fluorescence recovery after photobleaching

Summary

Summary: A method used to study the lateral movement of MEMBRANE PROTEINS and LIPIDS. A small area of a cell membrane is bleached by laser light and the amount of time necessary for unbleached fluorescent marker-tagged proteins to diffuse back into the bleached site is a measurement of the cell membrane's fluidity. The diffusion coefficient of a protein or lipid in the membrane can be calculated from the data. (From Segen, Current Med Talk, 1995).

Top Publications

  1. Winey M, Stemm Wolf A, Giddings T, Pearson C. Cytological analysis of Tetrahymena thermophila. Methods Cell Biol. 2012;109:357-78 pubmed publisher
    ..in live cells, as well as for tracking the dynamic behavior of proteins using pulse labeling and fluorescence recovery after photobleaching. For electron microscopy, cellular and antigenic preservation has been improved with the use of ..
  2. Brody Y, Neufeld N, Bieberstein N, Causse S, Böhnlein E, Neugebauer K, et al. The in vivo kinetics of RNA polymerase II elongation during co-transcriptional splicing. PLoS Biol. 2011;9:e1000573 pubmed publisher
    ..This suggests that the polymerase is released from chromatin prior to the completion of splicing, and the pre-mRNA is post-transcriptionally processed while still tethered to chromatin near the gene end...
  3. Carisey A, Stroud M, Tsang R, Ballestrem C. Fluorescence recovery after photobleaching. Methods Mol Biol. 2011;769:387-402 pubmed publisher
    This chapter describes the use of microscope-based fluorescence recovery after photobleaching (FRAP)...
  4. Erdel F, Schubert T, Marth C, Längst G, Rippe K. Human ISWI chromatin-remodeling complexes sample nucleosomes via transient binding reactions and become immobilized at active sites. Proc Natl Acad Sci U S A. 2010;107:19873-8 pubmed publisher
    ..Due to the relatively high intranuclear remodeler concentrations cellular response times for repositioning a given nucleosome were calculated to be in the range of tens of seconds to minutes. ..
  5. Luu D, Martinière A, Sorieul M, Runions J, Maurel C. Fluorescence recovery after photobleaching reveals high cycling dynamics of plasma membrane aquaporins in Arabidopsis roots under salt stress. Plant J. 2012;69:894-905 pubmed publisher
    ..GFP fusions with AtPIP1;2 and AtPIP2;1, two prototypic PM aquaporins, were used to develop a fluorescence recovery after photobleaching (FRAP) approach...
  6. Cizmecioglu O, Arnold M, Bahtz R, Settele F, Ehret L, Haselmann Weiss U, et al. Cep152 acts as a scaffold for recruitment of Plk4 and CPAP to the centrosome. J Cell Biol. 2010;191:731-9 pubmed publisher
    ..Our results suggest that Cep152 recruits Plk4 and CPAP to the centrosome to ensure a faithful centrosome duplication process. ..
  7. Kuhn T, Ihalainen T, Hyväluoma J, Dross N, Willman S, Langowski J, et al. Protein diffusion in mammalian cell cytoplasm. PLoS ONE. 2011;6:e22962 pubmed publisher
    ..The cytosol results were found to be in very good agreement with those by FCS. ..
  8. Mika J, Krasnikov V, van den Bogaart G, de Haan F, Poolman B. Evaluation of pulsed-FRAP and conventional-FRAP for determination of protein mobility in prokaryotic cells. PLoS ONE. 2011;6:e25664 pubmed publisher
    Macromolecule mobility is often quantified with Fluorescence Recovery After Photobleaching (FRAP)...
  9. Sugaya K, Seto S, Tsujimura K, Koide Y. Mobility of late endosomal and lysosomal markers on phagosomes analyzed by fluorescence recovery after photobleaching. Biochem Biophys Res Commun. 2011;410:371-5 pubmed publisher
    ..We investigated the mobility of Rab7 and LAMP1 on the phagosomes in macrophages by fluorescence recovery after photobleaching (FRAP) analysis...

