Статті в журналах: "Fluorescent recovery after photobleaching (FRAP)"

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Cadar, Adrian G., Tromondae K. Feaster, Kevin R. Bersell, Lili Wang, TingTing Hong, Joseph A. Balsamo, Zhentao Zhang, et al. "Real-time visualization of titin dynamics reveals extensive reversible photobleaching in human induced pluripotent stem cell-derived cardiomyocytes." American Journal of Physiology-Cell Physiology 318, no. 1 (January 1, 2020): C163—C173. http://dx.doi.org/10.1152/ajpcell.00107.2019.

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Fluorescence recovery after photobleaching (FRAP) has been useful in delineating cardiac myofilament biology, and innovations in fluorophore chemistry have expanded the array of microscopic assays used. However, one assumption in FRAP is the irreversible photobleaching of fluorescent proteins after laser excitation. Here we demonstrate reversible photobleaching regarding the photoconvertible fluorescent protein mEos3.2. We used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSCs) to knock-in mEos3.2 into the COOH terminus of titin to visualize sarcomeric titin incorporation and turnover. Upon cardiac induction, the titin-mEos3.2 fusion protein is expressed and integrated in the sarcomeres of hiPSC-derived cardiomyocytes (CMs). STORM imaging shows M-band clustered regions of bound titin-mEos3.2 with few soluble titin-mEos3.2 molecules. FRAP revealed a baseline titin-mEos3.2 fluorescence recovery of 68% and half-life of ~1.2 h, suggesting a rapid exchange of sarcomeric titin with soluble titin. However, paraformaldehyde-fixed and permeabilized titin-mEos3.2 hiPSC-CMs surprisingly revealed a 55% fluorescence recovery. Whole cell FRAP analysis in paraformaldehyde-fixed, cycloheximide-treated, and untreated titin-mEos3.2 hiPSC-CMs displayed no significant differences in fluorescence recovery. FRAP in fixed HEK 293T expressing cytosolic mEos3.2 demonstrates a 58% fluorescence recovery. These data suggest that titin-mEos3.2 is subject to reversible photobleaching following FRAP. Using a mouse titin-eGFP model, we demonstrate that no reversible photobleaching occurs. Our results reveal that reversible photobleaching accounts for the majority of titin recovery in the titin-mEos3.2 hiPSC-CM model and should warrant as a caution in the extrapolation of reliable FRAP data from specific fluorescent proteins in long-term cell imaging.

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Combs, Christian A., and Robert S. Balaban. "Enzyme-Dependant Fluorescence Recovery After Photobleaching (ED-FRAP): Application to Imaging Dehydrogenase Activity in Living Single Cells." Microscopy and Microanalysis 7, S2 (August 2001): 18–19. http://dx.doi.org/10.1017/s1431927600026167.

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Fluorescent recovery from photobleaching coupled with confocal microscopy was explored as a potential high-resolution method of imaging the distribution of enzyme activity in single living cardiac myocytes without relying on steady state measurements of fluorescence. On a fundamental level, much remains to be determined regarding how local conditions within a cell affect metabolism. Many studies have suggested that energy metabolism in muscle cells cannot be accurately described assuming a homogenous system of enzymatic reactions [1,2]. The autofluorescence of NADH has been used in many studies as a quantitative assay of mitochondrial energy metabolism [3,4], but studies of steady state fluorescence cannot distinguish between changes in energy production or utilization.In this study conditions were created where the fluorescent recovery of a probe would be solely dependent on cellular enzymatic activity (Enzyme Dependent Fluorescence Recovery after Photobleaching (ED-FRAP)). Experiments examining the inherent fluorescence of NADH (351nm excitation, 450 nm emission) were conducted on small droplets (less than 325 μm diameter) containing NADH alone, in droplets containing an enzyme system capable of synthesis of NADH (Figure 1A) and on isolated rabbit cardiac myocytes (Figure 1D). Photobleaching of the entire cell or droplet eliminated diffusion or bulk transport of NADH from non-bleached regions. Droplets containing NADH alone did not recover, while droplets containing enzyme were shown to recover exponentially (Figure 1B) with a rate constant of fluorescent recovery (kf) that was proportional to enzyme concentration (Figure 1C).

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Srikantha, Nishanthan, Yurema Teijeiro-Gonzalez, Andrew Simpson, Naba Elsaid, Satyanarayana Somavarapu, Klaus Suhling, and Timothy L. Jackson. "Determining vitreous viscosity using fluorescence recovery after photobleaching." PLOS ONE 17, no. 2 (February 10, 2022): e0261925. http://dx.doi.org/10.1371/journal.pone.0261925.

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Purpose Vitreous humor is a complex biofluid whose composition determines its structure and function. Vitreous viscosity will affect the delivery, distribution, and half-life of intraocular drugs, and key physiological molecules. The central pig vitreous is thought to closely match human vitreous viscosity. Diffusion is inversely related to viscosity, and diffusion is of fundamental importance for all biochemical reactions. Fluorescence Recovery After Photobleaching (FRAP) may provide a novel means of measuring intravitreal diffusion that could be applied to drugs and physiological macromolecules. It would also provide information about vitreous viscosity, which is relevant to drug elimination, and delivery. Methods Vitreous viscosity and intravitreal macromolecular diffusion of fluorescently labelled macromolecules were investigated in porcine eyes using fluorescence recovery after photobleaching (FRAP). Fluorescein isothiocyanate conjugated (FITC) dextrans and ficolls of varying molecular weights (MWs), and FITC-bovine serum albumin (BSA) were employed using FRAP bleach areas of different diameters. Results The mean (±standard deviation) viscosity of porcine vitreous using dextran, ficoll and BSA were 3.54 ± 1.40, 2.86 ± 1.13 and 4.54 ± 0.13 cP respectively, with an average of 3.65 ± 0.60 cP. Conclusions FRAP is a feasible and practical optical method to quantify the diffusion of macromolecules through vitreous.

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Braga, José, Joana M. P. Desterro, and Maria Carmo-Fonseca. "Intracellular Macromolecular Mobility Measured by Fluorescence Recovery after Photobleaching with Confocal Laser Scanning Microscopes." Molecular Biology of the Cell 15, no. 10 (October 2004): 4749–60. http://dx.doi.org/10.1091/mbc.e04-06-0496.

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

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Lorén, Niklas, Joel Hagman, Jenny K. Jonasson, Hendrik Deschout, Diana Bernin, Francesca Cella-Zanacchi, Alberto Diaspro, et al. "Fluorescence recovery after photobleaching in material and life sciences: putting theory into practice." Quarterly Reviews of Biophysics 48, no. 3 (August 2015): 323–87. http://dx.doi.org/10.1017/s0033583515000013.

