Signal amplification between Gbetagamma release and PI3Kgamma-mediated PI(3,4,5)P3 formation monitored by a fluorescent Gbetagamma biosensor protein and repetitive two component total internal reflection/fluorescence redistribution after photobleaching analysis

Phosphoinositide 3-kinase gamma (PI3Kgamma) is activated by Gbetagamma release after stimulation of Galpha i -coupled receptors, involving a recruitment of the enzyme to the plasma membrane via interaction of the regulatory subunit p101 or p87 with Gbetagamma. The receptor-mediated release of Gbetagamma was, however, insufficient to elicit a translocation of p101 observable by classical fluorescence microscopy approaches. Since the mobilities of plasma membrane-associated and cytosolic proteins differ strongly, small changes in the amount of plasma membrane association should be detectable by an altered diffusional behavior. Here, changes in mobility were monitored by fluorescence redistribution after photobleaching (FRAP) which was repetitively applied before and after stimulation of cells. To combine the advantages of total internal reflection (TIR) illumination, which preferentially excites fluorophors located at or near the plasma membrane, with that provided by the mobility information, we developed a combined TIR/FRAP setup which enabled us to point bleach parts of an image that was observed under TIR illumination. For FRAP data analysis, we introduce a convolution-based method and a global two component model. Using this TIR/FRAP approach, an increased plasma membrane association of the fluorescent Gbetagamma-binding domain of p101 after Gbetagamma release by G protein-coupled receptor stimulation could be detected and quantified. By comparing the translocation efficiency of this domain with that of YFP-GRP1(PH), a biosensor for the PI3Kgamma product PI(3,4,5)P3, we evaluate the signal amplification between Gbetagamma release and PI(3,4,5)P3 formation after activation of Galpha i -coupled receptors.     

Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes

Fluorescence recovery after photobleaching (FRAP) is a widely used tool for estimating mobility parameters of fluorescently tagged molecules in cells. Despite the widespread use of confocal laser scanning microscopes (CLSMs) to perform photobleaching experiments, quantitative data analysis has been limited by lack of appropriate practical models. Here, we present a new approximate FRAP model for use on any standard CLSM. The main novelty of the method is that it takes into account diffusion of highly mobile molecules during the bleach phase. In fact, we show that by the time the first postbleach image is acquired in a CLSM a significant fluorescence recovery of fast-moving molecules has already taken place. The model was tested by generating simulated FRAP recovery curves for a wide range of diffusion coefficients and immobile fractions. The method was further validated by an experimental determination of the diffusion coefficient of fluorescent dextrans and green fluorescent protein. The new FRAP method was used to compare the mobility rates of fluorescent dextrans of 20, 40, 70, and 500 kDa in aqueous solution and in the nucleus of living HeLa cells. Diffusion coefficients were lower in the nucleoplasm, particularly for higher molecular weight dextrans. This is most likely caused by a sterical hindrance effect imposed by nuclear components. Decreasing the temperature from 37 to 22 degrees C reduces the dextran diffusion rates by approximately 30% in aqueous solution but has little effect on mobility in the nucleoplasm. This suggests that spatial constraints to diffusion of dextrans inside the nucleus are insensitive to temperature.     

Evidence for lack of damage during photobleaching measurements of the lateral mobility of cell surface components.

Fluorescence recovery after photobleaching (FRAP) experiments to measure the mobility of cell surface components require a brief, but intense, pulse of light to photobleach the fluorescence in a restricted area of the cell. We studied possible photodamage to the cell surface during the photobleaching step using light and scanning electron microscopy (SEM) and various FRAP measurements themselves. The cell membrane was left impermeable to trypan blue after photobleaching. SEM studies show that the morphology of the cell surface is not altered by photobleaching. Cells can be repeatedly photobleached and/or photobleached using longer bleach times and greater intensities without systematically altering FRAP kinetics. Singlet oxygen quenchers or free radical traps designed to inhibit putative photoreagents produced during photobleaching do not markedly affect the results. Fluorescein and rhodamine labels give similar results. All of these results, obtained with several different monolayer cultures, suggest that photodamage induced during photobleaching is not a serious artefact in the cellular FRAP results obtained to date.     

