — understanding its potential while avoiding pitfalls

International audienceThe applications of Förster resonance energy transfer (FRET) grow with each year. However, different FRET techniques are not applied consistently, nor are results uniformly presented, which makes implementing and reproducing FRET experiments challenging. We discuss important considerations for designing and evaluating ensemble FRET experiments. Alongside a primer on FRET basics, we provide guidelines for making experimental design choices such as the donor-acceptor pair, instrumentation and labeling chemistries; selecting control experiments to unambiguously demonstrate FRET and validate that the experiments provide meaningful data about the biomolecular process in question; analyzing raw data and assessing the results; and reporting data and experimental details in a manner that easily allows for reproducibility. Some considerations are also given for FRET assays and FRET imaging, especially with fluorescent proteins. Our goal is to motivate and empower all biologists to consider FRET for the powerful research tool it can be

In Vivo Monitoring of Calpain Activity by Forster Resonance Energy Transfer

International audienc

Chasing the signaling run by tri-molecular time-lapse FRET microscopy

Abstract A feasible design is made to measure three protein/protein interactions to visualize signal pathways by time-lapse Förster resonance energy transfer (FRET) microscopy. When interacting proteins are in close proximity, excitation energy is provided to allow the energy flow from the first molecule to excite the second, followed by energy transfer to the third. By phorbol ester/calcium ionophore stimulation, for example, a real-time complex formation of ectopic IκBα/ERK/WWOX occurs as measured by FRET microscopy, indicative of an ongoing functional signaling. Hyaluronan induces membrane Hyal-2 signaling, which allows FRET measurement of the complex formation of ectopic Smad4/WWOX/Hyal-2 for causing bubbling cell death. If ectopic p53 is recruited to replace Hyal-2, the resulting ectopic Smad4/WWOX/p53 complex induces membrane blebbing without cell death. Together, in this perspective review article, we demonstrate the utilization of time-lapse FRET microscopy to visualize the signaling event via the tri-molecular protein complex formation and their biological outcomes. We show an initial two-protein binding to form the driving force to jumpstart the tri-molecular execution for the signal pathway

Improved split fluorescent proteins for endogenous protein labeling

Split fluorescent proteins (FPs) have been widely used to visualise proteins in cells. Here the authors develop a screen for engineering new split FPs, and report a yellow-green split-mNeonGreen2 with reduced background, a red split-sfCherry2 for multicolour labeling, and its photoactivatable variant for super-resolution use