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AbstractFluorescence recovery after photobleaching (FRAP) is a versatile tool for determining diffusion and interaction/binding properties in biological and material sciences. An understanding of the mechanisms controlling the diffusion requires a deep understanding of structure–interaction–diffusion relationships. In cell biology, for instance, this applies to the movement of proteins and lipids in the plasma membrane, cytoplasm and nucleus. In industrial applications related to pharmaceutics, foods, textiles, hygiene products and cosmetics, the diffusion of solutes and solvent molecules contributes strongly to the properties and functionality of the final product. All these systems are heterogeneous, and accurate quantification of the mass transport processes at the local level is therefore essential to the understanding of the properties of soft (bio)materials. FRAP is a commonly used fluorescence microscopy-based technique to determine local molecular transport at the micrometer scale. A brief high-intensity laser pulse is locally applied to the sample, causing substantial photobleaching of the fluorescent molecules within the illuminated area. This causes a local concentration gradient of fluorescent molecules, leading to diffusional influx of intact fluorophores from the local surroundings into the bleached area. Quantitative information on the molecular transport can be extracted from the time evolution of the fluorescence recovery in the bleached area using a suitable model. A multitude of FRAP models has been developed over the years, each based on specific assumptions. This makes it challenging for the non-specialist to decide which model is best suited for a particular application. Furthermore, there are many subtleties in performing accurate FRAP experiments. For these reasons, this review aims to provide an extensive tutorial covering the essential theoretical and practical aspects so as to enable accurate quantitative FRAP experiments for molecular transport measurements in soft (bio)materials.

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Golebiewska, Urszula, Jason G. Kay, Thomas Masters, Sergio Grinstein, Wonpil Im, Richard W. Pastor, Suzanne Scarlata, and Stuart McLaughlin. "Evidence for a fence that impedes the diffusion of phosphatidylinositol 4,5-bisphosphate out of the forming phagosomes of macrophages." Molecular Biology of the Cell 22, no. 18 (September 15, 2011): 3498–507. http://dx.doi.org/10.1091/mbc.e11-02-0114.

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To account for the many functions of phosphatidylinositol 4,5-bisphosphate (PIP2), several investigators have proposed that there are separate pools of PIP2 in the plasma membrane. Recent experiments show the surface concentration of PIP2 is indeed enhanced in regions where phagocytosis, exocytosis, and cell division occurs. Kinases that produce PIP2 are also concentrated in these regions. However, how is the PIP2 produced by these kinases prevented from diffusing rapidly away? First, proteins could act as “fences” around the perimeter of these regions. Second, some factor could markedly decrease the diffusion coefficient, D, of PIP2 within these regions. We used fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) to investigate these two possibilities in the forming phagosomes of macrophages injected with fluorescent PIP2. FCS measurements show that PIP2 diffuses rapidly (D ∼ 1 μm2/s) in both the forming phagosomes and unengaged plasma membrane. FRAP measurements show that the fluorescence from PIP2 does not recover (>100 s) after photobleaching the entire forming phagosome but recovers rapidly (∼10 s) in a comparable area of membrane outside the cup. These results (and similar data for a plasma membrane–anchored green fluorescent protein) support the hypothesis that a fence impedes the diffusion of PIP2 into and out of forming phagosomes.

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Verma, Sanjay K., Pratibha Kumari, Shagufi Naz Ansari, Mohd Ovais Ansari, Dondinath Deori, and Shaikh M. Mobin. "A novel mesoionic carbene based highly fluorescent Pd(ii) complex as an endoplasmic reticulum tracker in live cells." Dalton Transactions 47, no. 44 (2018): 15646–50. http://dx.doi.org/10.1039/c8dt02778a.

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Wagner, Stefan, Simion Chiosea, Maria Ivshina, and Jeffrey A. Nickerson. "In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160." Journal of Cell Biology 164, no. 6 (March 15, 2004): 843–50. http://dx.doi.org/10.1083/jcb.200307002.

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We present a new in vitro system for characterizing the binding and mobility of enhanced green fluorescent protein (EGFP)–labeled nuclear proteins by fluorescence recovery after photobleaching in digitonin-permeabilized cells. This assay reveals that SRm160, a splicing coactivator and component of the exon junction complex (EJC) involved in RNA export, has an adenosine triphosphate (ATP)–dependent mobility. Endogenous SRm160, lacking the EGFP moiety, could also be released from sites at splicing speckled domains by an ATP-dependent mechanism. A second EJC protein, RNPS1, also has an ATP-dependent mobility, but SRm300, a protein that binds to SRm160 and participates with it in RNA splicing, remains immobile after ATP supplementation. This finding suggests that SRm160-containing RNA export, but not splicing, complexes have an ATP-dependent mobility. We propose that RNA export complexes have an ATP-regulated mechanism for release from binding sites at splicing speckled domains. In vitro fluorescence recovery after photobleaching is a powerful tool for identifying cofactors required for nuclear binding and mobility.

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Wadsworth, P., and E. D. Salmon. "Analysis of the treadmilling model during metaphase of mitosis using fluorescence redistribution after photobleaching." Journal of Cell Biology 102, no. 3 (March 1, 1986): 1032–38. http://dx.doi.org/10.1083/jcb.102.3.1032.

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One recent hypothesis for the mechanism of chromosome movement during mitosis predicts that a continual, uniform, poleward flow or "treadmilling" of microtubules occurs within the half-spindle between the chromosomes and the poles during mitosis (Margolis, R. L., and L. Wilson, 1981, Nature (Lond.), 293:705-711). We have tested this treadmilling hypothesis using fluorescent analog cytochemistry and measurements of fluorescence redistribution after photobleaching to examine microtubule behavior during metaphase of mitosis. Mitotic BSC 1 mammalian tissue culture cells or newt lung epithelial cells were microinjected with brain tubulin labeled with 5-(4,6-dichlorotriazin-2-yl) amino fluorescein (DTAF) to provide a fluorescent tracer of the endogenous tubulin pool. Using a laser microbeam, fluorescence in the half-spindle was photobleached in either a narrow 1.6 micron wide bar pattern across the half-spingle or in a circular area of 2.8 or 4.5 micron diameter. Fluorescence recovery in the spindle fibers, measured using video microscopy or photometric techniques, occurs as bleached DTAF-tubulin subunits within the microtubules are exchanged for unbleached DTAF-tubulin in the cytosol by steady-state microtubule assembly-disassembly pathways. Recovery of 75% of the bleached fluorescence follows first-order kinetics and has an average half-time of 37 sec, at 31-33 degrees C. No translocation of the bleached bar region could be detected during fluorescence recovery, and the rate of recovery was independent of the size of the bleached spot. These results reveal that, for 75% of the half-spindle microtubules, FRAP does not occur by a synchronous treadmilling mechanism.

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Kindermann, Stefan, and Štěpán Papáček. "On Data Space Selection and Data Processing for Parameter Identification in a Reaction-Diffusion Model Based on FRAP Experiments." Abstract and Applied Analysis 2015 (2015): 1–17. http://dx.doi.org/10.1155/2015/859849.

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Fluorescence recovery after photobleaching (FRAP) is a widely used measurement technique to determine the mobility of fluorescent molecules within living cells. While the experimental setup and protocol for FRAP experiments are usually fixed, data (pre)processing represents an important issue. The aim of this paper is twofold. First, we formulate and solve the problem ofrelevantFRAP data selection. The theoretical findings are illustrated by the comparison of the results of parameter identification when the full data set was used and the case when theirrelevant data set(data with negligible impact on the confidence interval of the estimated parameters) was removed from the data space. Second, we analyze and compare two approaches of FRAP data processing. Our proposition, surprisingly for the FRAP community, claims that the data set represented by the FRAP recovery curves in form of a time series (integrated data approachcommonly used by the FRAP community) leads to a larger confidence interval compared to thefull(spatiotemporal)data approach.