More Information

Publications62

  1. van den Wildenberg S, Bollen Y, Peterman E. How to quantify protein diffusion in the bacterial membrane. Biopolymers. 2011;95:312-21 pubmed publisher
    ..using various experimental techniques, including fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), and particle tracking using single-molecule fluorescence (SMF) microscopy...
  2. Hemmerich P, Schmiedeberg L, Diekmann S. Dynamic as well as stable protein interactions contribute to genome function and maintenance. Chromosome Res. 2011;19:131-51 pubmed publisher
    ..Our new "induced stability" model suggests that self-organization, self-assembly, and assisted assembly contribute to nuclear architecture and function. ..
  3. van de Giessen M, van der Laan A, Hendriks E, Vidorreta M, Reiber J, Jost C, et al. Fully automated attenuation measurement and motion correction in FLIP image sequences. IEEE Trans Med Imaging. 2012;31:461-73 pubmed publisher
    ..The proposed method is fully automatic, and runs in approximately one minute per sequence, making it suitable for unsupervised batch processing of large data series. ..
  4. de Graaf P, Mousson F, Geverts B, Scheer E, Tora L, Houtsmuller A, et al. Chromatin interaction of TATA-binding protein is dynamically regulated in human cells. J Cell Sci. 2010;123:2663-71 pubmed publisher
    ..Here, we investigate the dynamic behaviour of TBP by a combination of fluorescence recovery after photobleaching (FRAP) and biochemical assays using human cell lines of different origin...
  5. Kaya A, Ugur O, Altuntaş O, Sayar K, Onaran H. Long and short distance movements of ?(2)-adrenoceptor in cell membrane assessed by photoconvertible fluorescent protein dendra2-?(2)-adrenoceptor fusion. Biochim Biophys Acta. 2011;1813:1511-24 pubmed publisher
    ..We also showed that intracellular compartments and plasma membrane are kinetically connected even at steady-state. ..
  6. Kang M, Day C, Dibenedetto E, Kenworthy A. A quantitative approach to analyze binding diffusion kinetics by confocal FRAP. Biophys J. 2010;99:2737-47 pubmed publisher
    ..We validate this method by measuring kinetic rate constants for the CAAX-mediated binding of Ras to membranes of the endoplasmic reticulum, obtaining binding constants of k(on) ? 255/s and k(off) ? 31/s. ..
  7. Schütz M, Scimemi P, Majumder P, De Siati R, Crispino G, Rodriguez L, et al. The human deafness-associated connexin 30 T5M mutation causes mild hearing loss and reduces biochemical coupling among cochlear non-sensory cells in knock-in mice. Hum Mol Genet. 2010;19:4759-73 pubmed publisher
    ..Our findings link hearing loss to decreased biochemical coupling due to the point-mutated Cx30 in mice...
  8. Mazza D, Abernathy A, Golob N, Morisaki T, McNally J. A benchmark for chromatin binding measurements in live cells. Nucleic Acids Res. 2012;40:e119 pubmed publisher
    ..benchmark we measured binding of the transcription factor p53 to chromatin by three approaches: fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and single-molecule tracking (SMT)...
  9. Mai J, Trump S, Ali R, Schiltz R, Hager G, Hanke T, et al. Are assumptions about the model type necessary in reaction-diffusion modeling? A FRAP application. Biophys J. 2011;100:1178-88 pubmed publisher
    At present, fluorescence recovery after photobleaching (FRAP) data are interpreted using various types of reaction-diffusion (RD) models: the model type is usually fixed first, and corresponding model parameters are inferred subsequently...
  10. Smisdom N, Braeckmans K, Deschout H, VandeVen M, Rigo J, De Smedt S, et al. Fluorescence recovery after photobleaching on the confocal laser-scanning microscope: generalized model without restriction on the size of the photobleached disk. J Biomed Opt. 2011;16:046021 pubmed publisher
    b>Fluorescence recovery after photobleaching (FRAP) carried out on a confocal laser-scanning microscope (CLSM) performs well for photobleached disks that are large compared to the resolution of the bleaching beam...
