Fluorescence Recovery After Photobleaching Experiments Research Articles

Efficient Estimates of Surface Diffusion Parameters for Spatio-Temporally Resolved Virus Replication Dynamics.

Advanced methods of treatment are needed to fight the threats of virus-transmitted diseases and pandemics. Often, they are based on an improved biophysical understanding of virus replication strategies and processes in their host cells. For instance, an essential component of the replication of the hepatitis C virus (HCV) proceeds under the influence of nonstructural HCV proteins (NSPs) that are anchored to the endoplasmatic reticulum (ER), such as the NS5A protein. The diffusion of NSPs has been studied by in vitro fluorescence recovery after photobleaching (FRAP) experiments. The diffusive evolution of the concentration field of NSPs on the ER can be described by means of surface partial differential equations (sufPDEs). Previous work estimated the diffusion coefficient of the NS5A protein by minimizing the discrepancy between an extended set of sufPDE simulations and experimental FRAP time-series data. Here, we provide a scaling analysis of the sufPDEs that describe the diffusive evolution of the concentration field of NSPs on the ER. This analysis provides an estimate of the diffusion coefficient that is based only on the ratio of the membrane surface area in the FRAP region to its contour length. The quality of this estimate is explored by a comparison to numerical solutions of the sufPDE for a flat geometry and for ten different 3D embedded 2D ER grids that are derived from fluorescence z-stack data of the ER. Finally, we apply the new data analysis to the experimental FRAP time-series data analyzed in our previous paper, and we discuss the opportunities of the new approach.

Salt-Induced Diffusion of Star and Linear Polyelectrolytes within Multilayer Films.

This study explores the effect of salt on the diffusivity of polyelectrolytes of varied molecular architecture in layer-by-layer (LbL) films in directions parallel and perpendicular to the substrate using fluorescence recovery after photobleaching (FRAP) and neutron reflectivity (NR) techniques, respectively. A family of linear, 4-arm, 6-arm, and 8-arm poly(methacrylic acids) (LPMAA, 4PMAA, 6PMAA, and 8PMAA, respectively) of matched molecular weights were synthesized using atom transfer radical polymerization and assembled with a linear polycation, poly[2-(trimethylammonium)ethyl methacrylate chloride] (QPC). NR studies involving deuterated QPC revealed ∼10-fold higher polycation mobility for the 8PMAA/QPC system compared to all-linear LbL films upon exposure to 0.25 M NaCl solutions at pH 6. FRAP experiments showed, however, that lateral diffusion of star PMAAs was lower than LPMAA at NaCl concentrations below ∼0.22 M NaCl, with a crossover to higher mobility of star polymers in more concentrated salt solutions. The stronger response of diffusion of star PMAA to salt is discussed in the context of several theories previously suggested for diffusivity of polyelectrolyte chains in multilayer films and coacervates.

CM-FRAP-Continuum Mechanics-Based Fluorescence Recovery After Photobleaching.

Fluorescence recovery after photobleaching (FRAP) is widely used to evaluate intracellular molecular turnover or repeated translocation of molecules using confocal laser scanning microscopy. While numerous models have been developed for the analysis of FRAP responses, in which chemical interactions and/or fast diffusion processes are involved, it is inherently difficult to evaluate the long-term behavior of molecular turnover because of the presence of intracellular flow and microscopic deformation of bleached regions. To overcome these difficulties, we have developed a novel continuum mechanics-based FRAP (CM-FRAP) approach that enables simultaneous evaluation of long-term molecular turnover and intracellular flow/deformation. Here we demonstrate the utility of CM-FRAP by using actin molecules associated with stress fibers in rat aortic smooth muscle cells with clarification of the experimental setup and data analysis. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Plasmid construction and sample preparation Basic Protocol 2: How to perform FRAP experiments Basic Protocol 3: Data analysis based on CM-FRAP.