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Cai, Ning, Alvin Chi-Keung Lai, Kin Liao, Peter R. Corridon, David J. Graves, and Vincent Chan. "Recent Advances in Fluorescence Recovery after Photobleaching for Decoupling Transport and Kinetics of Biomacromolecules in Cellular Physiology." Polymers 14, no. 9 (May 7, 2022): 1913. http://dx.doi.org/10.3390/polym14091913.

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Among the new molecular tools available to scientists and engineers, some of the most useful include fluorescently tagged biomolecules. Tools, such as green fluorescence protein (GFP), have been applied to perform semi-quantitative studies on biological signal transduction and cellular structural dynamics involved in the physiology of healthy and disease states. Such studies focus on drug pharmacokinetics, receptor-mediated endocytosis, nuclear mechanobiology, viral infections, and cancer metastasis. In 1976, fluorescence recovery after photobleaching (FRAP), which involves the monitoring of fluorescence emission recovery within a photobleached spot, was developed. FRAP allowed investigators to probe two-dimensional (2D) diffusion of fluorescently-labelled biomolecules. Since then, FRAP has been refined through the advancements of optics, charged-coupled-device (CCD) cameras, confocal microscopes, and molecular probes. FRAP is now a highly quantitative tool used for transport and kinetic studies in the cytosol, organelles, and membrane of a cell. In this work, the authors intend to provide a review of recent advances in FRAP. The authors include epifluorescence spot FRAP, total internal reflection (TIR)/FRAP, and confocal microscope-based FRAP. The underlying mathematical models are also described. Finally, our understanding of coupled transport and kinetics as determined by FRAP will be discussed and the potential for future advances suggested.

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Waharte, François, Karine Steenkeste, Romain Briandet, and Marie-Pierre Fontaine-Aupart. "Diffusion Measurements inside Biofilms by Image-Based Fluorescence Recovery after Photobleaching (FRAP) Analysis with a Commercial Confocal Laser Scanning Microscope." Applied and Environmental Microbiology 76, no. 17 (July 16, 2010): 5860–69. http://dx.doi.org/10.1128/aem.00754-10.

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ABSTRACT Research about the reactional and structural dynamics of biofilms at the molecular level has made great strides, owing to efficient fluorescence imaging methods in terms of spatial resolution and fast acquisition time but also to noninvasive conditions of observation consistent with in situ biofilm studies. In addition to conventional fluorescence intensity imaging, the fluorescence recovery after photobleaching (FRAP) module can now be routinely implemented on commercial confocal laser scanning microscopes (CLSMs). This method allows measuring of local diffusion coefficients in biofilms and could become an alternative to fluorescence correlation spectroscopy (FCS). We present here an image-based FRAP protocol to improve the accuracy of FRAP measurements inside “live” biofilms and the corresponding analysis. An original kymogram representation allows control of the absence of perturbing bacterial movement during image acquisition. FRAP data analysis takes into account molecular diffusion during the bleach phase and uses the image information to extract molecular diffusion coefficients. The fluorescence spatial intensity profile analysis used here for the first time with biofilms is supported both by our own mathematical model and by a previously published one. This approach was validated to FRAP experiments on fluorescent-dextran diffusion inside Lactoccocus lactis and Stenotrophomonas maltophilia biofilms, and the results were compared to previously published FCS measurements.

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Dallon, J. C., Cécile Leduc, Christopher P. Grant, Emily J. Evans, Sandrine Etienne-Manneville, and Stéphanie Portet. "Using Fluorescence Recovery After Photobleaching data to uncover filament dynamics." PLOS Computational Biology 18, no. 9 (September 26, 2022): e1010573. http://dx.doi.org/10.1371/journal.pcbi.1010573.

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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.

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Waters, Jennifer C. "Accuracy and precision in quantitative fluorescence microscopy." Journal of Cell Biology 185, no. 7 (June 29, 2009): 1135–48. http://dx.doi.org/10.1083/jcb.200903097.

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The light microscope has long been used to document the localization of fluorescent molecules in cell biology research. With advances in digital cameras and the discovery and development of genetically encoded fluorophores, there has been a huge increase in the use of fluorescence microscopy to quantify spatial and temporal measurements of fluorescent molecules in biological specimens. Whether simply comparing the relative intensities of two fluorescent specimens, or using advanced techniques like Förster resonance energy transfer (FRET) or fluorescence recovery after photobleaching (FRAP), quantitation of fluorescence requires a thorough understanding of the limitations of and proper use of the different components of the imaging system. Here, I focus on the parameters of digital image acquisition that affect the accuracy and precision of quantitative fluorescence microscopy measurements.

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Vikstrom, K. L., S. S. Lim, R. D. Goldman, and G. G. Borisy. "Steady state dynamics of intermediate filament networks." Journal of Cell Biology 118, no. 1 (July 1, 1992): 121–29. http://dx.doi.org/10.1083/jcb.118.1.121.

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We have conducted experiments to examine the dynamic exchange between subunit and polymer of vimentin intermediate filaments (IF) at steady state through the use of xrhodamine-labeled vimentin in fluorescence recovery after photobleaching (FRAP) analysis. The xrhodamine-vimentin incorporated into the endogenous vimentin IF network after microinjection into fibroblasts and could be visualized with a cooled charge-coupled device (CCD) camera and digital imaging fluorescence microscopy. Bar shaped regions were bleached in the fluorescent IF network using a beam from an argon ion laser and the cells were monitored at various times after bleaching to assess recovery of fluorescence in the bleached zones. We determined that bleached vimentin fibers can recover their fluorescence over relatively short time periods. Vimentin fibers in living cells also can exhibit significant movements, but the recovery of fluorescence was not dependent upon movement of fibers. Fluorescence recovery within individual fibers did not exhibit any marked polarity and was most consistent with a steady state exchange of vimentin subunits along the lengths of IF.

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Kure, Jakob L., Camilla B. Andersen, Thomas E. Rasmussen, B. Christoffer Lagerholm, and Eva C. Arnspang. "Defining the Diffusion in Model Membranes Using Line Fluorescence Recovery after Photobleaching." Membranes 10, no. 12 (December 17, 2020): 434. http://dx.doi.org/10.3390/membranes10120434.

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In this study, we explore the use of line FRAP to detect diffusion in synthetic lipid membranes. The study of the dynamics of these membrane lipids can, however, be challenging. The diffusion in two different synthetic membranes consisting of the lipid mixtures 1:1 DOPC:DPPC and 2:2:1 DOPC:DPPC:Cholesterol was studied with line FRAP. A correlation between diffusion coefficient and temperature was found to be dependent on the morphology of the membrane. We suggest line FRAP as a promising accessible and simple technique to study diffusion in plasma membranes.

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Yuste, S. B., E. Abad, and K. Lindenberg. "A reaction–subdiffusion model of fluorescence recovery after photobleaching (FRAP)." Journal of Statistical Mechanics: Theory and Experiment 2014, no. 11 (November 10, 2014): P11014. http://dx.doi.org/10.1088/1742-5468/2014/11/p11014.

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Berk, D. A., M. A. Swartz, A. J. Leu, and R. K. Jain. "Transport in lymphatic capillaries. II. Microscopic velocity measurement with fluorescence photobleaching." American Journal of Physiology-Heart and Circulatory Physiology 270, no. 1 (January 1, 1996): H330—H337. http://dx.doi.org/10.1152/ajpheart.1996.270.1.h330.

