From fixed to FRAP: measuring protein mobility and activity in living cells

Experiments with fluorescence recovery after photobleaching (FRAP) started 30 years ago to visualize the lateral mobility and dynamics of fluorescent proteins in living cells. Its popularity increased when non-invasive fluorescent tagging became possible with the green fluorescent protein (GFP). Many researchers use GFP to study the localization of fusion proteins in fixed or living cells, but the same fluorescent proteins can also be used to study protein mobility in living cells. Here we review the potential of FRAP to study protein dynamics and activity within a single living cell. These measurements can be made with most standard confocal laser-scanning microscopes equipped with photobleaching protocols.     

Reversible photobleaching of enhanced green fluorescent proteins

Color variants of green fluorescent protein (GFP) are increasingly used for multicolor imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP). Here we show that experimental settings commonly used in these imaging experiments may induce an as yet uncharacterized reversible photobleaching of fluorescent proteins, which is more pronounced at acidic pH. Whereas the reversible photobleaching spectrum of eCFP corresponds to its absorption spectrum, reversible photobleaching spectra of yellow variants resemble absorption spectra of their protonated states. Fluorescence intensities recover spontaneously with time constants of 25-58 s. The recovery of eCFP can be further accelerated by illumination. The resulting steady-state fluorescence reflects a variable equilibrium between reversible photobleaching, spontaneous recovery, and light-induced recovery. These processes can cause significant artifacts in commonly applied imaging techniques, photobleach-based FRET determinations, and FRAP assays.     

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.     

Genetic tags for labelling live cells: gap junctions and beyond

The availability of green fluorescent protein (GFP) as a tracer for observing proteins in living cells has revolutionized cell biology and spurred an intensive search for GFP variants with novel characteristics, additional autofluorescent proteins and alternative techniques of protein labelling. Two recent studies - one on tagging with tetracysteine motifs and labelling with biarsenic fluorophores of different colours, and the other on GFP tagging and fluorescence recovery after photobleaching (FRAP) - show how membrane channels are added and removed from gap junctions by using different fluorescent tags to distinguish between newly synthesized and older protein populations.     

Live-cell nucleocytoplasmic protein shuttle assay utilizing laser confocal microscopy and FRAP.

In eukaryotes, protein trafficking to and from the nucleus, or shuttling, has been demonstrated to be an important function for proteins that have vital roles in one or both subcellular compartments. Current techniques of detecting protein nuclear shuttling are extremely labor intensive and only statically visualize evidence of shuttling. Fluorescence recovery after photobleaching (FRAP), or fluorescence microphotolysis, has proven to be an effective method of analyzing protein dynamics in live cells, especially when coupled to GFP technology. Here, we describe a relatively simple in vivo protein nuclear shuttling assay that utilizes red fluorescent and green fluorescent protein fusions as substrates for FRAP using a laser confocal microscope. This technique is less time consuming than established shuttle assays, is internally controlled, and visualizes nucleocytoplasmic shuttling in living cells of the same species and cell type. This technique can be potentially used to detect the ability of any nuclear protein to shuttle from the nucleus to any other subcellular compartment for any eukaryotic species in which GFP or dsRed1 fusion protein can be expressed.     

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.     

Existence of periplasmic barriers preventing green fluorescent protein diffusion from cell to cell in the cyanobacterium Anabaena sp. strain PCC 7120

When deprived of combined nitrogen, the filamentous cyanobacterium Anabaena PCC 7120 relies on intercellular cooperation involving two cell types: nitrogen-fixing heterocysts and photosynthetic vegetative cells. Heterocysts send fixed nitrogen to vegetative cells over long distances along the filament, receiving a reduced carbon source from them. These intercellular exchanges might involve a continuous periplasm along the filament or cytoplasm-to-cytoplasm conduits or both. In the present study, the green fluorescent protein (GFP) was fused to a twin-arginine translocation signal sequence, which exported GFP to the periplasm of either a heterocyst using the heterocyst-specific promoters PhepA and PpatB or to the periplasm of vegetative cells using the vegetative cell-specific promoter PrbcL. Using the techniques of FRAP (fluorescence recovery after photobleaching) and FLIP (fluorescence loss in photobleaching), we found no evidence for intercellular diffusion of GFP through the periplasm, either from a heterocyst to vegetative cells or vice versa, or among vegetative cells. GFP could diffuse within the periplasm of the producing cell, but the diffusion stopped at the cell border. GFP diffusion could occur between two dividing cells before septum closure. This study indicates that barriers exist at the periplasmic space to prevent free GFP diffusion across cell border along the filament.     