Long-term molecular turnover of actin stress fibers revealed by advection-reaction analysis in fluorescence recovery after photobleaching

Fluorescence recovery after photobleaching (FRAP) is a versatile technique to evaluate the intracellular molecular exchange called turnover. Mechanochemical models of FRAP typically consider the molecular diffusion and chemical reaction that simultaneously occur on a time scale of seconds to minutes. Particularly for long-term measurements, however, a mechanical advection effect can no longer be ignored, which transports the proteins in specific directions within the cells and accordingly shifts the spatial distribution of the local chemical equilibrium. Nevertheless, existing FRAP models have not considered the spatial shift, and as such, the turnover rate is often analyzed without considering the spatiotemporally updated chemical equilibrium. Here we develop a new FRAP model aimed at long-term measurements to quantitatively determine the two distinct effects of the advection and chemical reaction, i.e., the different major sources of the change in fluorescence intensity. To validate this approach, we carried out FRAP experiments on actin in stress fibers over a time period of more than 900 s, and the advection rate was shown to be comparable in magnitude to the chemical dissociation rate. We further found that the actin-myosin interaction and actin polymerization differently affect the advection and chemical dissociation. Our results suggest that the distinction between the two effects is indispensable to extract the intrinsic chemical properties of the actin cytoskeleton from the observations of complicated turnover in cells.

Using Fluorescence Recovery After Photobleaching data to uncover filament dynamics.

Fluorescence Recovery After Photobleaching (FRAP) has been extensively used to understand molecular dynamics in cells. This technique when applied to soluble, globular molecules driven by diffusion is easily interpreted and well understood. However, the classical methods of analysis cannot be applied to anisotropic structures subjected to directed transport, such as cytoskeletal filaments or elongated organelles transported along microtubule tracks. A new mathematical approach is needed to analyze FRAP data in this context and determine what information can be obtain from such experiments. To address these questions, we analyze fluorescence intensity profile curves after photobleaching of fluorescently labelled intermediate filaments anterogradely transported along microtubules. We apply the analysis to intermediate filament data to determine information about the filament motion. Our analysis consists of deriving equations for fluorescence intensity profiles and developing a mathematical model for the motion of filaments and simulating the model. Two closed forms for profile curves were derived, one for filaments of constant length and one for filaments with constant velocity, and three types of simulation were carried out. In the first type of simulation, the filaments have random velocities which are constant for the duration of the simulation. In the second type, filaments have random velocities which instantaneously change at random times. In the third type, filaments have random velocities and exhibit pausing between velocity changes. Our analysis shows: the most important distribution governing the shape of the intensity profile curves obtained from filaments is the distribution of the filament velocity. Furthermore, filament length which is constant during the experiment, had little impact on intensity profile curves. Finally, gamma distributions for the filament velocity with pauses give the best fit to asymmetric fluorescence intensity profiles of intermediate filaments observed in FRAP experiments performed in polarized migrating astrocytes. Our analysis also shows that the majority of filaments are stationary. Overall, our data give new insight into the regulation of intermediate filament dynamics during cell migration.

In preprints: morphogens in motion.

In preprints: morphogens in motion.

Residency time of agonists does not affect the stability of GPCR-arrestin complexes.

The interaction of arrestins with G-protein coupled receptors (GPCRs) desensitizes agonist-dependent receptor responses and often leads to receptor internalization. GPCRs that internalize without arrestin have been classified as "class A" GPCRs whereas "class B" GPCRs co-internalize with arrestin into endosomes. The interaction of arrestins with GPCRs requires both agonist activation and receptor phosphorylation. Here, we ask the question whether agonists with very slow off-rates can cause the formation of particularly stable receptor-arrestin complexes. The stability of GPCR-arrestin-3 complexes at two class A GPCRs, the β2 -adrenoceptor and the μ opioid receptor, was assessed using two different techniques, fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) employing several ligands with very different off-rates. Arrestin trafficking was determined by confocal microscopy. Upon agonist washout, GPCR-arrestin-3 complexes showed markedly different dissociation rates in single-cell FRET experiments. In FRAP experiments, however, all full agonists led to the formation of receptor-arrestin complexes of identical stability whereas the complex between the μ receptor and arrestin-3 induced by the partial agonist morphine was less stable. Agonists with very slow off-rates could not mediate the co-internalization of arrestin-3 with class A GPCRs into endosomes. Agonist off-rates do not affect the stability of GPCR-arrestin complexes but phosphorylation patterns do. Our results imply that orthosteric agonists are not able to pharmacologically convert class A into class B GPCRs.