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Despite its relevance to the physiology of lymph formation and propulsion, the instantaneous flow velocity in single lymphatic capillaries has not been measured to date. The method of fluorescence recovery after photobleaching (FRAP) was adapted for this purpose and used to characterize flow in the lymphatic capillaries in tail skin of anesthetized mice during a constant-pressure intradermal injection of fluorescein isothiocyanate-dextran (mol wt 2 x 10(6). The median lymph flow velocity was 4.7 microns/s, and the velocity magnitude ranged from 0 to 29 microns/s. The direction of flow was generally proximal, but stasis and backflow toward the site of injection was also detected. Evidence for oscillatory flow was detected in some FRAP experiments, and in separate experiments a periodicity of approximately 120 min-1, directly correlated to respiration frequency, was measured by tracking the motion of fluorescent latex microspheres (1 micron diam) introduced into the lymphatic capillary network. The velocity magnitude showed a correlation with duration of infusion but not with distance from injection site. It is speculated that the temporal decay of mean velocity magnitude could be related to the relaxation of local pressure gradients as partially collapsed vessels expand during the infusion.

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Day, Charles A., and Minchul Kang. "The Utility of Fluorescence Recovery after Photobleaching (FRAP) to Study the Plasma Membrane." Membranes 13, no. 5 (May 2, 2023): 492. http://dx.doi.org/10.3390/membranes13050492.

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The plasma membrane of mammalian cells is involved in a wide variety of cellular processes, including, but not limited to, endocytosis and exocytosis, adhesion and migration, and signaling. The regulation of these processes requires the plasma membrane to be highly organized and dynamic. Much of the plasma membrane organization exists at temporal and spatial scales that cannot be directly observed with fluorescence microscopy. Therefore, approaches that report on the membrane’s physical parameters must often be utilized to infer membrane organization. As discussed here, diffusion measurements are one such approach that has allowed researchers to understand the subresolution organization of the plasma membrane. Fluorescence recovery after photobleaching (or FRAP) is the most widely accessible method for measuring diffusion in a living cell and has proven to be a powerful tool in cell biology research. Here, we discuss the theoretical underpinnings that allow diffusion measurements to be used in elucidating the organization of the plasma membrane. We also discuss the basic FRAP methodology and the mathematical approaches for deriving quantitative measurements from FRAP recovery curves. FRAP is one of many methods used to measure diffusion in live cell membranes; thus, we compare FRAP with two other popular methods: fluorescence correlation microscopy and single-particle tracking. Lastly, we discuss various plasma membrane organization models developed and tested using diffusion measurements.

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Rayan, Gamal, Jean-Erik Guet, Nicolas Taulier, Frederic Pincet, and Wladimir Urbach. "Recent Applications of Fluorescence Recovery after Photobleaching (FRAP) to Membrane Bio-Macromolecules." Sensors 10, no. 6 (June 10, 2010): 5927–48. http://dx.doi.org/10.3390/s100605927.

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Barsony, J., J. Carroll, W. McKoy, I. Renyi, D. L. Gould, H. Htun, and G. Hager. "Intracellular Traffic of Glucocorticoid Receptors: Studies With Green Fluorescent Protein Chimeras in Living Cells." Microscopy and Microanalysis 3, S2 (August 1997): 131–32. http://dx.doi.org/10.1017/s1431927600007546.

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As ligand-regulated transcription factors, glucocorticoid receptors (GR) must traffic through the cytoplasm, traverse the nuclear pores, and subsequently traffic within the the nucleus to reach their target genes. Due to technical difficulties with immunocytology, little is known about the translocation process or the intranuclear localization. The recent characterization of a chromophore, green fluorescent protein (GFP), provided a general tool to fluorescently label proteins in living cells. With the development of a transcriptionally active GFP-GR chimera, it became possible to visualize GR translocation and intranuclear distribution in living cells.This chimeric receptor was transiently transfected into mouse adenocarcinoma cells, allowing the direct visualization of GR using real-time video and confocal laser scanning microscopy. Mobility of GFP-GR was analyzed with fluorescent recovery after photobleaching (FRAP).The hormone-free GFP-GR was localized in the cytoplasm figure 1). Dexamethasone (lOnM) initiated GFP-GR translocation into the nucleus (Figure 2 and 3). The translocation rate was dose- and temperature-dependent, and occurred in a pulsatile manner along cytoplasmic fibrillar structures (Figure 2). FRAP experiments showed that GFP-GR remained in motion within the nucleus after translocation.

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Worden, Austin N. "The Alphabet Soup of Microscopy: An Introduction to Advanced Imaging Techniques. Part II: What the FLIP, FLAP, FRAP, FRET, and FLIM is Going On?" Microscopy Today 32, no. 5 (September 2024): 53–57. http://dx.doi.org/10.1093/mictod/qaae065.

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Abstract The use of fluorescence in imaging has been a pivotal factor in advancing our scientific understanding. As microscopy continues to evolve, the terminology used to describe these techniques becomes increasingly complex, often resulting in a bewildering array of acronyms that resemble alphabet soup. Among the most prominent acronyms are those associated with advanced fluorescence microscopy techniques: Fluorescence Loss in Photobleaching (FLIP), Fluorescence Localization after Photobleaching (FLAP), Fluorescence Recovery after Photobleaching (FRAP), Förster Resonance Energy Transfer (FRET), and Fluorescence Lifetime Imaging Microscopy (FLIM). Each of these methods provides invaluable insights into molecular dynamics and interactions within living cells and tissues. This article, Part 2 of the Alphabet Soup of Microscopy series, aims to clarify these widely used fluorescence microscopy techniques and to illuminate their contributions to our understanding of cellular processes.

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Cardarelli, Francesco, Luca Tosti, Michela Serresi, Fabio Beltram, and Ranieri Bizzarri. "Fluorescent Recovery after Photobleaching (FRAP) Analysis of Nuclear Export Rates Identifies Intrinsic Features of Nucleocytoplasmic Transport." Journal of Biological Chemistry 287, no. 8 (December 21, 2011): 5554–61. http://dx.doi.org/10.1074/jbc.m111.304899.

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24

Huang, Zhenqiu, Sabine Kaltenbrunner, Eva Šimková, David Stanĕk, Julius Lukeš, and Hassan Hashimi. "Dynamics of Mitochondrial RNA-Binding Protein Complex in Trypanosoma brucei and Its Petite Mutant under Optimized Immobilization Conditions." Eukaryotic Cell 13, no. 9 (July 25, 2014): 1232–40. http://dx.doi.org/10.1128/ec.00149-14.

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ABSTRACT There are a variety of complex metabolic processes ongoing simultaneously in the single, large mitochondrion of Trypanosoma brucei . Understanding the organellar environment and dynamics of mitochondrial proteins requires quantitative measurement in vivo . In this study, we have validated a method for immobilizing both procyclic stage (PS) and bloodstream stage (BS) T. brucei brucei with a high level of cell viability over several hours and verified its suitability for undertaking fluorescence recovery after photobleaching (FRAP), with mitochondrion-targeted yellow fluorescent protein (YFP). Next, we used this method for comparative analysis of the translational diffusion of mitochondrial RNA-binding protein 1 (MRP1) in the BS and in T. b. evansi . The latter flagellate is like petite mutant Saccharomyces cerevisiae because it lacks organelle-encoded nucleic acids. FRAP measurement of YFP-tagged MRP1 in both cell lines illuminated from a new perspective how the absence or presence of RNA affects proteins involved in mitochondrial RNA metabolism. This work represents the first attempt to examine this process in live trypanosomes.