荧光漂白后恢复技术及其在活细胞分子机制研究中的应用

荧光漂白后恢复（FRAP）是一项利用荧光探针研究活体细胞中各类分子迁移特性的技术。简要介绍了FRAP技术的原理和具体实施要求,列出了动态比和扩散系数的计算公式,并例举了近几年FRAP技术在细胞分子机制研究中的应用。     

Phycobiliprotein diffusion in chloroplasts of cryptophyte Rhodomonas CS24

Unicellular cryptophyte algae employ antenna proteins with phycobilin chromophores in their photosynthetic machinery. The mechanism of light harvesting in these organisms is significantly different than the energy funneling processes in phycobilisomes utilized by cyanobacteria and red algae. One of the most striking features of cryptophytes is the location of the water-soluble phycobiliproteins, which are contained within the intrathylakoid spaces and are not on the stromal side of the lamellae as in the red algae and cyanobacteria. Studies of mobility of phycobiliproteins at the lumenal side of the thylakoid membranes and how their diffusional behavior may influence the energy funneling steps in light harvesting are reported. Confocal microscopy and fluorescence recovery after photobleaching (FRAP) are used to measure the diffusion coefficient of phycoerythrin 545 (PE545), the primary light harvesting protein of Rhodomonas CS24, in vivo. It is concluded that the diffusion of PE545 in the lumen is inhibited, suggesting possible membrane association or aggregation as a potential source of mobility hindrance. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Investigation of binding mechanisms of nuclear proteins using confocal scanning laser microscopy and FRAP

Fluorescence recovery after photobleaching (FRAP) measurements offer an important tool towards analysing diffusion processes within living biological cells. A model is presented that aims to provide a rigorous theoretical framework from which binding information of proteins from FRAP data can be extracted. A single binding reaction is considered and a set of mathematical equations is introduced that incorporates the concentration of free proteins, vacant binding sites and bound complexes in addition to the on- and off-rates of the proteins. To allow a realistic FRAP model, characteristics of the instruments used to perform FRAP measurements are included in the equation. The proposed model has been designed to be applied to biological samples with a confocal scanning laser microscope (CSLM) equipped with the feature to bleach regions characterised by a radially Gaussian distributed profile. Binding information emerges from FRAP simulations considering the diffusion coefficient, radial extent of the bleached volume and bleach constant as parameters derived from experimental data. The proposed model leads to FRAP curves that depend on the on- and off-rates. Analytical expressions are used to define the boundaries of on- and off-rate parameter space in simplified cases when molecules can move on an infinite domain. A similar approach is ensued when movement is restricted in a compartment with a finite size. The theoretical model can be used in conjunction to experimental data acquired by CSLM to investigate the biophysical properties of proteins in living cells.     

Using FRAP and mathematical modeling to determine the in vivo kinetics of nuclear proteins

Fluorescence recovery after photobleaching (FRAP) has become a popular technique to investigate the behavior of proteins in living cells. Although the technique is relatively old, its application to studying endogenous intracellular proteins in living cells is relatively recent and is a consequence of the newly developed fluorescent protein-based living cell protein tags. This is particularly true for nuclear proteins, in which endogenous protein mobility has only recently been studied. Here we examine the experimental design and analysis of FRAP experiments. Mathematical modeling of FRAP data enables the experimentalist to extract information such as the association and dissociation constants, distribution of a protein between mobile and immobilized pools, and the effective diffusion coefficient of the molecule under study. As experimentalists begin to dissect the relative influence of protein domains within individual proteins, this approach will allow a quantitative assessment of the relative influences of different molecular interactions on the steady-state distribution and protein function in vivo.     