α-Amylase interaction with soluble fibre: Insights from diffusion experiment using fluorescence recovery after photobleaching (FRAP) and permeation experiment using ultrafiltration membrane

The modulation of starch hydrolysis by soluble fibres has been reported in both in vitro and in vivo conditions. Several mechanisms have been put forward including increased gastric retention time, and altered gastric digesta rheology affecting overall mass transfer (enzyme, substrate and hydrolysed products). The direct inhibition of amylase activity by soluble fibres has also been reported but little is known on the mechanisms. Herein we studied the interaction of porcine α-amylase with soluble fibre at low concentrations (to reduce the effect of viscosity). Two methods were performed, free diffusion of α-amylase in fibre solution using fluorescence recovery after photobleaching (FRAP) and forced elution of α-amylase in fibre solution through ultrafiltration membrane. The results provided the evidence that α-amylase potentially binds with soluble fibre. The degree of diffusion (D/D0 from FRAP experiments) by arabinoxylan and β-glucan at the concentration level of 0.05% and 0.5% w/v is significantly lower than that of pectin. The permeation experiment using ultrafilter membrane showed that the effect of arabinoxylan on intensity retention of fluorescence-labelled α-amylase was found to be in between β-glucan and pectin; pectin has a higher level of intensity retention than arabinoxylan and β-glucan. The results suggested that the interaction (primarily due to binding) could be more pronounced in neutral fibre (β-glucan and arabinoxylan) than the charged polymer (pectin). Overall, this short communication provided a new perspective of soluble fibre-enzyme interactions and experimental approaches for further research.

Use of Fluorescence Recovery After Photobleaching (FRAP) to Measure In Vivo Dynamics of Cell Junction-Associated Polarity Proteins.

Here, we present a detailed protocol for fluorescence recovery after photobleaching (FRAP) to measure the dynamics of junctional populations of proteins in living tissue. Specifically, we describe how to perform FRAP in Drosophila pupal wings on fluorescently tagged core planar polarity proteins, which exhibit relatively slow junctional turnover. We provide a step-by-step practical guide to performing FRAP, and list a series of controls and optimizations to do before conducting a FRAP experiment. Finally, we describe how to present the FRAP data for publication.

Destination active chromatin: RAG2 regions that pave the way to V(D)J recombination

Abstract V(D)J recombination is required to establish the diversity of antigen receptors in the adaptive immune system. Recombination activating gene 2 (RAG2), a protein subunit of the nuclear V(D)J recombinase, rearranges gene segments to generate functional genes that encode antigen receptors. Based on our previous results, we hypothesized that a negatively charged region (the acidic hinge) in RAG2 functions to regulate localization of the V(D)J recombinase with chromatin. To test this, we generated the acidic hinge mutant 1 (A1; D/E 370–383 A), acidic hinge mutant 2 (A2; D/E 404–410 A), and D400H (a known mutation that causes immunodeficiency) to determine impact on chromatin association and V(D)J recombination activity. Dynamics of RAG2 mobility with the chromatin were determined with fluorescence recovery after photobleaching (FRAP) experiments with stably expressed GFP-tagged wild type (WT) or mutant RAG2 in RAG2−/− pro-B cells. Changes in V(D)J recombination activity and in RAG2 localization was determined for cells expressing the WT versus mutant RAG2 proteins. FRAP experiments indicated that the acidic hinge mutants had more tightly bound interactions with chromatin, as compared to WT RAG2. A1 resulted in increased V(D)J recombination, whereas, A2 and D400H led to decreased V(D)J recombination. Interestingly, these results correlated with an increased and a decreased nuclear localization of A1 and A2, respectively, relative to WT RAG2. These results indicate that the acidic region has a role in the cellular localization of the V(D)J recombinase, affecting its recombination efficiency. However, D400H localized similarly to WT RAG2, indicating the residue has a more direct role in the nuclear function of the V(D)J recombinase.