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25

Scardigli, M., C. Crocini, C. Ferrantini, T. Gabbrielli, L. Silvestri, R. Coppini, C. Tesi, et al. "Quantitative assessment of passive electrical properties of the cardiac T-tubular system by FRAP microscopy." Proceedings of the National Academy of Sciences 114, no. 22 (May 15, 2017): 5737–42. http://dx.doi.org/10.1073/pnas.1702188114.

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Well-coordinated activation of all cardiomyocytes must occur on every heartbeat. At the cell level, a complex network of sarcolemmal invaginations, called the transverse-axial tubular system (TATS), propagates membrane potential changes to the cell core, ensuring synchronous and uniform excitation–contraction coupling. Although myocardial conduction of excitation has been widely described, the electrical properties of the TATS remain mostly unknown. Here, we exploit the formal analogy between diffusion and electrical conductivity to link the latter with the diffusional properties of TATS. Fluorescence recovery after photobleaching (FRAP) microscopy is used to probe the diffusion properties of TATS in isolated rat cardiomyocytes: A fluorescent dextran inside TATS lumen is photobleached, and signal recovery by diffusion of unbleached dextran from the extracellular space is monitored. We designed a mathematical model to correlate the time constant of fluorescence recovery with the apparent diffusion coefficient of the fluorescent molecules. Then, apparent diffusion is linked to electrical conductivity and used to evaluate the efficiency of the passive spread of membrane depolarization along TATS. The method is first validated in cells where most TATS elements are acutely detached by osmotic shock and then applied to probe TATS electrical conductivity in failing heart cells. We find that acute and pathological tubular remodeling significantly affect TATS electrical conductivity. This may explain the occurrence of defects in action potential propagation at the level of single T-tubules, recently observed in diseased cardiomyocytes.

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Favard, Cyril. "Numerical Simulation and FRAP Experiments Show That the Plasma Membrane Binding Protein PH-EFA6 Does Not Exhibit Anomalous Subdiffusion in Cells." Biomolecules 8, no. 3 (September 5, 2018): 90. http://dx.doi.org/10.3390/biom8030090.

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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.

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27

Yoon, Kyeong Han, Miri Yoon, Robert D. Moir, Satya Khuon, Frederick W. Flitney, and Robert D. Goldman. "Insights into the Dynamic Properties of Keratin Intermediate Filaments in Living Epithelial Cells." Journal of Cell Biology 153, no. 3 (April 24, 2001): 503–16. http://dx.doi.org/10.1083/jcb.153.3.503.

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The properties of keratin intermediate filaments (IFs) have been studied after transfection with green fluorescent protein (GFP)-tagged K18 and/or K8 (type I/II IF proteins). GFP-K8 and -K18 become incorporated into tonofibrils, which are comprised of bundles of keratin IFs. These tonofibrils exhibit a remarkably wide range of motile and dynamic activities. Fluorescence recovery after photobleaching (FRAP) analyses show that they recover their fluorescence slowly with a recovery t1/2 of ∼100 min. The movements of bleach zones during recovery show that closely spaced tonofibrils (<1 μm apart) often move at different rates and in different directions. Individual tonofibrils frequently change their shapes, and in some cases these changes appear as propagated waveforms along their long axes. In addition, short fibrils, termed keratin squiggles, are seen at the cell periphery where they move mainly towards the cell center. The motile properties of keratin IFs are also compared with those of type III IFs (vimentin) in PtK2 cells. Intriguingly, the dynamic properties of keratin tonofibrils and squiggles are dramatically different from those of vimentin fibrils and squiggles within the same cytoplasmic regions. This suggests that there are different factors regulating the dynamic properties of different types of IFs within the same cytoplasmic regions.

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Hori, Masatoshi, Jeffrey D. Jones, Lee Janson, Keith Ragsdale, and Katherine Luby-Phelps. "Light – microscopic analysis of the physical properties of cytoplasm in living cells." Proceedings, annual meeting, Electron Microscopy Society of America 54 (August 11, 1996): 734–35. http://dx.doi.org/10.1017/s0424820100166130.

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We have used a variety of optical techniques to explore the intracellular constraints on diffusion, cytoplasmic compartmentalization and biomechanics of living tissue culture cells. Fluorescence ratio imaging measurements of solvent viscosity in single cells indicate that the solvent viscosity of cytoplasm does not differ detectably from bulk water. Nevertheless, data obtained by fluorescence recovery after photobleaching (FRAP) of inert tracer molecules suggest that macromolecular crowding and molecular sieving retard the long-range translational diffusion of protein-sized molecules from 4 to 50-fold relative to their diffusion in dilute aqueous solution.Transient binding interactions with intracellular components may additionally retard the diffusion of proteins. Recent FRAP studies of fluorescent analogs of calmodulin suggest that as little as 5% of this protein is freely diffusing, even in unstimulated cells where intracellular [Ca2+] is below the threshold for activation of calmodulin binding to calcium-dependent targets in vitro. Preliminary data show that binding is mitigated or abolished by charge reversal mutations in the central helix of calmodulin or by a point mutation in calcium binding loop 4 that greatly reduces the affinity of calmodulin for Ca2+.

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29

Orekhov, Fedor K., Arthur G. Jablokov, and Andrew A. Skrynnik. "Ybridization of laser-induced spectrofluorescence analysis (lifs), matrix-assisted laser desorption / ionization mass spectrometry (maldi), fluorescence recovery after photobleaching (frap) anf fluorescence loss in photobleaching (flip) microtechnics." Journal of Biomedical Technologies, no. 2 (December 2016): 42–52. http://dx.doi.org/10.15393/j6.art.2016.3702.

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Watanabe, Yumi, Masahito Hayashi, Toshiki Yagi, and Ritsu Kamiya. "Turnover of Actin in Chlamydomonas Flagella Detected by Fluorescence Recovery After Photobleaching (FRAP)." Cell Structure and Function 29, no. 3 (2004): 67–72. http://dx.doi.org/10.1247/csf.29.67.

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Kipper, Franciele Cristina, Alessandra Sayuri Kikuchi Tamajusuku, Darlan Conterno Minussi, José Eduardo Vargas, Ana Maria Oliveira Battastini, Elzbieta Kaczmarek, Simon Christopher Robson, Guido Lenz, and Márcia Rosângela Wink. "Analysis of NTPDase2 in the cell membrane using fluorescence recovery after photobleaching (FRAP)." Cytometry Part A 93, no. 2 (January 24, 2018): 232–38. http://dx.doi.org/10.1002/cyto.a.23317.

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32

Storrie, B., R. Pepperkok, E. H. Stelzer, and T. E. Kreis. "The intracellular mobility of a viral membrane glycoprotein measured by confocal microscope fluorescence recovery after photobleaching." Journal of Cell Science 107, no. 5 (May 1, 1994): 1309–19. http://dx.doi.org/10.1242/jcs.107.5.1309.