Diffusion of green fluorescent protein in three cell environments in Escherichia coli

Surprisingly little is known about the physical environment inside a prokaryotic cell. Knowledge of the rates at which proteins and other cell components can diffuse is crucial for the understanding of a cell as a physical system. There have been numerous measurements of diffusion coefficients in eukaryotic cells by using fluorescence recovery after photobleaching (FRAP) and related techniques. Much less information is available about diffusion coefficients in prokaryotic cells, which differ from eukaryotic cells in a number of significant respects. We have used FRAP to observe the diffusion of green fluorescent protein (GFP) in cells of Escherichia coli elongated by growth in the presence of cephalexin. GFP was expressed in the cytoplasm, exported into the periplasm using the twin-arginine translocation (Tat) system, or fused to an integral plasma membrane protein (TatA). We show that TatA-GFP diffuses in the plasma membrane with a diffusion coefficient comparable to that of a typical eukaryotic membrane protein. A previous report showed a very low rate of protein diffusion in the E. coli periplasm. However, we measured a GFP diffusion coefficient only slightly smaller in the periplasm than that in the cytoplasm, showing that both cell compartments are relatively fluid environments.     

Modulation of nucleocytoplasmic trafficking by retention in cytoplasm or nucleus

Nuclear protein transport processes have largely been studied using in vitro semi‐intact cell systems where high concentrations of nuclear localizing substrates are used, and cytoplasmic components such as the microtubule (MT) network, are either absent or damaged. Here we use the fluorescence recovery after photobleaching (FRAP) technique to analyze the nucleocytoplasmic flux of distinct fluorescently tagged proteins over time in living cultured cells. FRAP was performed in different parts of the cell to analyze the kinetics of nucleocytoplasmic trafficking and intranuclear/cytoplasmic mobility of the tumor suppressor Rb protein and a SV40 large tumor antigen (T‐ag) derivative containing the nuclear localization sequence (NLS), both fused to green fluorescent protein (GFP). The results indicate that proteins carrying the T‐ag NLS are highly mobile in the nucleus and cytoplasm. Rb, in contrast, is largely immobile in both cellular compartments, with similar nuclear import and export kinetics. Rb nuclear export was CRM‐1‐mediated, with its reduced mobility in the cytoplasm in part due to association with MTs. Overall our results show that nuclear and cytoplasm retention modulates the rates of nuclear protein import and export in intact cells. J. Cell. Biochem. 107: 1160–1167, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

Photoswitching-Free FRAP Analysis with a Genetically Encoded Fluorescent Tag

Fluorescence recovery after photobleaching (FRAP) is a widely used imaging technique for measuring protein dynamics in live cells that has provided many important biological insights. Although FRAP presumes that the conversion of a fluorophore from a bright to a dark state is irreversible, GFP as well as other genetically encoded fluorescent proteins now in common use can also exhibit a reversible conversion known as photoswitching. Various studies have shown how photoswitching can cause at least four different artifacts in FRAP, leading to false conclusions about various biological phenomena, including the erroneous identification of anomalous diffusion or the overestimation of the freely diffusible fraction of a cellular protein. Unfortunately, identifying and then correcting these artifacts is difficult. Here we report a new characteristic of an organic fluorophore tetramethylrhodamine bound to the HaloTag protein (TMR-HaloTag), which like GFP can be genetically encoded, but which directly and simply overcomes the artifacts caused by photoswitching in FRAP. We show that TMR exhibits virtually no photoswitching in live cells under typical imaging conditions for FRAP. We also demonstrate that TMR eliminates all of the four reported photoswitching artifacts in FRAP. Finally, we apply this photoswitching-free FRAP with TMR to show that the chromatin decondensation following UV irradiation does not involve loss of nucleosomes from the damaged DNA. In sum, we demonstrate that the TMR Halo label provides a genetically encoded fluorescent tag very well suited for accurate FRAP experiments.     

GFP-labelled Rubisco and aspartate aminotransferase are present in plastid stromules and traffic between plastids

Plastid stromules are membrane-bound protrusions of the plastid envelope that contain soluble stroma. Stromules are often found connecting plastids within a cell and fluorescence recovery after photobleaching (FRAP) experiments have demonstrated that green fluorescent protein (GFP) can move between plastids via these connections. In this report, the ability of endogenous plastid proteins to travel through stromules was investigated. The motility of GFP-labelled plastid aspartate aminotransferase and the Rubisco small subunit was studied in stromules by FRAP. Both fusion proteins assemble into protein complexes that appear to behave similarly to their endogenous counterparts. In addition, both enzymes are capable of trafficking between plastids via stromules.     

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after merging (FRAM). FRAM adopts the same principles as the fluorescence recovery after photobleaching (FRAP) technique, especially for measuring fluorescence recovery after bleaching a specific area, but FRAM uses a drop coalescence instead of photobleaching dye molecules to induce a chemical potential gradient of dye molecules. Our technique has several advantages over FRAP: it only requires a fluorescence microscope rather than a confocal microscope equipped with high power lasers; it is essentially free from the selection of fluorescence dyes; and it has far more freedom to define the measured diffusion area. Furthermore, FRAM potentially provides a route for studying the mixing or inter-diffusion of two different surfactants, when the monolayers at a surface of droplet and at a flat air/water interface are prepared with different species, independently.     