Tension-Dependent Myosin Dynamics on Contractile Actomyosin Structures

Dynamic cellular processes, such as cell migration and division, are powered by mechanical tension generated by the actomyosin cytoskeleton, where myosin molecular motors pull on actin filaments. To maintain proper mechanical tension, myosin dynamics is regulated by several factors, such as phosphorylation on the regulatory light chain. Mechanical tension is also proposed to regulate myosin dynamics. In vitro studies have shown that myosin detaches slower from F-actin under resisting load. However, it is unclear whether mechanical tension plays a role in regulating myosin dynamics in cells. In this work, we assess the role of mechanical tension in myosin turnover dynamics on stress fibers by conducting fluorescence recovery after photobleaching (FRAP) experiments. To perturb cellular tension, cells are treated with non-saturating levels of the Rho-kinase inhibitor Y27632, where the total traction force decreases while the overall actomyosin architecture is preserved. Consistent with past literature, myosin recovers much faster after photobleaching, which is thought to be due to an increase in diffusive myosin monomers. However, the recovery profile suggests a reaction-dominant process, suggesting that myosin dissociates faster due to decreased tension. Furthermore, super-resolution live-cell imaging revealed quasi-sarcomeric myosin bands on individual stress fibers recovering after photobleaching. Under normal levels of cellular tension, myosin always recovers on established myosin bands. In constrast, under reduced tension, myosin bands become more disordered and dynamic. This suggests that myosin turnover by monomers associating with or dissociating from an existing minifilament. The core of myosin bands is stable under tension, resulting in the immobile fraction in FRAP experiments. Upon tension reduction, these cores become mobile and can turn over. Together with experiments that directly modulates tension on stress fibers, such as stress fiber rupturing, our data sheds light on the role of mechanical tension regulating myosin dynamics.

DeepFRAP: Fast fluorescence recovery after photobleaching data analysis using deep neural networks.

Conventional analysis of fluorescence recovery after photobleaching (FRAP) data for diffusion coefficient estimation typically involves fitting an analytical or numerical FRAP model to the recovery curve data using non‐linear least squares. Depending on the model, this can be time consuming, especially for batch analysis of large numbers of data sets and if multiple initial guesses for the parameter vector are used to ensure convergence. In this work, we develop a completely new approach, DeepFRAP, utilizing machine learning for parameter estimation in FRAP. From a numerical FRAP model developed in previous work, we generate a very large set of simulated recovery curve data with realistic noise levels. The data are used for training different deep neural network regression models for prediction of several parameters, most importantly the diffusion coefficient. The neural networks are extremely fast and can estimate the parameters orders of magnitude faster than least squares. The performance of the neural network estimation framework is compared to conventional least squares estimation on simulated data, and found to be strikingly similar. Also, a simple experimental validation is performed, demonstrating excellent agreement between the two methods. We make the data and code used publicly available to facilitate further development of machine learning‐based estimation in FRAP.Lay descriptionFluorescence recovery after photobleaching (FRAP) is one of the most frequently used methods for microscopy‐based diffusion measurements and broadly used in materials science, pharmaceutics, food science and cell biology. In a FRAP experiment, a laser is used to photobleach fluorescent particles in a region. By analysing the recovery of the fluorescence intensity due to the diffusion of still fluorescent particles, the diffusion coefficient and other parameters can be estimated. Typically, a confocal laser scanning microscope (CLSM) is used to image the time evolution of the recovery, and a model is fit using least squares to obtain parameter estimates. In this work, we introduce a new, fast and accurate method for analysis of data from FRAP. The new method is based on using artificial neural networks to predict parameter values, such as the diffusion coefficient, effectively circumventing classical least squares fitting. This leads to a dramatic speed‐up, especially noticeable when analysing large numbers of FRAP data sets, while still producing results in excellent agreement with least squares. Further, the neural network estimates can be used as very good initial guesses for least squares estimation in order to make the least squares optimization convergence much faster than it otherwise would. This provides for obtaining, for example, diffusion coefficients as soon as possible, spending minimal time on data analysis. In this fashion, the proposed method facilitates efficient use of the experimentalist's time which is the main motivation to our approach. The concept is demonstrated on pure diffusion. However, the concept can easily be extended to the diffusion and binding case. The concept is likely to be useful in all application areas of FRAP, including diffusion in cells, gels and solutions.

Fluorescence recovery after photobleaching: direct measurement of diffusion anisotropy.

Fluorescence recovery after photobleaching (FRAP) is a widely used technique for studying diffusion in biological tissues. Most of the existing approaches for the analysis of FRAP experiments assume isotropic diffusion, while only a few account for anisotropic diffusion. In fibrous tissues, such as articular cartilage, tendons and ligaments, diffusion, the main mechanism for molecular transport, is anisotropic and depends on the fibre alignment. In this work, we solve the general diffusion equation governing a FRAP test, assuming an anisotropic diffusivity tensor and using a general initial condition for the case of an elliptical (thereby including the case of a circular) bleaching profile. We introduce a closed-form solution in the spatial coordinates, which can be applied directly to FRAP tests to extract the diffusivity tensor. We validate the approach by measuring the diffusivity tensor of [Formula: see text] FITC-Dextran in porcine medial collateral ligaments. The measured diffusion anisotropy was [Formula: see text] (SE), which is in agreement with that reported in the literature. The limitations of the approach, such as the size of the bleached region and the intensity of the bleaching, are studied using COMSOL simulations.