  11. Kawamata N, Pennella M, Woo J, Berk A, Koeffler H. Dominant-negative mechanism of leukemogenic PAX5 fusions. Oncogene. 2012;31:966-77 pubmed publisher
    ..PAX5-C20S is a tetramer, which interacts extraordinarily stably with chromatin as determined by Fluorescence Recovery After Photobleaching in living cells...
  12. Rayan G, Guet J, Taulier N, Pincet F, Urbach W. Recent applications of fluorescence recovery after photobleaching (FRAP) to membrane bio-macromolecules. Sensors (Basel). 2010;10:5927-48 pubmed publisher
    This review examines some recent applications of fluorescence recovery after photobleaching (FRAP) to biopolymers, while mainly focusing on membrane protein studies...
  13. Berkovich R, Wolfenson H, Eisenberg S, Ehrlich M, Weiss M, Klafter J, et al. Accurate quantification of diffusion and binding kinetics of non-integral membrane proteins by FRAP. Traffic. 2011;12:1648-57 pubmed publisher
    ..Despite a fair number of approximate fitting functions for analyzing fluorescence recovery after photobleaching (FRAP) data, no approach was able to cope with the full diffusion-exchange problem...
  14. Stasevich T, Mueller F, Michelman Ribeiro A, Rosales T, Knutson J, McNally J. Cross-validating FRAP and FCS to quantify the impact of photobleaching on in vivo binding estimates. Biophys J. 2010;99:3093-101 pubmed publisher
    ..To address this uncertainty, we compare fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) measurements of the binding kinetics of a ..
  15. Maiuri P, Knezevich A, De Marco A, Mazza D, Kula A, McNally J, et al. Fast transcription rates of RNA polymerase II in human cells. EMBO Rep. 2011;12:1280-5 pubmed publisher
    ..The calculated transcription rate above 50 kb min(-1) points towards a wide dynamic range of RNAPII velocities in living cells...
  16. Goehring N, Chowdhury D, Hyman A, Grill S. FRAP analysis of membrane-associated proteins: lateral diffusion and membrane-cytoplasmic exchange. Biophys J. 2010;99:2443-52 pubmed publisher
    Obtaining quantitative kinetic parameters from fluorescence recovery after photobleaching (FRAP) experiments generally requires a theoretical analysis of protein mobility and appropriate solutions for FRAP recovery derived for a given ..
  17. Mueller F, Karpova T, Mazza D, McNally J. Monitoring dynamic binding of chromatin proteins in vivo by fluorescence recovery after photobleaching. Methods Mol Biol. 2012;833:153-76 pubmed publisher
    b>Fluorescence recovery after photobleaching (FRAP) has now become widely used to investigate nuclear protein binding to chromatin in live cells...
  18. Bizzarri R, Cardarelli F, Serresi M, Beltram F. Fluorescence recovery after photobleaching reveals the biochemistry of nucleocytoplasmic exchange. Anal Bioanal Chem. 2012;403:2339-51 pubmed publisher
    b>Fluorescence recovery after photobleaching (FRAP) can help unveil subtle dynamical and biochemical properties of intracellular components...
  19. Machado M, Mitchell C. Temporal changes in microvessel leakiness during wound healing discriminated by in vivo fluorescence recovery after photobleaching. J Physiol. 2011;589:4681-96 pubmed publisher
    ..vessels, angiogenic plexus and blind-ended vessels (BEVs)) was quantified using in vivo fluorescence recovery after photobleaching (FRAP) and linear regression analysis of recovery profiles...
  20. Huang J, Huang L, Chen Y, Austin E, Devor C, Roegiers F, et al. Differential regulation of adherens junction dynamics during apical-basal polarization. J Cell Sci. 2011;124:4001-13 pubmed publisher
    ..assay the in vivo dynamics of biosynthetic turnover and membrane redistribution by fluorescence recovery after photobleaching (FRAP) assays...
  21. Gause M, Misulovin Z, Bilyeu A, Dorsett D. Dosage-sensitive regulation of cohesin chromosome binding and dynamics by Nipped-B, Pds5, and Wapl. Mol Cell Biol. 2010;30:4940-51 pubmed publisher
    ..We addressed this question by in vivo fluorescence recovery after photobleaching (FRAP) with Drosophila salivary glands...
