GFP-based Fluorescence Resonance Energy Transfer Research Articles

Fluorescence Resonance Energy Transfer (FRET)-based Detection of Profilin–VASP Interaction

Profilins belong to a family of small G-actin binding proteins which are thought to assist in F-actin elongation at the leading edge of migrating cells through their interactions with a host of actin-binding proteins including Ena (enabled)/VASP (vasodilator stimulated phosphoprotein). Profilin's interactions with the major actin regulators have been studied almost exclusively using biochemical methods. Therefore spatiotemporal features of these protein-protein interactions have not been resolved so far. In this paper, we for the first time demonstrate the feasibility of GFP-based fluorescence resonance energy transfer (FRET) technique to detect VASP's interaction with profilin-1, a ubiquitously expressed member of profilin family of genes. Specifically, we performed acceptor photobleaching FRET in MDA-MB-231 breast cancer cells to show prominent VASP-Pfn1 interaction at the membrane ruffles near the leading edge.

An experimental study of GFP‐based FRET, with application to intrinsically unstructured proteins

We have experimentally studied the fluorescence resonance energy transfer (FRET) between green fluorescent protein (GFP) molecules by inserting folded or intrinsically unstructured proteins between CyPet and Ypet. We discovered that most of the enhanced FRET signal previously reported for this pair was due to enhanced dimerization, so we engineered a monomerizing mutation into each. An insert containing a single fibronectin type III domain (3.7 nm end-to-end) gave a moderate FRET signal while a two-domain insert (7.0 nm) gave no FRET. We then tested unstructured proteins of various lengths, including the charged-plus-PQ domain of ZipA, the tail domain of alpha-adducin, and the C-terminal tail domain of FtsZ. The structures of these FRET constructs were also studied by electron microscopy and sedimentation. A 12 amino acid linker and the N-terminal 33 amino acids of the charged domain of the ZipA gave strong FRET signals. The C-terminal 33 amino acids of the PQ domain of the ZipA and several unstructured proteins with 66-68 amino acids gave moderate FRET signals. The 150 amino acid charged-plus-PQ construct gave a barely detectable FRET signal. FRET efficiency was calculated from the decreased donor emission to estimate the distance between donor and acceptor. The donor-acceptor distance varied for unstructured inserts of the same length, suggesting that they had variable stiffness (persistence length). We conclude that GFP-based FRET can be useful for studying intrinsically unstructured proteins, and we present a range of calibrated protein inserts to experimentally determine the distances that can be studied.

Mutation in the SH1 helix reduces the activation energy of the ATP-induced conformational transition of myosin

The SH1 helix is a joint that links the converter subdomain to the rest of the myosin motor domain. Recently, we showed that a mutation within the SH1 helix in Dictyostelium myosin II (R689H) reduced the elasticity and thermal stability of the protein. To reveal the involvement of the SH1 helix in ATP-dependent conformational changes of the motor domain, we have investigated the effects of the R689H mutation on the conformational changes of the converter, using a GFP-based fluorescence resonance energy transfer method. Although the mutation does not seem to strongly affect conformations, we found that it significantly reduced the activation energy required for the ATP-induced conformational transition corresponding to the recovery stroke. Given the effects of the mutation on the mechanical properties of myosin, we propose that the SH1 helix plays an important role in the mechanochemical energy conversion underlying the conformational change of the myosin motor domain.

GFP-based FRET analysis in live cells

Fluorescence resonance energy transfer (FRET) is a widely utilized optical technique for measuring small distances of 1–10 nm in live cells. In recent years, its application has been greatly popularized by the discovery of green fluorescent protein (GFP) and many improved variants which make good donor–acceptor fluorophore pairs. GFP-based proteins are structurally stable, relatively inert, and can be reliably attached to points of interest. The combination of easy access to the GFP-based FRET technique and its obvious usefulness in many applications can lead to complacency. Potential problems such as light contaminants, e.g., bleed-through and cross-talk, and inconsistent donor and acceptor concentrations are easily overlooked and can lead to errors in FRET calculation and data interpretation. In this article, we outline possible pitfalls of GFP-based FRET and approaches that address these issues, including a “Spectra FRET” technique that can be easily applied to live cell studies.

Monitoring spatio-temporal regulation of Ras and Rho GTPases with GFP-based FRET probes

GFP-based fluorescence resonance energy transfer (FRET) probes that visualize local activity-changes of Ras and Rho GTPases in living cells are now available for examining the spatio-temporal regulation of these proteins. This article describes principles and strategies to develop intramolecular FRET probes for Ras- and Rho-family GTPases. The procedure for characterizing candidate probes, and image acquisition and processing are also explained. An optimal FRET probe should have (i) a wide dynamic range (which means a high sensitivity), (ii) a high fluorescence intensity, (iii) target specificity, and (iv) a minimal perturbation to endogenous signaling cascades. Although an improvement of FRET probes should be executed in a trial-and-error manner, practical tips for optimization are provided here. In addition, we illustrate some applications of FRET probes for neuronal cells, which are composed of diverse subcellular compartments with different functions; thus, tools to decipher the dynamics of GTPase activity in each compartment have long been desired.

Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins.

Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for various cellular functions. Although most indicators have cyan- and yellow-emitting fluorescent proteins (CFP and YFP) as FRET donor and acceptor, their poor dynamic range often prevents detection of subtle but significant signals. Here, we optimized the relative orientation of the two chromophores in the Ca(2+) indicator, yellow cameleon (YC), by fusing YFP at different angles. We generated circularly permuted YFPs (cpYFPs) that showed efficient maturation and acid stability. One of the cpYFPs incorporated in YC absorbs a great amount of excited energy from CFP in its Ca(2+)-saturated form, thereby increasing the Ca(2+)-dependent change in the ratio of YFP/CFP by nearly 600%. Both in cultured cells and in the nervous system of transgenic mice, the new YC enables visualization of subcellular Ca(2+) dynamics with better spatial and temporal resolution than before. Our study provides an important guide for the development and improvement of indicators using GFP-based FRET.

Visualization of glucocorticoid receptor and mineralocorticoid receptor interactions in living cells with GFP-based fluorescence resonance energy transfer.

Adrenal corticosteroids readily enter the brain and exert markedly diverse effects, including stress responses in the target neural cells via two receptor systems, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). It has been shown that the GR and MR are highly colocalized in the hippocampus. Given the differential action of the MR and GR in the hippocampal region, it is important to elucidate how these receptors interact with each other in response to corticosteroids. We investigated the heterodimerization of the MR and GR with green fluorescent protein-based fluorescence resonance energy transfer (FRET) microscopy in living cells with spatiotemporal manner. FRET was evaluated in three ways: (1) ratio imaging; (2) emission spectra; and (3) acceptor photobleaching. FRET analysis demonstrated that cyan fluorescent protein-GR and yellow fluorescent protein-MR form heterodimers after corticosterone (CORT) treatment both in the nucleus of cultured hippocampal neurons and COS-1 cells, whereas they do not form heterodimers in the cytoplasm. The content of the GR-MR heterodimer was higher at 10(-6) m CORT than at 10(-9) m CORT and reached a maximum level after 60 min of CORT treatment in both cultured hippocampal neurons and COS-1 cells. The distribution pattern of heterodimers in the nucleus of cultured hippocampal neurons was more restricted than that in COS-1 cells. The present study using mutant fusion proteins in nuclear localization signal showed that these corticosteroid receptors are not translocated into the nucleus in the form of heterodimers even after treatment with ligand and thus allow no heterodimerization to take place in the cytoplasm. These results obtained with FRET analyses give new insights into the sites, time course, and effects of ligand concentration on heterodimersization of the GR and MR.

Visualizing the Signal Transduction Pathways in Living Cells with GFP-Based FRET Probes

Visualizing how signals are transmitted within a living cell has long been a goal of molecular biologists, which has now been realized by probes based on the principle of fluorescence resonance energy transfer (FRET). Variants of green fluorescent protein (GFP) enabled the preparation of genetically-encoded FRET probes, and their application has been expanded for use in many areas of biology. The GFP-based FRET probes can be classified as belonging to one of two types, intermolecular and intramolecular FRET probes. The merit of the intermolecular FRET probe lies in the ease of preparation of the probes, whereas the merit of the intramolecular FRET probe lies in the high signal-to-noise ratio. Although these GFP-based probes are powerful tools for the visualization of signal transduction cascades, numerous pitfalls remain associated with this technique. Here, we provide an overview of the GFP-based FRET probes and discuss these issues.

The use of green fluorescent proteins to view association between phospholipase C beta and G protein subunits in cells.

A major advance in biology is the ability to attach either green fluorescence protein (GFP) or one of its variants to a target protein and follow its cellular localization and interaction with other partners by fluorescence microscopy. Our laboratory has previously developed fluorescence energy-transfer methods to measure the kinetics and affinities of the lateral association between phospholipase C (PLC) and G protein subunits on membrane surfaces. We are currently developing methods to view these associations in living cells using fluorescence resonance energy transfer (FRET) between GFP-based chimeras. Because the improvements and variations of these GFP-based FRET techniques has continued on a rapid pace, we focus only on the basic principles behind these measurements and the methods used, which may continue to be applicable as improvements become available.

Application of the Fluorescence Resonance Energy Transfer Method for Studying the Dynamics of Caspase-3 Activation during UV-Induced Apoptosis in Living HeLa Cells

Activation of caspase-3 is a central event in apoptosis. We have developed a GFP-based FRET (fluorescence resonance energy transfer) probe that is highly sensitive to the activation of caspase-3 in intact living cells. This probe was constructed by fusing a CFP (cyan fluorescent protein) and a YFP (yellow fluorescent protein) with a specialized linker containing the caspase-3 cleavage sequence: DEVD. The linker design was optimized to produce a large FRET effect. Using purified protein, we observed a fivefold change in the fluorescence emission ratio when the probe was cleaved by caspase-3. To demonstrate the usefulness of this method, we introduced this FRET probe into HeLa cells by both transient and stable transfection. We observed that during UV-induced apoptosis, the activation of caspase-3 varied significantly between different cells; but once the caspase was activated, the enzyme within the cell became fully active within a few minutes. This technique will be highly useful for correlating the caspase-3 activation with other apoptotic events and for rapid-screening of potential drugs that may target the apoptotic process.

Green Fluorescent Protein-based Probes.

The green fluorescent protein (GFP) of the jelly fish Aequorea victoria is a spontaneously fluorescent protein that can be incorporated into other proteins by genetic fusion. One of our approaches is to use two GFPs of different colors to permit fluorescence resonance energy transfer (FRET), which is highly sensitive to the relative orientation and distance between the two fluorophores and alters the ratio of their emission intensities. For example, cameleons are genetically-encoded fluorescent indicators for calcium. I describe here potentially unique features of GFP-based FRET, which would allow us to make probes for many intracellular signaling events that are currently assayed by grinding millions of cells.