Photobleaching approaches to investigate diffusional mobility and trafficking of Ras in living cells

Recent advances in our understanding of the intracellular trafficking, membrane microenvironment, and subcellular sites of signaling of Ras have been driven by observations of GFP-tagged Ras in living cells. Here, we describe methods to gain further insight into the regulation of these events through the use of quantitative fluorescence microscopy. We focus on three techniques, fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), and selective photobleaching. While all of these techniques exploit photobleaching as a tool to monitor protein dynamics, they each provide a unique subset of information. In particular, FRAP provides measurements of protein mobility via lateral diffusion by monitoring recovery of fluorescence into a region following a single photobleaching event. FLIP assesses the level of continuity and communication between subcellular compartments by repetitively photobleaching a region of interest and following concomitant loss of fluorescence from other areas in the cell. Selective photobleaching reveals kinetic information about active and passive transport of proteins into organelles such as the Golgi complex or between areas of protein enrichment such as caveolae. We describe how to implement these techniques using commercially available confocal microscopes and outline methods for data analysis. Finally, we discuss how these approaches are being used to provide new insights into the mechanisms of membrane microdomain localization, vesicular versus non-vesicular transport, and kinetics of exchange of Ras on and off of cell membranes.     

Lateral diffusion anisotropy and membrane lipid/skeleton interaction in outer hair cells

The organization of the plasma membrane of cells in lipid domains affects the way the membrane interacts with the underlying protein skeleton, which in turn affects the lateral mobility of lipid and protein molecules in the membrane. Membrane fluidity properties can be monitored by various approaches, the most versatile of which is fluorescence recovery after photobleaching (FRAP). We extended previous FRAP experiments on isolated cochlear outer hair cells (OHCs) by analyzing the two-dimensional pattern of lipid diffusion in the lateral membrane of these cells. We found that membrane lipid mobility in freshly isolated OHCs is orthotropic, diffusion being faster in the axial direction of the cell and slower in the circumferential direction. Increasing the cell's turgor pressure by osmotic challenge reduced the axial diffusion constant, but had only a slight effect on circumferential diffusion. Our results suggest that lipid mobility in the OHC plasma membrane is affected by the presence of the cell's orthotropic membrane skeleton. This effect could reflect interaction with spectrin filaments or with other membrane skeletal proteins. We also performed a number of FRAP measurements in temporal bone preparations preserving the structural integrity of the hearing organ. The diffusion rates measured for OHCs in this preparation were in good agreement with those obtained in isolated OHCs, and comparable to the mobility rates measured on the sensory inner hair cells. These observations support the idea that the plasma membranes of both types of hair cells share similar highly fluid phases in the intact organ. Lipid mobility was significantly slower in the membranes of supporting cells of the organ of Corti, which could reflect differences in lipid phase or stronger hindrance by the cytoskeleton in these membranes.     

Methods to measure the lateral diffusion of membrane lipids and proteins

In this chapter, we discuss methods to measure lateral mobility of membrane lipids and proteins using techniques based on the light microscope. These methods typically sample lateral mobility in very small, micron-sized regions of the membrane so that they can be used to measure diffusion in regions of single cells. The methods are based on fluorescence from the molecules of interest or from light scattered from particles attached to single or small groups of membrane lipids or proteins. Fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and Single particle tracking (SPT) are presented in that order. FRAP and FCS methodologies are described for a dedicated wide field microscope although many confocal microscopes now have software permitting these measurement to be made; nevertheless, the principles of the measurement are the same for a wide field or confocal microscope. SPT can be applied to trace the movements of single fluorescent molecules in membranes but this aspect will not be treated in detail.     

Minimizing the impact of photoswitching of fluorescent proteins on FRAP analysis

Fluorescence recovery after photobleaching (FRAP) is a widely used imaging technique for measuring the mobility of fluorescently tagged proteins in living cells. Although FRAP presumes that high-intensity illumination causes only irreversible photobleaching, reversible photoswitching of many fluorescent molecules, including GFP, can also occur. Here, we show that this photoswitching is likely to contaminate many FRAPs of GFP, and worse, the size of its contribution can be up to 60% under different experimental conditions, making it difficult to compare FRAPs from different studies. We develop a procedure to correct FRAPs for photoswitching and apply it to FRAPs of the GFP-tagged histone H2B, which, depending on the precise photobleaching conditions exhibits apparent fast components ranging from 9-36% before correction and ～1% after correction. We demonstrate how this ～1% fast component of H2B-GFP can be used as a benchmark both to estimate the role of photoswitching in previous FRAP studies of TATA binding proteins (TBP) and also as a tool to minimize the contribution of photoswitching to tolerable levels in future FRAP experiments. In sum, we show how the impact of photoswitching on FRAP can be identified, minimized, and corrected.     

