Convolution-based one and two component FRAP analysis: theory and application

The method of fluorescence redistribution after photobleaching (FRAP) is increasingly receiving interest in biological applications as it is nowadays used not only to determine mobility parameters per se, but to investigate dynamic changes in the concentration or distribution of diffusing molecules. Here, we develop a new simple convolution-based approach to analyze FRAP data using the whole image information. This method does not require information about the timing and localization of the bleaching event but uses the first image acquired directly after photobleaching to calculate the intensity distributions, instead. Changes in pools of molecules with different velocities, which are monitored by applying repetitive FRAP experiments within a single cell, can be analyzed by means of a global model by assuming two global diffusion coefficients with changing portions. We validate the approach by simulation and show that translocation of the YFP-fused PH-domain of phospholipase Cδ1 can be quantitatively monitored by FRAP analysis in a time-resolved manner. The new FRAP data analysis procedure may be applied to investigate signal transduction pathways using biosensors that change their mobility. An altered mobility in response to the activation of signaling cascades may result either from an altered size of the biosensor, e.g. due to multimerization processes or from translocation of the sensor to an environment with different viscosity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tracking microdomain dynamics in cell membranes

Studies of the diffusion of proteins and lipids in the plasma membrane of cells have long pointed to the presence of membrane domains. A major challenge in the field of membrane biology has been to characterize the various cellular structures and mechanisms that impede free diffusion in cell membranes and determine the consequences that membrane compartmentalization has on cellular biology. In this review, we will provide a brief summary of the classes of domains that have been characterized to date, focusing on recent efforts to identify the properties of lipid rafts in cells through measurements of protein and lipid diffusion.     

Optimization of laser beams in FRAP experiments of microscopical objects

In fluorescence recovery after photobleaching (FRAP) experiments the sample is irradiated on a small spot, the diameter of which must be related to the sample size and the diffusion constant to be measured. This paper considers the conventional FRAP set-up where a laser beam is directed through a microscope vertical illuminator to the sample. The requirements of an intermediate optical system producing a Gaussian beam with a waist of given radius in the microscope object plane are considered, and the optical parameters determined.     

Unrestricted Diffusion of Exogenous and Endogenous PIP2 in Baby Hamster Kidney and Chinese Hamster Ovary Cell Plasmalemma

We used two approaches to characterize the lateral mobility of phosphatidylinositol 4,5-bisphosphate (PIP₂) in the plasmalemma of baby hamster kidney and Chinese hamster ovary fibroblasts. First, nitrobenzoxadiazole-labeled C6-phosphatidylcholine and C16-PIP₂ were incorporated into plasma membrane “lawns” (∼20 × 30 μm) from these cells and into the outer monolayer of intact cells. Diffusion coefficients determined by fluorescence recovery after photobleaching were similar for the two lipids and were higher in lawns, ∼0.3 μm²/s, than on the cell surface, ∼0.1 μm²/s. For membrane lawns, the fractional recoveries (75–90%) were close to those expected from the fraction of total membrane bleached, and labeling by the probes was several times greater than for intact cells. Second, we analyzed cells expressing M1 muscarinic receptors and green fluorescent protein fused with PIP₂-binding pleckstrin-homology domains, Tubby domains or diacylglycerol (DAG)-binding C1 domains. On-cell gigaseal patches were formed with pipette tips >5 μm in diameter. When the agonist carbachol (0.3 mm) was applied either within or outside of the pipette, lipid signals crossed the pipette barrier rapidly in both directions and membrane blebbing occurred on both membrane sides. Accurate simulations of lipid gradients required diffusion coefficients >1 μm²/s. Exogenous DAG also crossed the pipette barrier rapidly. In summary, we found no evidence for restricted diffusion of signaling lipids in these cells. The lower mobility and incorporation of phospholipid at the extracellular leaflet may reflect a more ordered and condensed extracellular monolayer, as expected from previous studies. An erratum to this article can be found at     

