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.     

Characterization of Cell Boundary and Confocal Effects Improves Quantitative FRAP Analysis

Fluorescence recovery after photobleaching (FRAP) is an important tool used by cell biologists to study the diffusion and binding kinetics of vesicles, proteins, and other molecules in the cytoplasm, nucleus, or cell membrane. Although many FRAP models have been developed over the past decades, the influence of the complex boundaries of 3D cellular geometries on the recovery curves, in conjunction with regions of interest and optical effects (imaging, photobleaching, photoswitching, and scanning), has not been well studied. Here, we developed a 3D computational model of the FRAP process that incorporates particle diffusion, cell boundary effects, and the optical properties of the scanning confocal microscope, and validated this model using the tip-growing cells of Physcomitrella patens. We then show how these cell boundary and optical effects confound the interpretation of FRAP recovery curves, including the number of dynamic states of a given fluorophore, in a wide range of cellular geometries—both in two and three dimensions—namely nuclei, filopodia, and lamellipodia of mammalian cells, and in cell types such as the budding yeast, Saccharomyces pombe, and tip-growing plant cells. We explored the performance of existing analytical and algorithmic FRAP models in these various cellular geometries, and determined that the VCell VirtualFRAP tool provides the best accuracy to measure diffusion coefficients. Our computational model is not limited only to these cells types, but can easily be extended to other cellular geometries via the graphical Java-based application we also provide. This particle-based simulation—called the Digital Confocal Microscopy Suite or DCMS—can also perform fluorescence dynamics assays, such as number and brightness, fluorescence correlation spectroscopy, and raster image correlation spectroscopy, and could help shape the way these techniques are interpreted.     

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.     

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.     

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.     

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.     

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.     

Characterizing fluorescence recovery curves for nuclear proteins undergoing binding events

Fluorescence recovery after photobleaching (FRAP) is an experimental technique used to measure the mobility of proteins within the cell nucleus. After proteins of interest are fluorescently tagged for their visualization and monitoring, a small region of the nucleus is photobleached. The experimental FRAP data are obtained by recording the recovery of the fluorescence in this region over time. In this paper, we characterize the fluorescence recovery curves for diffusing nuclear proteins undergoing binding events with an approximate spatially homogeneous structure. We analyze two mathematical models for interpreting the experimental FRAP data, namely a reaction-diffusionmodel and a compartmental model. Perturbation analysis leads to a clear explanation of two important limiting dynamical types of behavior exhibited by experimental recovery curves, namely, (1) a reduced diffusive recovery, and (2) a biphasic recovery characterized by a fast phase and a slow phase. We show how the two models, describing the same type of dynamics using different approaches, relate and share common ground. The results can be used to interpret experimental FRAP data in terms of protein dynamics and to simplify the task of parameter estimation. Application of the results is demonstrated for nuclear actin and type H1 histone.     

Simplified Equation to Extract Diffusion Coefficients from Confocal FRAP Data

Quantitative measurements of diffusion can provide important information about how proteins and lipids interact with their environment within the cell and the effective size of the diffusing species. Confocal fluorescence recovery after photobleaching (FRAP) is one of the most widely accessible approaches to measure protein and lipid diffusion in living cells. However, straightforward approaches to quantify confocal FRAP measurements in terms of absolute diffusion coefficients are currently lacking. Here, we report a simplified equation that can be used to extract diffusion coefficients from confocal FRAP data using the half time of recovery and effective bleach radius for a circular bleach region, and validate this equation for a series of fluorescently labeled soluble and membrane‐bound proteins and lipids. We show that using this approach, diffusion coefficients ranging over three orders of magnitude can be obtained from confocal FRAP measurements performed under standard imaging conditions, highlighting its broad applicability.     