A Generalization of Theory for Two-Dimensional Fluorescence Recovery after Photobleaching Applicable to Confocal Laser Scanning Microscopes

Fluorescence recovery after photobleaching (FRAP) using confocal laser scanning microscopes (confocal FRAP) has become a valuable technique for studying the diffusion of biomolecules in cells. However, two-dimensional confocal FRAP sometimes yields results that vary with experimental setups, such as different bleaching protocols and bleaching spot sizes. In addition, when confocal FRAP is used to measure diffusion coefficients (D) for fast diffusing molecules, it often yields D-values that are one or two orders-of-magnitude smaller than that predicted theoretically or measured by alternative methods such as fluorescence correlation spectroscopy. Recently, it was demonstrated that this underestimation of D can be corrected by taking diffusion during photobleaching into consideration. However, there is currently no consensus on confocal FRAP theory, and no efforts have been made to unify theories on conventional and confocal FRAP. To this end, we generalized conventional FRAP theory to incorporate diffusion during photobleaching so that analysis by conventional FRAP theory for a circular region of interest is easily applicable to confocal FRAP. Finally, we demonstrate the accuracy of these new (to our knowledge) formulae by measuring D for soluble enhanced green fluorescent protein in aqueous glycerol solution and in the cytoplasm and nucleus of COS7 cells.     

Measuring diffusion and binding kinetics by contact area FRAP

The immunological synapse is a stable intercellular structure that specializes in substance and signal transfer from one immune cell to another. Its formation is regulated in part by the diffusion of adhesion and signaling molecules into, and their binding of countermolecules in the contact area. The stability of immunological synapses allows receptor-ligand interactions to approximate chemical equilibrium despite other dynamic aspects. We have developed a mathematical model that describes the coupled reaction-diffusion process in an established immunological synapse. In this study, we extend a previously described contact area fluorescence recovery after photobleaching (FRAP) experiment to test the validity of the model. The receptor binding activity and lateral mobility of fluorescently labeled, lipid-anchored ligands in the bilayer resulted in their accumulation, as revealed by a much higher fluorescence intensity inside the contact area than outside. After complete photobleaching of the synapse, fluorescence recovery requires ligands to dissociate and rebind, and to diffuse in and out of the contact area. Such a FRAP time course consequently provides information on reaction and diffusion, which can be extracted by fitting the model solution to the data. Surprisingly, reverse rates in the two-dimensional contact area were at least 100-fold slower than in three-dimensional solution. As previously reported in immunological synapses, a significant nonrecoverable fraction of fluorescence was observed with one of two systems studied, suggesting some ligands either dissociated or diffused much more slowly compared with other ligands in the same synapse. The combined theory and experiment thus provides a new method for in situ measurements of kinetic rates, diffusion coefficients, and nonrecoverable fractions of interacting molecules in immunological synapses and other stable cell-bilayer junctions.     

Spatial Fourier analysis of video photobleaching measurements. Principles and optimization.

The major use of the fluorescence recovery after photobleaching (FRAP) technique is to measure the translational motion of the molecular components in various condensed media. In a conventional laser spot photobleaching experiment, a photomultiplier is used to measure the total brightness levels of the bleached region in the sample, so no spatial information can be directly obtained. In video-FRAP, a series of images after photobleaching is acquired, allowing the spatial character of the recovery to be determined; this permits direct detection of both anisotropic diffusion and flow. To utilize all of the available image data to determine the transport coefficients, a two-dimensional spatial Fourier transform analysis of the images after photobleaching was employed. The change in the transform between two time points reflects the action of diffusion during the interim. An important advantage of this method, which involves taking the ratio of image transforms at different time points, is that it does not require a specific initial condition to be created by laser photobleaching. The ability of the analysis to extract transport coefficients from computer-simulated diffusional recovery is assessed in the presence of increasing amounts of noise. Experimental data analysis from the diffusion of proteins in viscous solutions and from the diffusion of protein receptors on cell surfaces demonstrate the feasibility of the Fourier analysis to obtain transport coefficients from the video FRAP measurement.