Dynamics of putative raft-associated proteins at the cell surface

Lipid rafts are conceptualized as membrane microdomains enriched in cholesterol and glycosphingolipid that serve as platforms for protein segregation and signaling. The properties of these domains in vivo are unclear. Here, we use fluorescence recovery after photobleaching to test if raft association affects a protein's ability to laterally diffuse large distances across the cell surface. The diffusion coefficients (D) of several types of putative raft and nonraft proteins were systematically measured under steady-state conditions and in response to raft perturbations. Raft proteins diffused freely over large distances (> 4 microm), exhibiting Ds that varied 10-fold. This finding indicates that raft proteins do not undergo long-range diffusion as part of discrete, stable raft domains. Perturbations reported to affect lipid rafts in model membrane systems or by biochemical fractionation (cholesterol depletion, decreased temperature, and cholesterol loading) had similar effects on the diffusional mobility of raft and nonraft proteins. Thus, raft association is not the dominant factor in determining long-range protein mobility at the cell surface.     

Thermobifida fusca cellulases exhibit limited surface diffusion on bacterial micro‐crystalline cellulose

Elucidation of cellulase–cellulose interactions is key to modeling biomass deconstruction and in understanding the processes that lead to cellulase inactivation. Here, fluorescence recovery after photobleaching and single molecule tracking (SMT) experiments are used to assess the surface diffusion of Thermobifida fusca cellulases on bacterial micro‐crystalline cellulose. Our results show that cellulases exhibit limited surface diffusion when bound to crystalline cellulose and that a large fraction of the cellulases remain immobile even at temperatures optimal for catalysis. A comparison of our experimental results to Monte Carlo (MC) simulations, which use published diffusion coefficients to model cellulase displacements, shows that even those enzymes that are mobile on the cellulose surface exhibit significantly slower diffusive motions than previously reported. In addition, it is observed that the enzymes that show significant displacements exhibit complex, non‐steady surface motions, which suggest that cellulose–bound cellulases exist in molecular states with different diffusive characteristics. These results challenge the notion that cellulases can freely diffuse over cellulose surfaces without catalyzing bond cleavage. Biotechnol. Bioeng. 2013; 110: 47–56. © 2012 Wiley Periodicals, Inc.     

Anomalously slow mobility of fluorescent lipid probes in the plasma membrane of the yeastSaccharomyces cerevisiae

Summary We measured the lateral mobility of two fluorescent lipid probes dioctadecylindocarbocyanine (dil) and tetramethyl rhodamine phosphatidylethanolamine (R-PE) in the plasma mem branesof Saccharomyces cerevisiae inol andopi 3 spheroplasts. These are well-characterized strains with mutations in the inositol and phosphatidylcholine biosynthetic pathways. Membrane phospholipid composition was altered by growing these mutants in the presence or absence of inositol and choline. Lateral mobil ity was measured by fluorescence recovery after photobleaching (FRAP). Microscopic fluorescence polarization employing CCD digital imaging produced an ordered orientation distribution of the lipid probe dil, confirming that at least one of the probes was largely incorporated into the bilayer membrane. Our results demonstrated anomalously slow mobility of both lipid probes for both mutants, regardless of whether the lipid composition was near normal or dramatically altered in relative composition of phosphatidylinositol and phosphatidylcholine. Trypsinization of the spheroplasts to remove surface proteins resulted in markedly increased lateral mobility. However, even in trypsinized sphero plasts, mobility was still somewhat lower than the mobility ob served in the membrane of mammalian cells, such as rat smooth muscle culture cells tested here for comparison.     