  22. Cardarelli F, Tosti L, Serresi M, Beltram F, Bizzarri R. Fluorescent recovery after photobleaching (FRAP) analysis of nuclear export rates identifies intrinsic features of nucleocytoplasmic transport. J Biol Chem. 2012;287:5554-61 pubmed publisher
    ..These findings suggest differential gating at the NPC level. ..
  23. Daniels B, Perkins E, Dobrowsky T, Sun S, Wirtz D. Asymmetric enrichment of PIE-1 in the Caenorhabditis elegans zygote mediated by binary counterdiffusion. J Cell Biol. 2009;184:473-9 pubmed publisher
  24. González Muñoz E, Lopez Iglesias C, Calvo M, Palacin M, Zorzano A, Camps M. Caveolin-1 loss of function accelerates glucose transporter 4 and insulin receptor degradation in 3T3-L1 adipocytes. Endocrinology. 2009;150:3493-502 pubmed publisher
    ..We propose that caveolin-1/caveolae control insulin action in adipose cells...
  25. Guo L, Zhou D, Pryse K, Okunade A, Su X. Fatty acid 2-hydroxylase mediates diffusional mobility of Raft-associated lipids, GLUT4 level, and lipogenesis in 3T3-L1 adipocytes. J Biol Chem. 2010;285:25438-47 pubmed publisher
    ..of FA2H appeared to increase raft lipid mobility as it significantly accelerated the rates of fluorescence recovery after photobleaching measurements of lipid rafts labeled with Alexa 488-conjugated cholera toxin subunit B...
  26. Stasevich T, Mueller F, Brown D, McNally J. Dissecting the binding mechanism of the linker histone in live cells: an integrated FRAP analysis. EMBO J. 2010;29:1225-34 pubmed publisher
    ..Using a novel fluorescence recovery after photobleaching (FRAP) procedure, we have measured the affinities of these regions individually, in pairs, and ..
  27. Saulière Nzeh Ndong A, Saulière Nzeh A, Millot C, Corbani M, Mazeres S, Lopez A, et al. Agonist-selective dynamic compartmentalization of human Mu opioid receptor as revealed by resolutive FRAP analysis. J Biol Chem. 2010;285:14514-20 pubmed publisher
    ..Here, we report fluorescence recovery after photobleaching (FRAP) measurements performed at variable spot radius for human mu opioid (hMOP) receptors on SH-..
  28. Al Tanoury Z, Schaffner Reckinger E, Halavatyi A, Hoffmann C, Moes M, Hadzic E, et al. Quantitative kinetic study of the actin-bundling protein L-plastin and of its impact on actin turn-over. PLoS ONE. 2010;5:e9210 pubmed publisher
    ..with GFP-coupled WT-L-plastin, Ser5 substitution variants (S5/A, S5/E) or actin and analyzed by fluorescence recovery after photobleaching (FRAP)...
  29. Siriaco G, Deuring R, Chioda M, Becker P, Tamkun J. Drosophila ISWI regulates the association of histone H1 with interphase chromosomes in vivo. Genetics. 2009;182:661-9 pubmed publisher
    ..Our findings suggest that ISWI regulates higher-order chromatin structure by modulating the interaction of H1 with interphase chromosomes. ..
  30. de Beco S, Gueudry C, Amblard F, Coscoy S. Endocytosis is required for E-cadherin redistribution at mature adherens junctions. Proc Natl Acad Sci U S A. 2009;106:7010-5 pubmed publisher
    ..the dynamics of E-cadherin were quantitatively analyzed by a new approach combining 2-photon fluorescence recovery after photobleaching (FRAP) and fast 3D wide-field fluorescence microscopy...
  31. Hayashi Takanaka Y, Yamagata K, Nozaki N, Kimura H. Visualizing histone modifications in living cells: spatiotemporal dynamics of H3 phosphorylation during interphase. J Cell Biol. 2009;187:781-90 pubmed publisher
    ..Visualizing histone modifications in living cells will facilitate future epigenetic and cell regulation studies. ..