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Fluorescence recovery after photobleaching (FRAP) has been a powerful tool for characterizing the mobility of cell surface membrane proteins. However, the application of FRAP to the study of intracellular membrane proteins has been hampered by the lack of specific probes and their physical inaccessibility in the cytoplasm. We have measured the mobility of a model transmembrane protein, the temperature-sensitive vesicular stomatitis viral membrane glycoprotein (ts-O45-G), in transit from the endoplasmic reticulum (ER) to the Golgi complex. ts-O45-G accumulates in the ER at nonpermissive temperature (39.5 degrees C) and is transported via the Golgi complex to the surface upon shifting cells to the permissive temperature (31 degrees C). Rhodamine-labeled Fab fragments against a cytoplasmic epitope of ts-O45-G (rh-P5D4-Fabs) were microinjected into cells to visualize the intracellular viral membrane protein and to determine its mobility by FRAP with a confocal microscope. Moreover, we have measured the effects of microinjected antibodies against beta-COP on the mobility of ts-O45-G following release of the temperature block. FRAP was essentially complete when rh-P5D4-Fab-injected cells were bleached either following release of labeled ts-O45-G from the ER or upon its accumulation at 20 degrees C in the trans-Golgi network (TGN). In contrast, recovery was reduced by about one third when infected cells had been injected with antibodies that bind to beta-COP in vivo. The diffusion constant of mobile ts-O45-G under all conditions was approximately 10 × 10(−10) cm2/s. These results validate the feasibility of FRAP for the study of an intracellular transmembrane protein and provide the first evidence that such a protein is highly mobile.

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Brandon, Elizabeth, Tomasz Szul, Cecilia Alvarez, Robert Grabski, Ronald Benjamin, Ryoichi Kawai, and Elizabeth Sztul. "On and Off Membrane Dynamics of the Endoplasmic Reticulum–Golgi Tethering Factor p115 In Vivo." Molecular Biology of the Cell 17, no. 7 (July 2006): 2996–3008. http://dx.doi.org/10.1091/mbc.e05-09-0862.

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The mechanisms regulating membrane recruitment of the p115 tethering factor in vivo are unknown. Here, we describe cycling of p115 between membranes and cytosol and document the effects of Golgi matrix proteins, Rab1, and soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) on this process. Rapid membrane/cytosol exchange is shown by swift (t1/2 ∼20 s) loss of Golgi-localized p115-green fluorescent protein (GFP) after repeated photobleaching of cell periphery and rapid (t1/2 ∼13 s) fluorescence recovery after photobleaching Golgi-localized p115-GFP. p115 mutant missing the GM130/giantin binding site exhibits analogous fluorescence recovery after photobleaching (FRAP) (t1/2 ∼13 s), suggesting that GM130 and giantin are not major determinants of p115 membrane dynamics. In contrast, p115-GFP exchanges more rapidly (t1/2 ∼8 s) in cells expressing the inactive Rab1/N121I mutant, indicating that p115 cycling is influenced by Rab1. p115-GFP dynamics is also influenced by the assembly status of SNAREs. In cells expressing an ATPase-deficient NSF/E329Q mutant that inhibits SNARE complex disassembly, the cycling kinetics of p115-GFP are significantly slower (t1/2 ∼21 s). In contrast, in cells incubated at reduced temperature (10°C) that inhibits vesicular traffic, the cycling kinetics of p115-GFP are faster (t1/2 ∼7 s). These data suggest that p115-binding sites on the membrane are provided by unassembled SNAREs. In agreement, biochemical studies show increased p115 recruitment to membranes in the presence of NSF and α-SNAP. Our data support a model in which recruitment of tethers is directly regulated by the assembly status of SNAREs.

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Saito, Takumi, Daiki Matsunaga, and Shinji Deguchi. "Long-term molecular turnover of actin stress fibers revealed by advection-reaction analysis in fluorescence recovery after photobleaching." PLOS ONE 17, no. 11 (November 7, 2022): e0276909. http://dx.doi.org/10.1371/journal.pone.0276909.

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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.

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35

Mullineaux, Conrad W., Anja Nenninger, Nicola Ray, and Colin Robinson. "Diffusion of Green Fluorescent Protein in Three Cell Environments in Escherichia Coli." Journal of Bacteriology 188, no. 10 (May 15, 2006): 3442–48. http://dx.doi.org/10.1128/jb.188.10.3442-3448.2006.

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ABSTRACT 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.

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Engel, Stephanie, Silvia Scolari, Bastian Thaa, Nils Krebs, Thomas Korte, Andreas Herrmann, and Michael Veit. "FLIM-FRET and FRAP reveal association of influenza virus haemagglutinin with membrane rafts." Biochemical Journal 425, no. 3 (January 15, 2010): 567–73. http://dx.doi.org/10.1042/bj20091388.

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It has been supposed that the HA (haemagglutinin) of influenza virus must be recruited to membrane rafts to perform its function in membrane fusion and virus budding. In the present study, we aimed at substantiating this association in living cells by biophysical methods. To this end, we fused the cyan fluorescent protein Cer (Cerulean) to the cytoplasmic tail of HA. Upon expression in CHO (Chinese-hamster ovary) cells HA–Cer was glycosylated and transported to the plasma membrane in a similar manner to authentic HA. We measured FLIM-FRET (Förster resonance energy transfer by fluorescence lifetime imaging microscopy) and showed strong association of HA–Cer with Myr-Pal–YFP (myristoylated and palmitoylated peptide fused to yellow fluorescent protein), an established marker for rafts of the inner leaflet of the plasma membrane. Clustering was significantly reduced when rafts were disintegrated by cholesterol extraction and when the known raft-targeting signals of HA, the palmitoylation sites and amino acids in its transmembrane region, were removed. FRAP (fluorescence recovery after photobleaching) showed that removal of raft-targeting signals moderately increased the mobility of HA in the plasma membrane, indicating that the signals influence access of HA to slowly diffusing rafts. However, Myr-Pal–YFP exhibited a much faster mobility compared with HA–Cer, demonstrating that HA and the raft marker do not diffuse together in a stable raft complex for long periods of time.

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Moir, Robert D., Miri Yoon, Satya Khuon, and Robert D. Goldman. "Nuclear Lamins a and B1." Journal of Cell Biology 151, no. 6 (December 11, 2000): 1155–68. http://dx.doi.org/10.1083/jcb.151.6.1155.

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At the end of mitosis, the nuclear lamins assemble to form the nuclear lamina during nuclear envelope formation in daughter cells. We have fused A- and B-type nuclear lamins to the green fluorescent protein to study this process in living cells. The results reveal that the A- and B-type lamins exhibit different pathways of assembly. In the early stages of mitosis, both lamins are distributed throughout the cytoplasm in a diffusible (nonpolymerized) state, as demonstrated by fluorescence recovery after photobleaching (FRAP). During the anaphase-telophase transition, lamin B1 begins to become concentrated at the surface of the chromosomes. As the chromosomes reach the spindle poles, virtually all of the detectable lamin B1 has accumulated at their surfaces. Subsequently, this lamin rapidly encloses the entire perimeter of the region containing decondensing chromosomes in each daughter cell. By this time, lamin B1 has assembled into a relatively stable polymer, as indicated by FRAP analyses and insolubility in detergent/high ionic strength solutions. In contrast, the association of lamin A with the nucleus begins only after the major components of the nuclear envelope including pore complexes are assembled in daughter cells. Initially, lamin A is found in an unpolymerized state throughout the nucleoplasm of daughter cell nuclei in early G1 and only gradually becomes incorporated into the peripheral lamina during the first few hours of this stage of the cell cycle. In later stages of G1, FRAP analyses suggest that both green fluorescent protein lamins A and B1 form higher order polymers throughout interphase nuclei.