Parvalbumin is freely mobile in axons, somata and nuclei of cerebellar Purkinje neurones

The Ca(2+) -binding protein (CaBP) parvalbumin (PV) is strongly expressed in cerebellar Purkinje neurones (PNs). It is considered a pure Ca(2+) buffer, lacking any Ca(2+) sensor function. Consistent with this notion, no PV ligand was found in dendrites of PNs. Recently, however, we observed for a related CaBP that ligand-targeting differs substantially between dendrites and axons. Thus, here we quantified the diffusion of dye-labelled PV in axons, somata and nuclei of PNs by two-photon fluorescence recovery after photobleaching (FRAP). In all three compartments the fluorescence rapidly returned to baseline, indicating that no large or immobile PV ligand was present. In the axon, FRAP was well described by a one-dimensional diffusion equation and a diffusion coefficient (D) of 12 (IQR 6-20) micro m(2)/s. For the soma and nucleus a three-dimensional model yielded similar D values. The diffusional mobility in these compartments was approximately 3 times smaller than in dendrites. Based on control experiments with fluorescein dextrans, we attributed this reduced mobility of PV to different cytoplasmic properties rather than to specific PV interactions in these compartments. Our findings support the notion that PV functions as a pure Ca(2+) buffer and will aid simulations of neuronal Ca(2+) signalling.     

Semi-automated program for analysis of local Ca2+ spark release with application for classification of heart cell type

Local Ca²⁺ spark releases are essential to the Ca²⁺ cycling process. Thus, they play an important role in ventricular and atrial cell contraction, as well as in sinoatrial cell automaticity. Characterizing their properties in healthy cells from different regions in the heart can reveal the basic biophysical differences among these regions. We designed a semi-automatic Matlab Graphical User Interface (called Sparkalyzer) to characterize parameters of Ca²⁺ spark release from any major cardiac tissue, as recorded in line-scan mode with a confocal laser-scanning microscope. We validated the algorithm on experimental images from rabbit sinoatrial, atrial, and ventricular cells loaded with Fluo-4 AM. The program characterizes general image parameters of Ca²⁺ transients and sparks: spark duration, which indicates for how long the spark provides Ca²⁺ to the closed intracellular mechanisms (typical value: 25 ± 1, 23 ± 1, 26 ± 1 ms for sinoatrial, atrial, and ventricular cells, respectively); spark amplitude, which indicates the amount of Ca²⁺ released by a single spark (1.6 ± 0.1, 1.6 ± 0.2, 1.4 ± 0.1 F/F₀ for sinoatrial, atrial, and ventricular cells, respectively); spark length, which is the length of the Ca²⁺ wavelets fired out of a row of ryanodine receptors (5 ± 0.1, 5 ± 0.2, 3.4 ± 0.3 μm for sinoatrial, atrial, or ventricular cells, respectively) and number of sparks (0.14 ± 0.02, 0.025 ± 0.01, 0.02 ± 0.01 for 1 μm in 1 s for sinoatrial, atrial, and ventricular cells, respectively). This method is reliable for Ca²⁺ spark analysis of sinoatrial, atrial, or ventricular cells. Moreover, by examining the average value of Ca²⁺ spark characteristics and their scattering around the mean, atrial, ventricular and sinoatrial cells can be differentiated.     

Lipid diffusion in planar membranes investigated by fluorescence correlation spectroscopy

Investigation of lipid lateral mobility in biological membranes and their artificial models provides information on membrane dynamics and structure; methods based on optical microscopy are very convenient for such investigations. We focus on fluorescence correlation spectroscopy (FCS), explain its principles and review its state of the art versions such as 2-focus, Z-scan or scanning FCS, which overcome most artefacts of standard FCS (especially those resulting from the need for an external calibration) making it a reliable and versatile method. FCS is also compared to single particle tracking and fluorescence photobleaching recovery and the applicability and the limitations of the methods are briefly reviewed. We discuss several key questions of lateral mobility investigation in planar lipid membranes, namely the influence which membrane and aqueous phase composition (ionic strength and sugar content), choice of a fluorescent tracer molecule, frictional coupling between the two membrane leaflets and between membrane and solid support (in the case of supported membranes) or presence of membrane inhomogeneities has on the lateral mobility of lipids. The recent FCS studies addressing those questions are reviewed and possible explanations of eventual discrepancies are mentioned.     

