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A Guide to Fluorescent Protein FRET Pairs
Förster or fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide a powerful tool for visualizing signaling molecules in live cells with high spatiotemporal resolution. Fluorescent proteins (FPs) are most commonly used as both donor and acceptor fluorophores in FRET biosensors, especially since FPs are genetically encodable and live-cell compatible. In this review, we will provide an overview of methods to measure FRET changes in biological contexts, discuss the palette of FP FRET pairs developed and their relative strengths and weaknesses, and note important factors to consider when using FPs for FRET studies
Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM
Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research
Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs
Förster resonance energy transfer (FRET) is a powerful method for obtaining information about small-scale lengths between biomacromolecules. Visible fluorescent proteins (VFPs) are widely used as spectrally different FRET pairs, where one VFP acts as a donor and another VFP as an acceptor. The VFPs are usually fused to the proteins of interest, and this fusion product is genetically encoded in cells. FRET between VFPs can be determined by analysis of either the fluorescence decay properties of the donor molecule or the rise time of acceptor fluorescence. Time-resolved fluorescence spectroscopy is the technique of choice to perform these measurements. FRET can be measured not only in solution, but also in living cells by the technique of fluorescence lifetime imaging microscopy (FLIM), where fluorescence lifetimes are determined with the spatial resolution of an optical microscope. Here we focus attention on time-resolved fluorescence spectroscopy of purified, selected VFPs (both single VFPs and FRET pairs of VFPs) in cuvette-type experiments. For quantitative interpretation of FRET–FLIM experiments in cellular systems, details of the molecular fluorescence are needed that can be obtained from experiments with isolated VFPs. For analysis of the time-resolved fluorescence experiments of VFPs, we have utilised the maximum entropy method procedure to obtain a distribution of fluorescence lifetimes. Distributed lifetime patterns turn out to have diagnostic value, for instance, in observing populations of VFP pairs that are FRET-inactiv
A practical review on the measurement tools for cellular adhesion force
Cell cell and cell matrix adhesions are fundamental in all multicellular
organisms. They play a key role in cellular growth, differentiation, pattern
formation and migration. Cell-cell adhesion is substantial in the immune
response, pathogen host interactions, and tumor development. The success of
tissue engineering and stem cell implantations strongly depends on the fine
control of live cell adhesion on the surface of natural or biomimetic
scaffolds. Therefore, the quantitative and precise measurement of the adhesion
strength of living cells is critical, not only in basic research but in modern
technologies, too. Several techniques have been developed or are under
development to quantify cell adhesion. All of them have their pros and cons,
which has to be carefully considered before the experiments and interpretation
of the recorded data. Current review provides a guide to choose the appropriate
technique to answer a specific biological question or to complete a biomedical
test by measuring cell adhesion
Fluorescence energy transfer enhancement in aluminum nanoapertures
Zero-mode waveguides (ZMWs) are confining light into attoliter volumes,
enabling single molecule fluorescence experiments at physiological micromolar
concentrations. Among the fluorescence spectroscopy techniques that can be
enhanced by ZMWs, F\"{o}rster resonance energy transfer (FRET) is one of the
most widely used in life sciences. Combining zero-mode waveguides with FRET
provides new opportunities to investigate biochemical structures or follow
interaction dynamics at micromolar concentration with single molecule
resolution. However, prior to any quantitative FRET analysis on biological
samples, it is crucial to establish first the influence of the ZMW on the FRET
process. Here, we quantify the FRET rates and efficiencies between individual
donor-acceptor fluorophore pairs diffusing in aluminum zero-mode waveguides.
Aluminum ZMWs are important structures thanks to their commercial availability
and the large literature describing their use for single molecule fluorescence
spectroscopy. We also compare the results between ZMWs milled in gold and
aluminum, and find that while gold has a stronger influence on the decay rates,
the lower losses of aluminum in the green spectral region provide larger
fluorescence brightness enhancement factors. For both aluminum and gold ZMWs,
we observe that the FRET rate scales linearly with the isolated donor decay
rate and the local density of optical states (LDOS). Detailed information about
FRET in ZMWs unlocks their application as new devices for enhanced single
molecule FRET at physiological concentrations
Single molecule studies on the dynamics of the transcription initiation complex of yeast mitochondria
Department of Biomedical EngineeringThe transcription initiation complex in the yeast mitochondria of Saccharomyces cerevisiae comprises the RNA polymerase, Rpo41, the initiation factor, Mtf1, and the DNA including 6 base pair promoter sequence. The Mtf1 is known to recognize and help to open the promoter region during the initiation stage, but its exact role and mechanism still remains unclear. We designed a multi-color single molecule FRET assay to directly measure the dynamics of the complex during transcription initiation. The labels on the DNA report on its opening-closing dynamics, while the label on Mtf1 report on the recruitment, dynamics, and dissociation of the initiation factor. From these measurements, we can correlate the promoter opening dynamics, factor binding/dissociation, and the transition to the elongation phase. Mtf1 is also associated with controlling the production of abortive RNA transcripts. We observed the scrunching motion during transcription by stepping along the DNA template with various combinations of nucleotide substrates. The FRET distribution shifted toward the high FRET region as we stepped further. From these observations, we propose a mechanistic model of the transcription initiation in the yeast mitochondria.ope
Nanophotonic enhancement of the F\"orster resonance energy transfer rate on single DNA molecules
Nanophotonics achieves accurate control over the luminescence properties of a
single quantum emitter by tailoring the light-matter interaction at the
nanoscale and modifying the local density of optical states (LDOS). This
paradigm could also benefit to F\"orster resonance energy transfer (FRET) by
enhancing the near-field electromagnetic interaction between two fluorescent
emitters. Despite the wide applications of FRET in nanosciences, using
nanophotonics to enhance FRET remains a debated and complex challenge. Here, we
demonstrate enhanced energy transfer within single donor-acceptor fluorophore
pairs confined in gold nanoapertures. Experiments monitoring both the donor and
the acceptor emission photodynamics at the single molecule level clearly
establish a linear dependence of the FRET rate on the LDOS in nanoapertures.
These findings are applied to enhance the FRET rate in nanoapertures up to six
times, demonstrating that nanophotonics can be used to intensify the near-field
energy transfer and improve the biophotonic applications of FRET
The Rac-FRET mouse reveals tight spatiotemporal control of Rac activity in primary cells and tissues
The small G protein family Rac has numerous regulators that integrate extracellular signals into tight spatiotemporal maps of its activity to promote specific cell morphologies and responses. Here, we have generated a mouse strain, Rac-FRET, which ubiquitously expresses the Raichu-Rac biosensor. It enables FRET imaging and quantification of Rac activity in live tissues and primary cells without affecting cell properties and responses. We assessed Rac activity in chemotaxing Rac-FRET neutrophils and found enrichment in leading-edge protrusions and unexpected longitudinal shifts and oscillations during protruding and stalling phases of migration. We monitored Rac activity in normal or disease states of intestinal, liver, mammary, pancreatic, and skin tissue, in response to stimulation or inhibition and upon genetic manipulation of upstream regulators, revealing unexpected insights into Rac signaling during disease development. The Rac-FRET strain is a resource that promises to fundamentally advance our understanding of Rac-dependent responses in primary cells and native environments
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