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Floury, J., M. N. Madec, F. Waharte, S. Jeanson, and S. Lortal. "First assessment of diffusion coefficients in model cheese by fluorescence recovery after photobleaching (FRAP)." Food Chemistry 133, no. 2 (July 2012): 551–56. http://dx.doi.org/10.1016/j.foodchem.2012.01.030.

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Wahl, Philippe, and Fouad Azizi. "Fluorescent recovery after photobleaching (FRAP) of a fluorescent transferrin internalized in the late transferrin endocytic compartment of living A431 cells: Theory." Biochimica et Biophysica Acta (BBA) - Biomembranes 1327, no. 1 (July 1997): 69–74. http://dx.doi.org/10.1016/s0005-2736(97)00045-x.

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40

Handwerger, Korie E., Christine Murphy, and Joseph G. Gall. "Steady-state dynamics of Cajal body components in the Xenopus germinal vesicle." Journal of Cell Biology 160, no. 4 (February 17, 2003): 495–504. http://dx.doi.org/10.1083/jcb.200212024.

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Cajal bodies (CBs) are evolutionarily conserved nuclear organelles that contain many factors involved in the transcription and processing of RNA. It has been suggested that macromolecular complexes preassemble or undergo maturation within CBs before they function elsewhere in the nucleus. Most such models of CB function predict a continuous flow of molecules between CBs and the nucleoplasm, but there are few data that directly support this view. We used fluorescence recovery after photobleaching (FRAP) on isolated Xenopus oocyte nuclei to measure the steady-state exchange rate between the nucleoplasm and CBs of three fluorescently tagged molecules: U7 small nuclear RNA, coilin, and TATA-binding protein (TBP). In the nucleoplasm, the apparent diffusion coefficients for the three molecules ranged from 0.26 to 0.40 μm2 s−1. However, in CBs, fluorescence recovery was markedly slower than in the nucleoplasm, and there were at least three kinetic components. The recovery rate within CBs was independent of bleach spot diameter and could not be attributed to high CB viscosity or density. We propose that binding to other molecules and possibly assembly into larger complexes are the rate-limiting steps for FRAP of U7, coilin, and TBP inside CBs.

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41

Redman, C. A., and J. R. Kusel. "Distribution and biophysical properties of fluorescent lipids on the surface of adult Schistosoma mansoni." Parasitology 113, no. 2 (August 1996): 137–43. http://dx.doi.org/10.1017/s0031182000066385.

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SUMMARYThe properties of 4 fluorescent lipid compounds in the surface membrane of adult male Schistosoma mansoni worms were examined by fluorescent microscopy and fluorescent recovery after photobleaching (FRAP). The data suggest that the probes N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl) sphingosine (BODIPY FL ceramide) and PKH2 pass through the outer membrane and enter structures in or below the membrane. In contrast 5-(N-octadecanoyl)aminofluorescein (AF18) and N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl) sphingosylphosphocholine (BODIPY FL sphingomyelin) insert into the outer monolayer. The DL values of these latter 2 compounds, 8·83 ± 2·35 × 10−9 cm2 s−1 and 2·76 ± 0·53 × 10−9cm2 s−1, respectively, suggest that they enter different domains. Furthermore, it was observed that both BODIPY FL ceramide and BODIPY FL sphingomyelin entered particular structures in or under the surface membrane. The possible nature of these particles is discussed.

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42

Azizi, Fouad, and Philippe Wahl. "Fluorescence recovery after photobleaching (FRAP) of a fluorescent transferrin internalized in the late transferrin endocytic compartment of living A431 cells: Experiments." Biochimica et Biophysica Acta (BBA) - Biomembranes 1327, no. 1 (July 1997): 75–88. http://dx.doi.org/10.1016/s0005-2736(97)00046-1.

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43

Govindaraj, Kannan, Jan Hendriks, Diane S. Lidke, Marcel Karperien, and Janine N. Post. "Changes in Fluorescence Recovery After Photobleaching (FRAP) as an indicator of SOX9 transcription factor activity." Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1862, no. 1 (January 2019): 107–17. http://dx.doi.org/10.1016/j.bbagrm.2018.11.001.

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44

Dewavrin, Jean-Yves, and Michael Raghunath. "Determining the Optimal Degree of Macromolecular Crowding in Solution Using Fluorescence Recovery after Photobleaching (FRAP)." Biophysical Journal 102, no. 3 (January 2012): 400a. http://dx.doi.org/10.1016/j.bpj.2011.11.2184.

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45

Zhao, Runchen, Siqi Cui, Zhuoxu Ge, Yuqi Zhang, Kaustav Bera, Lily Zhu, Sean X. Sun, and Konstantinos Konstantopoulos. "Hydraulic resistance induces cell phenotypic transition in confinement." Science Advances 7, no. 17 (April 2021): eabg4934. http://dx.doi.org/10.1126/sciadv.abg4934.

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Cells penetrating into confinement undergo mesenchymal-to-amoeboid transition. The topographical features of the microenvironment expose cells to different hydraulic resistance levels. How cells respond to hydraulic resistance is unknown. We show that the cell phenotype shifts from amoeboid to mesenchymal upon increasing resistance. By combining automated morphological tracking and wavelet analysis along with fluorescence recovery after photobleaching (FRAP), we found an oscillatory phenotypic transition that cycles from blebbing to short, medium, and long actin network formation, and back to blebbing. Elevated hydraulic resistance promotes focal adhesion maturation and long actin filaments, thereby reducing the period required for amoeboid-to-mesenchymal transition. The period becomes independent of resistance upon blocking the mechanosensor TRPM7. Mathematical modeling links intracellular calcium oscillations with actomyosin turnover and force generation and recapitulates experimental data. We identify hydraulic resistance as a critical physical cue controlling cell phenotype and present an approach for connecting fluorescent signal fluctuations to morphological oscillations.

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46

Seksek, Olivier, Joachim Biwersi, and A. S. Verkman. "Translational Diffusion of Macromolecule-sized Solutes in Cytoplasm and Nucleus." Journal of Cell Biology 138, no. 1 (July 14, 1997): 131–42. http://dx.doi.org/10.1083/jcb.138.1.131.