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。     

The human serotonin<Subscript>1A</Subscript> receptor exhibits G-protein-dependent cell surface dynamics

Seven transmembrane domain G-protein-coupled receptors constitute the largest family of proteins in mammals. Signal transduction events mediated by such receptors are the primary means by which cells communicate with and respond to their external environment. The major paradigm in this signal transduction process is that stimulation of the receptor leads to the recruitment and activation of heterotrimeric GTP-binding proteins. These initial events, which are fundamental to all types of G-protein-coupled receptor signaling, occur at the plasma membrane via protein–protein interactions. As a result, the dynamics of the activated receptor on cell surfaces represents an important determinant in its encounter with G-proteins, and has significant impact on the overall efficiency of the signal transduction process. We have monitored the cell surface dynamics of the serotonin_1A receptor, an important member of the G-protein-coupled receptor superfamily, in relation to its interaction with G-proteins. Fluorescence recovery after photobleaching experiments carried out with the receptor tagged to the enhanced yellow fluorescent protein indicate that G-protein activation alters the diffusion properties of the receptor in a manner suggesting the activation process leads to dissociation of G-proteins from the receptor. This result demonstrates that the cell surface dynamics of the serotonin_1A receptor is modulated in a G-protein-dependent manner. Importantly, this result could provide the basis for a sensitive and powerful approach to assess receptor/G-protein interaction in an intact cellular environment.     

The von Hippel-Lindau tumor suppressor protein influences microtubule dynamics at the cell periphery

The von Hippel-Lindau (VHL) protein protects microtubules (MTs) from destabilization by nocodazole treatment. Based on this fixed-cell assay with static end points, VHL has been reported to directly stabilize the MT cytoskeleton. To investigate the dynamic changes in MTs induced by VHL in living cells, we measured the influence of VHL on tubulin turnover using fluorescence recovery after photobleaching (FRAP). To this end, we engineered VHL-deficient renal cell carcinoma cells to constitutively incorporate fluorescently labeled tubulin and to inducibly express VHL. Induction of VHL in these cells resulted in a decrease of tubulin turnover as measured by FRAP at the cell periphery, while minimally influencing MT dynamics around the centrosome. Our data indicates that VHL changes the behavior of MTs dependent on their subcellular localization implying a role for VHL in cellular processes such as migration, polarization, and cell-cell interactions. Here we propose a complementary method to directly measure VHL-induced subcellular changes in microtubule dynamics, which may serve as a tool to study the effect of MT binding proteins such as VHL.     

The intracellular localisation and mobility of Type Igamma phosphatidylinositol 4P 5-kinase splice variants

There are three known splice variants of Type Iγ phosphatidylinositol 4-phosphate 5-kinase (PIPkin Iγ): PIPkins Iγ87, Iγ90, and the most recently cloned (Giudici, M.L., Emson, P.C. and Irvine, R.F. (2004) A novel neuronal-specific splice variant of Type I phosphatidylinositol 4-phosphate 5-kinase isoform gamma. Biochem. J. 379, 489–496) PIPkin IγC (here called PIPkin Iγ93). Here, we have explored the subcellular localisation and mobility of Type I PIPkins in transfected cells by confocal microscopy and flourescence recovery after photobleaching. The unique behaviour shown by PIPkin Iγ93 is consistent with its suggested distinct function. Moreover, the markedly different localisation and mobility of active versus inactive PIPkin Iγ93 provide insights into the factors that dictate cellular targeting of Type Iγ PIPkins.     