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.     

Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples

We present a truly quantitative fluorescence recovery after photobleaching (FRAP) model for use with the confocal laser scanning microscope based on the photobleaching of a long line segment. The line FRAP method is developed to complement the disk FRAP method reported before. Although being more subject to the influence of noise, the line FRAP model has the advantage of a smaller bleach region, thus allowing for faster and more localized measurements of the diffusion coefficient and mobile fraction. The line FRAP model is also very well suited to examine directly the influence of the bleaching power on the effective bleaching resolution. We present the outline of the mathematical derivation, leading to a final analytical expression to calculate the fluorescence recovery. We examine the influence of the confocal aperture and the bleaching power on the measured diffusion coefficient to find the optimal experimental conditions for the line FRAP method. This will be done for R-phycoerythrin and FITC-dextrans of various molecular weights. The ability of the line FRAP method to measure correctly absolute diffusion coefficients in three-dimensional samples will be evaluated as well. Finally we show the application of the method to the simultaneous measurement of free green fluorescent protein diffusion in the cytoplasm and nucleus of living A549 cells.     

Inferring a system model of subcellular topoisomerase II beta localization dynamics

Measuring the mobility of proteins in living cells has become critical to many studies in cell biology and forms the basis for discussion on sub-cellular dynamics. Increasingly localization networks are being put together into compartment models to represent the exchange of molecules, represented mathematically as ordinary differential equations (ODE). The set-up is based on published literature, the "knowledge" of the investigator and 3D visualization of the data. Here we demonstrate this method on the example of a simple distribution model of the molecule Topoisomerase II beta (Topo II beta), nuclear protein that modifies DNA topology. It is found in high concentration in the nucleolus and diffuse in the nucleoplasm, demonstrating a non-membranous inhomogeneity in its distribution. We expand on the simple model by adding additional components to fit fluorescence recovery after photobleaching (FRAP) experiments for protein (GFP) labeled Topo II beta to measure its mobility. This model is then validated by comparing it with alternative scenarios and shown to have predictive power.     

Parameters which affect the estimation of protein mobility by method FRAP in living cells on the example of protein fibrillarin

Method FRAP (fluorescence recovery after photobleaching) in combination with confocal laser scanning microscopy represents one of the principal approaches in studying the properties of proteins in living mammal cells. However, the data of different authors on the dynamic properties of the same protein and even in cells of the same type can differ greatly. The reasons of such discrepancies were not specifically analyzed yet. In the present work, on the example of the nucleolar protein fibrillarin fused to EGFP, was studied the impact of area of the region of interest (ROI) and temperature conditions on the main dynamic characteristics of the protein, such as mobile fraction and time for half-time of fluorescence recovery after photobleaching (t1/2). Obtained results suggest that both parameters have a great impact on the estimation of mobile properties of fibrillarin-EGFP in HeLa cells. Was concluded that during FRAP experiments the area of ROI has to be standardized and, as possible, minimized. Moreover, analyzing the dynamic properties of the nucleolar proteins, which take part in the temperature-sensitive reactions, the standard temperature conditions should also be standardized.     

LC3 fluorescent puncta in autophagosomes or in protein aggregates can be distinguished by FRAP analysis in living cells

LC3 is a marker protein that is involved in the formation of autophagosomes and autolysosomes, which are usually characterized and monitored by fluorescence microscopy using fluorescent protein-tagged LC3 probes (FP-LC3). FP-LC3 and even endogenous LC3 can also be incorporated into intracellular protein aggregates in an autophagy-independent manner. However, the dynamic process of LC3 associated with autophagosomes and autolysosomes or protein aggregates in living cells remains unclear. Here, we explored the dynamic properties of the two types of FP-LC3-containing puncta using fluorescence microscopy techniques, including fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET). The FRAP data revealed that the fluorescent signals of FP-LC3 attached to phagophores or in mature autolysosomes showed either minimal or no recovery after photobleaching, indicating that the dissociation of LC3 from the autophagosome membranes may be very slow. In contrast, FP-LC3 in the protein aggregates exhibited nearly complete recovery (more than 80%) and rapid kinetics of association and dissociation (half-time < 1 sec), indicating a rapid exchange occurs between the aggregates and cytoplasmic pool, which is mainly due to the transient interaction of LC3 and SQSTM1/p62. Based on the distinct dynamic properties of FP-LC3 in the two types of punctate structures, we provide a convenient and useful FRAP approach to distinguish autophagosomes from LC3-involved protein aggregates in living cells. Using this approach, we find the FP-LC3 puncta that adjacently localized to the phagophore marker ATG16L1 were protein aggregate-associated LC3 puncta, which exhibited different kinetics compared with that of autophagic structures.