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Fluorescence recovery after photobleaching (FRAP) was used to quantify the translational diffusion of microinjected FITC-dextrans and Ficolls in the cytoplasm and nucleus of MDCK epithelial cells and Swiss 3T3 fibroblasts. Absolute diffusion coefficients (D) were measured using a microsecond-resolution FRAP apparatus and solution standards. In aqueous media (viscosity 1 cP), D for the FITC-dextrans decreased from 75 to 8.4 × 10−7 cm2/s with increasing dextran size (4–2,000 kD). D in cytoplasm relative to that in water (D/Do) was 0.26 ± 0.01 (MDCK) and 0.27 ± 0.01 (fibroblasts), and independent of FITC-dextran and Ficoll size (gyration radii [RG] 40–300 Å). The fraction of mobile FITC-dextran molecules (fmob), determined by the extent of fluorescence recovery after spot photobleaching, was >0.75 for RG < 200 Å, but decreased to <0.5 for RG > 300 Å. The independence of D/Do on FITC-dextran and Ficoll size does not support the concept of solute “sieving” (size-dependent diffusion) in cytoplasm. Photobleaching measurements using different spot diameters (1.5–4 μm) gave similar D/Do, indicating that microcompartments, if present, are of submicron size. Measurements of D/Do and fmob in concentrated dextran solutions, as well as in swollen and shrunken cells, suggested that the low fmob for very large macromolecules might be related to restrictions imposed by immobile obstacles (such as microcompartments) or to anomalous diffusion (such as percolation). In nucleus, D/Do was 0.25 ± 0.02 (MDCK) and 0.27 ± 0.03 (fibroblasts), and independent of solute size (RG 40–300 Å). Our results indicate relatively free and rapid diffusion of macromolecule-sized solutes up to approximately 500 kD in cytoplasm and nucleus.

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47

Wüstner, Daniel. "Dynamic Mode Decomposition of Fluorescence Loss in Photobleaching Microscopy Data for Model-Free Analysis of Protein Transport and Aggregation in Living Cells." Sensors 22, no. 13 (June 23, 2022): 4731. http://dx.doi.org/10.3390/s22134731.

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The phase separation and aggregation of proteins are hallmarks of many neurodegenerative diseases. These processes can be studied in living cells using fluorescent protein constructs and quantitative live-cell imaging techniques, such as fluorescence recovery after photobleaching (FRAP) or the related fluorescence loss in photobleaching (FLIP). While the acquisition of FLIP images is straightforward on most commercial confocal microscope systems, the analysis and computational modeling of such data is challenging. Here, a novel model-free method is presented, which resolves complex spatiotemporal fluorescence-loss kinetics based on dynamic-mode decomposition (DMD) of FLIP live-cell image sequences. It is shown that the DMD of synthetic and experimental FLIP image series (DMD-FLIP) allows for the unequivocal discrimination of subcellular compartments, such as nuclei, cytoplasm, and protein condensates based on their differing transport and therefore fluorescence loss kinetics. By decomposing fluorescence-loss kinetics into distinct dynamic modes, DMD-FLIP will enable researchers to study protein dynamics at each time scale individually. Furthermore, it is shown that DMD-FLIP is very efficient in denoising confocal time series data. Thus, DMD-FLIP is an easy-to-use method for the model-free detection of barriers to protein diffusion, of phase-separated protein assemblies, and of insoluble protein aggregates. It should, therefore, find wide application in the analysis of protein transport and aggregation, in particular in relation to neurodegenerative diseases and the formation of protein condensates in living cells.

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Vámosi, György, Elza Friedländer-Brock, Shehu M. Ibrahim, Roland Brock, János Szöllősi, and György Vereb. "EGF Receptor Stalls upon Activation as Evidenced by Complementary Fluorescence Correlation Spectroscopy and Fluorescence Recovery after Photobleaching Measurements." International Journal of Molecular Sciences 20, no. 13 (July 9, 2019): 3370. http://dx.doi.org/10.3390/ijms20133370.

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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.

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Hansen, Jesper S., Nils J. Færgeman, Birthe B. Kragelund, and Jens Knudsen. "Acyl-CoA-binding protein (ACBP) localizes to the endoplasmic reticulum and Golgi in a ligand-dependent manner in mammalian cells." Biochemical Journal 410, no. 3 (February 27, 2008): 463–72. http://dx.doi.org/10.1042/bj20070559.

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In the present study, we microinjected fluorescently labelled liver bovine ACBP (acyl-CoA-binding protein) [FACI-50 (fluorescent acyl-CoA indicator-50)] into HeLa and BMGE (bovine mammary gland epithelial) cell lines to characterize the localization and dynamics of ACBP in living cells. Results showed that ACBP targeted to the ER (endoplasmic reticulum) and Golgi in a ligand-binding-dependent manner. A variant Y28F/K32A-FACI-50, which is unable to bind acyl-CoA, did no longer show association with the ER and became segregated from the Golgi, as analysed by intensity correlation calculations. Depletion of fatty acids from cells by addition of FAFBSA (fatty-acid-free BSA) significantly decreased FACI-50 association with the Golgi, whereas fatty acid overloading increased Golgi association, strongly supporting that ACBP associates with the Golgi in a ligand-dependent manner. FRAP (fluorescence recovery after photobleaching) showed that the fatty-acid-induced targeting of FACI-50 to the Golgi resulted in a 5-fold reduction in FACI-50 mobility. We suggest that ACBP is targeted to the ER and Golgi in a ligand-binding-dependent manner in living cells and propose that ACBP may be involved in vesicular trafficking.

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Olmsted, J. B., D. L. Stemple, W. M. Saxton, B. W. Neighbors, and J. R. McIntosh. "Cell cycle-dependent changes in the dynamics of MAP 2 and MAP 4 in cultured cells." Journal of Cell Biology 109, no. 1 (July 1, 1989): 211–23. http://dx.doi.org/10.1083/jcb.109.1.211.

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To examine the behavior of microtubule-associated proteins (MAPs) in living cells, MAP 4 and MAP 2 have been derivatized with 6-iodoacetamido-fluorescein, and the distribution of microinjected MAP has been analyzed using a low light level video system and fluorescence redistribution after photobleaching. Within 1 min following microinjection of fluoresceinated MAP 4 or MAP 2, fluorescent microtubule arrays were visible in interphase or mitotic PtK1 cells. After cold treatment of fluorescent MAP 2-containing cells (3 h, 4 degrees C), microtubule fluorescence disappeared, and the only fluorescence above background was located at the centrosomes; microtubule patterns returned upon warming. Loss of microtubule immunofluorescence after nocodozole treatment was similar in MAP-injected and control cells, suggesting that injected fluorescein-labeled MAP 2 did not stabilize microtubules. The dynamics of the MAPs were examined further by FRAP. FRAP analysis of interphase cells demonstrated that MAP 2 redistributed with half-times slightly longer (60 +/- 25 s) than those for MAP 4 (44 +/- 20 s), but both types of MAPs bound to microtubules in vivo exchanged with soluble MAPs at rates exceeding the rate of tubulin turnover. These data imply that microtubules in interphase cells are assembled with constantly exchanging populations of MAP. Metaphase cells at 37 degrees C or 26 degrees C showed similar mean redistribution half-times for both MAP 2 and MAP 4; these were 3-4 fold faster than the interphase rates (MAP 2, t1/2 = 14 +/- 6 s; MAP 4, t1/2 = 17 +/- 5 s). The extent of recovery of spindle fluorescence in MAP-injected cells was to 84-94% at either 26 or 37 degrees C. Although most metaphase tubulin, like the MAPs, turns over rapidly and completely under physiologic conditions, published work shows either reduced rates or extents of turnover at 26 degrees C, suggesting that the fast mitotic MAP exchange is not simply because of fast tubulin turnover. Exchange of MAP 4 bound to telophase midbodies occurred with dynamics comparable to those seen in metaphase spindles (t1/2 = approximately 27 s) whereas midbody tubulin exchange was slow (greater than 300 s). These data demonstrate that the rate of MAP exchange on microtubules is a function of time in the cell cycle.

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