Total internal reflection fluorescence microscopy: application to substrate-supported planar membranes

The use of total internal reflection illumination in fluorescence microscopy (TIRFM) is reviewed with emphasis on application to fluorescent macromolecules that specifically and reversibly bind to planar model membranes supported on glass or quartz substrates. Several methods for characterizing macromolecular motion and organization are discussed: the measurement of equilibrium binding curves to obtain values for equilibrium binding constants; the measurement of fluorescence photobleaching recovery curves to obtain values of kinetic rate constants and surface diffusion coefficients; and the measurement of fluorescence intensities as a function of the evanescent field polarization to characterize orientational order. Applications to cell-substrate contact regions are summarized and future directions of TIRFM are outlined. Correspondence to: N. L. Thompson     

A comparison of the translational diffusion of a normal and a membrane-spanning lipid in Lα phase 1-palmitoyl-2-oleoylphosphatidylcholine bilayers

We have used the fluorescence recovery after photobleaching technique to study the translational diffusion, in L phase multibilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), of fluorescent derivatives of 1-palmitoyl-2-oleoylphosphatidylethanolamine (NBD-POPE) and a membrane-spanning phosphatidylethanolamine (NBD-MSPE). The latter derivative was prepared from a membrane-spanning glycerol-dialkyl-glycerol tetraether lipid isolated from the thermophilic and acidophilic archaebacterium Sulfolobus solfataricus. The translational diffusion was examined between about 15° and 45°C. It is shown that over this temperature range the translational diffusion coefficient for NBD-MSPE is 2/3 that for NBD-POPE which spans only one monolayer of the bilayer. The result is interpreted in terms of existing models for translational diffusion in lipid membranes.Abbreviations D _t translational diffusion coefficient - FRAP fluorescence recovery after photobleaching - MSPE a membrane-spanning phosphatidylethanolamine derived from a glycerol-dialkyl-glycerol tetraether lipid isolated from Sulfolobus solfataricus - NBD 4-nitrobenz-2-oxa-1,3-diazolyl - PE phosphatidylethanolamine - POPC 1-palmitoyl-2-oleoylphosphatidylcholine - POPE 1-palmitoyl-2-oleoylphosphatidylethanolamine     

Active Galpha(q) subunits and M3 acetylcholine receptors promote distinct modes of association of RGS2 with the plasma membrane

RGS proteins accelerate the GTPase activity of heterotrimeric G proteins at the plasma membrane. Association of RGS proteins with the plasma membrane can be mediated by interactions with other membrane proteins and by direct interactions with the lipid bilayer. Here we use fluorescence recovery after photobleaching (FRAP) to characterize interactions between RGS2 and M3 acetylcholine receptors (M3Rs), Galpha subunits and the lipid bilayer. Active Galpha(q) and M3Rs both recruited RGS2-EGFP to the plasma membrane. RGS2-EGFP remained bound to the plasma membrane between interactions with active Galpha(q), but rapidly exchanged between membrane-associated and cytosolic pools when recruited by M3Rs.     

Challenges and artifacts in quantitative photobleaching experiments

Confocal fluorescence recovery after photobleaching (FRAP) is today the prevalent tool when studying the diffusional and kinetic properties of proteins in living cells. Obtaining quantitative data for diffusion coefficients via FRAP, however, is challenged by the fact that both bleaching and scanning take a finite time. Starting from an experimental case, it is shown by means of computer simulations that this intrinsic temporal limitation can lead to a gross underestimation of diffusion coefficients. Determining the binding kinetics of proteins to membranes with FRAP is further shown to be severely hampered by additional diffusional contributions, e.g. diffusion-limited binding. In some cases, the binding kinetics may even be masked entirely by diffusion. As current efforts to approach biological problems with biophysical models have to rely on experimentally determined model parameters, e.g. binding rates and diffusion constants, it is proposed that the accuracy in evaluating FRAP measurements can be improved by means of accompanying computer simulations.     

Bounding fluid viscosity and translational diffusion in a fluid lipid bilayer

Fluorescence recovery after photobleaching was used to investigate the translational diffusion of a fluorescent derivative of a membrane-spanning lipid in L phase multibilayers of 1-palmitoyl-2-oleoylphosphatidylcholine prepared in water and in glycerol. The translational diffusion coefficient in hydrated bilayers (D _w) ranged between 2 and 5x10^–8 cm²/s and in glycerinated bilayers (D _g) the range was between 3 and 24×10^–10 cm²/s between 10° and 40°C. These results are discussed in terms of models for diffusion in membranes.