Expanding the scope of quantitative FRAP analysis

In this study, new mathematical models were developed for analysis of fluorescence recovery after photobleaching (FRAP) data to account for features not represented in previous analysis: conical photobleaching geometry, spatial variations in binding of fluorescent molecules, and directed transport of fluorescent molecules. To facilitate computations in conical geometry, a fast computational method for calculation of fluorescence recovery is presented. Two approximations are presented to aid in FRAP analysis when binding varies spatially, one applying to cases of relatively fast diffusion and slow binding and the other to binding of molecules to small cellular structures. Numerical results show that using a model that represents the influential physical processes and that is formulated in the appropriate geometry can substantially improve the accuracy of FRAP calculations.     

Computation of FRAP recovery times for linker histone – chromatin binding on the basis of Brownian dynamics simulations

BackgroundFluorescence recovery after photobleaching (FRAP) studies can provide kinetic information about proteins in cells. Single point mutations can significantly affect the binding kinetics of proteins and result in variations in the recovery half time (t₅₀) measured in FRAP experiments. FRAP measurements of linker histone (LH) proteins in the cell nucleus have previously been reported by Brown et al. (2006) and Lele et al. (2006).MethodsWe performed Brownian dynamics (BD) simulations of the diffusional association of the wild-type and 38 single or double point mutants of the globular domain of mouse linker histone H1.0 (gH1.0) to a nucleosome. From these simulations, we calculated the bimolecular association rate constant (k_on), the Gibbs binding free energy (ΔG) and the dissociation rate constant (k_off) related to formation of a diffusional encounter complex between the nucleosome and the gH1.0.ResultsWe used these parameters, after application of a correction factor to account for the effects of the crowded environment of the nucleus, to compute FRAP recovery times and curves that are in good agreement with previously published, experimentally measured FRAP recovery time courses.ConclusionsOur computational analysis suggests that BD simulations can be used to predict the relative effects of single point mutations on FRAP recovery times related to protein binding.General SignificanceBD simulations assist in providing a detailed molecular level interpretation of FRAP data.     

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.     

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.     

Caenorhabditis elegans auxilin: a J-domain protein essential for clathrin-mediated endocytosis in vivo

The budding of clathrin-coated vesicles is essential for protein transport. After budding, clathrin must be uncoated before the vesicles can fuse with other membranous structures. In vitro, the molecular chaperone Hsc70 uncoats clathrin-coated vesicles in an ATP-dependent process that requires a specific J-domain protein such as auxilin. However, there is little evidence that either Hsc70 or auxilin is essential in vivo. Here we show that C. elegans has a single auxilin homologue that is identical to mammalian auxilin in its in vitro activity. When RNA-mediated interference (RNAi) is used to inhibit auxilin expression in C. elegans, oocytes show markedly reduced receptor-mediated endocytosis of yolk protein tagged with green fluorescent protein (GFP). In addition, most of these worms arrest during larval development, exhibit defective distribution of GFP-clathrin in many cell types, and show a marked change in clathrin dynamics, as determined by fluorescence recovery after photobleaching (FRAP). We conclude that auxilin is required for in vivo clathrin-mediated endocytosis and development in C. elegans.     

Photoactivated structural dynamics of fluorescent proteins

Proteins of the GFP (green fluorescent protein) family have revolutionized life sciences because they allow the tagging of biological samples in a non-invasive genetically encoded way. 'Phototransformable' fluorescent proteins, in particular, have recently attracted widespread interest, as their fluorescence state can be finely tuned by actinic light, a property central to the development of super-resolution microscopy. Beyond microscopy applications, phototransformable fluorescent proteins are also exquisite tools to investigate fundamental protein dynamics. Using light to trigger processes such as photoactivation, photoconversion, photoswitching, blinking and photobleaching allows the exploration of the conformational landscape in multiple directions. In the present paper, we review how structural dynamics of phototransformable fluorescent proteins can be monitored by combining X-ray crystallography, in crystallo optical spectroscopy and simulation tools such as quantum chemistry/molecular mechanics hybrid approaches. Besides their usefulness to rationally engineer better performing fluorescent proteins for nanoscopy and other biotechnological applications, these investigations provide fundamental insights into protein dynamics.