Local details versus effective medium approximation: A study of diffusion in microfluidic random networks made from Voronoi tessellations

We measured the effective diffusion coefficient in regions of microfluidic networks of controlled geometry using the fluorescence recovery after photobleaching (FRAP) technique. The geometry of the networks was based on Voronoi tessellations, and had varying characteristic length scale and porosity. For a fixed network, FRAP experiments were performed in regions of increasing size. Our results indicate that the boundary of the bleached region, and in particular the cumulative area of the channels that connect the bleached region to the rest of the network, are important in the measured value of the effective diffusion coefficient. We found that the statistical geometrical variations between different regions of the network decrease with the size of the bleached region as a power law, meaning that the statistical error of effective medium approximations decrease with the size of the studied medium with no characteristic length scale.

Quantifying Dynamics in Phase-Separated Condensates Using Fluorescence Recovery after Photobleaching

Cells contain numerous membraneless organelles that assemble by intracellular liquid-liquid phase separation. The viscous properties and associated biomolecular mobility within these condensed phase droplets, or condensates, are increasingly recognized as important for cellular function and also dysfunction, for example, in protein aggregation pathologies. Fluorescence recovery after photobleaching (FRAP) is widely used to assess condensate fluidity and to estimate protein diffusion coefficients. However, the models and assumptions utilized in FRAP analysis of protein condensates are often not carefully considered. Here, we combine FRAP experiments on both in vitro reconstituted droplets and intracellular condensates with systematic examination of different models that can be used to fit the data and evaluate the impact of model choice on measured values. A key finding is that model boundary conditions can give rise to widely divergent measured values. This has important implications, for example, in experiments that bleach subregions versus the entire condensate, two commonly employed experimental approaches. We suggest guidelines for determining the appropriate modeling framework and highlight emerging questions about the molecular dynamics at the droplet interface. The ability to accurately determine biomolecular mobility both in the condensate interior and at the interface is important for obtaining quantitative insights into condensate function, a key area for future research.

EGF Receptor Stalls upon Activation as Evidenced by Complementary Fluorescence Correlation Spectroscopy and Fluorescence Recovery after Photobleaching Measurements.

To elucidate the molecular details of the activation-associated clustering of epidermal growth factor receptors (EGFRs), the time course of the mobility and aggregation states of eGFP tagged EGFR in the membranes of Chinese hamster ovary (CHO) cells was assessed by in situ mobility assays. Fluorescence correlation spectroscopy (FCS) was used to probe molecular movements of small ensembles of molecules over short distances and time scales, and to report on the state of aggregation. The diffusion of larger ensembles of molecules over longer distances (and time scales) was investigated by fluorescence recovery after photobleaching (FRAP). Autocorrelation functions could be best fitted by a two-component diffusion model corrected for triplet formation and blinking. The slow, 100–1000 ms component was attributed to membrane localized receptors moving with free Brownian diffusion, whereas the fast, ms component was assigned to cytosolic receptors or their fragments. Upon stimulation with 50 nM EGF, a significant decrease from 0.11 to 0.07 μm2/s in the diffusion coefficient of membrane-localized receptors was observed, followed by recovery to the original value in ~20 min. In contrast, the apparent brightness of diffusing species remained the same. Stripe FRAP experiments yielded a decrease in long-range molecular mobility directly after stimulation, evidenced by an increase in the recovery time of the slow component from 13 to 21.9 s. Our observations are best explained by the transient attachment of ligand-bound EGFRs to immobile or slowly moving structures such as the cytoskeleton or large, previously photobleached receptor aggregates.

Determination of δ-opioid receptor molecules mobility in living cells plasma membrane by novel method of FRAP analysis

Fluorescence recovery after photobleaching (FRAP) is the preferred method for analyzing the lateral mobility of fluorescently-tagged proteins in the plasma membranes (PMs) of live cells. FRAP experiments are described as being easy to perform; however, the analysis of the acquired data can be difficult. The evaluation procedure must be properly combined with the imaging setup of the confocal microscope to provide unbiased results.With the aim of increasing the accuracy of determining the diffusion coefficient (D) and mobile fraction (Mf) of PM proteins, we developed a novel method for FRAP analysis in the equatorial plane of the cell. This method is based on the calculation of photobleaching characteristics, derived from the light intensity profile and optical parameters of the confocal microscope, and on the model of fluorescent molecule diffusion in PM regions outside of the focal plane. Furthermore, cell movement artifacts in the FRAP data are ameliorated by using a region of interest, which is not fixed but instead moves adaptively in coordination with the movement of cells.When this method was used to determine the mobility of the δ-opioid receptor-eYFP in HEK293 cells, a highly significant decrease in receptor mobility was detected in cholesterol-depleted cells. This decrease was fully reversible by the replenishment of cholesterol levels. Our results demonstrate the crucial role played by cholesterol in the dynamic organization of δ-opioid receptors in the PM under in vivo conditions. Our method may be applied for the determination of the D and Mf values of other PM proteins.

Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures.

Fluorescence recovery after photobleaching (FRAP)is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical FRAP experiment, steady-state fluorescence is observed by repeated imaging with low-intensity laser light. Subsequently, the fluorescent molecules are rapidly and irreversibly impaired via brief exposure to high-intensity laser light. Information about protein mobility is obtained by monitoring the recovery of fluorescence. We used FRAP to determine the mobility of p62 in aggresome-like induced structures (ALIS) in murine macrophages after stimulation with lipopolysaccharide (LPS). Because many existing FRAP protocols are either incomplete or complex, our goal was to provide a comprehensive, practical, and straightforward step-by-step protocol for FRAP experiments with live cells. Here, we describe RAW264.7 macrophage transfection with yellow fluorescent protein-p62 (YFP-p62), induction of ALIS by exposing the cells to LPS, and a step-by-step method for collecting prebleach and postbleach FRAP images and data analysis. Finally, we discuss important factors to consider when conducting a FRAP experiment.

A micropatterning platform for quantifying interaction kinetics between the T cell receptor and an intracellular binding protein

A complete understanding of signaling processes at the plasma membrane depends on a quantitative characterization of the interactions of the involved proteins. Fluorescence recovery after photobleaching (FRAP) is a widely used and convenient technique to obtain kinetic parameters on protein interactions in living cells. FRAP experiments to determine unbinding time constants for proteins at the plasma membrane, however, are often hampered by non-specific contributions to the fluorescence recovery signal. On the example of the interaction between the T cell receptor (TCR) and the Syk kinase ZAP70, we present here an approach based on protein micropatterning that allows the elimination of such non-specific contributions and considerably simplifies analysis of FRAP data. Specifically, detection and reference areas are created within single cells, each being either enriched or depleted in TCR, which permits the isolation of ZAP70-TCR binding in a straight-forward manner. We demonstrate the applicability of our method by comparing it to a conventional FRAP approach.

Numerical Simulation and FRAP Experiments Show That the Plasma Membrane Binding Protein PH-EFA6 Does Not Exhibit Anomalous Subdiffusion in Cells.

The fluorescence recovery after photobleaching (FRAP) technique has been used for decades to measure movements of molecules in two-dimension (2D). Data obtained by FRAP experiments in cell plasma membranes are assumed to be described through a means of two parameters, a diffusion coefficient, D (as defined in a pure Brownian model) and a mobile fraction, M. Nevertheless, it has also been shown that recoveries can be nicely fit using anomalous subdiffusion. Fluorescence recovery after photobleaching (FRAP) at variable radii has been developed using the Brownian diffusion model to access geometrical characteristics of the surrounding landscape of the molecule. Here, we performed numerical simulations of continuous time random walk (CTRW) anomalous subdiffusion and interpreted them in the context of variable radii FRAP. These simulations were compared to experimental data obtained at variable radii on living cells using the pleckstrin homology (PH) domain of the membrane binding protein EFA6 (exchange factor for ARF6, a small G protein). This protein domain is an excellent candidate to explore the structure of the interface between cytosol and plasma membrane in cells. By direct comparison of our numerical simulations to the experiments, we show that this protein does not exhibit anomalous diffusion in baby hamster kidney (BHK) cells. The non Brownian PH-EFA6 dynamics observed here are more related to spatial heterogeneities such as cytoskeleton fence effects.