  32. Edwin N, Hammer R, McCarley R, Russo P. Reversibility of beta-amyloid self-assembly: effects of pH and added salts assessed by fluorescence photobleaching recovery. Biomacromolecules. 2010;11:341-7 pubmed publisher
    ..Oligomeric aggregates were found by fluorescence photobleaching recovery to respond readily to pH and salt conditions. Changing these external cues leads to formation or disassembly of aggregates smaller than 100 nm within minutes...
  33. Sanger J, Wang J, Holloway B, Du A, Sanger J. Myofibrillogenesis in skeletal muscle cells in zebrafish. Cell Motil Cytoskeleton. 2009;66:556-66 pubmed publisher
    ..b>Fluorescence Recovery After Photobleaching showed that the exchange of proteins (actin, alpha-actinin, FATZ, myotilin and telethonin) ..
  34. Calamai M, Specht C, Heller J, Alcor D, Machado P, Vannier C, et al. Gephyrin oligomerization controls GlyR mobility and synaptic clustering. J Neurosci. 2009;29:7639-48 pubmed publisher
    ..Using fluorescence recovery after photobleaching, we studied the exchange kinetics of synaptic gephyrin clusters...
  35. Michelman Ribeiro A, Mazza D, Rosales T, Stasevich T, Boukari H, Rishi V, et al. Direct measurement of association and dissociation rates of DNA binding in live cells by fluorescence correlation spectroscopy. Biophys J. 2009;97:337-46 pubmed publisher
    ..We find that our FCS estimates are quantitatively consistent with our fluorescence recovery after photobleaching (FRAP) measurements on the same VBP domains...
  36. Tian A, Baumgart T. Sorting of lipids and proteins in membrane curvature gradients. Biophys J. 2009;96:2676-88 pubmed publisher
    ..The sorting of Cholera toxin subunit B is rationalized by statistical models. We discuss the implications of our findings for intracellular sorting mechanisms. ..
  37. Rose R, Briddon S, Holliday N. Bimolecular fluorescence complementation: lighting up seven transmembrane domain receptor signalling networks. Br J Pharmacol. 2010;159:738-50 pubmed publisher
    ..These capabilities suggest that BiFC techniques will become ever more useful in the analysis of ligand and 7TM receptor pharmacology at the molecular level of protein-protein interactions. ..
  38. Trembecka D, Kuzak M, Dobrucki J. Conditions for using FRAP as a quantitative technique--influence of the bleaching protocol. Cytometry A. 2010;77:366-70 pubmed publisher
    b>Fluorescence recovery after photobleaching (FRAP) is a tool widely used in studies of dynamic behavior of fluorescently-tagged proteins in live cells...
  39. Kumar M, Mommer M, Sourjik V. Mobility of cytoplasmic, membrane, and DNA-binding proteins in Escherichia coli. Biophys J. 2010;98:552-9 pubmed publisher
    ..Here we combined fluorescence recovery after photobleaching with numerical modeling to analyze mobility of a set of fluorescent protein fusions in the ..
  40. Mueller F, Mazza D, Stasevich T, McNally J. FRAP and kinetic modeling in the analysis of nuclear protein dynamics: what do we really know?. Curr Opin Cell Biol. 2010;22:403-11 pubmed publisher
    ..in live cells has been analyzed by kinetic modeling procedures applied to experimental data from fluorescence recovery after photobleaching (FRAP)...
  41. Lechertier T, Grob A, Hernandez Verdun D, Roussel P. Fibrillarin and Nop56 interact before being co-assembled in box C/D snoRNPs. Exp Cell Res. 2009;315:928-42 pubmed publisher
    ..In addition, no RNA seems required to maintain fibrillarin-Nop56 interaction. ..
  42. Hagen G, Caarls W, Lidke K, de Vries A, Fritsch C, Barisas B, et al. Fluorescence recovery after photobleaching and photoconversion in multiple arbitrary regions of interest using a programmable array microscope. Microsc Res Tech. 2009;72:431-40 pubmed publisher
    ..b>Fluorescence recovery after photobleaching (FRAP) is commonly used for the determination of lateral diffusion constants of membrane ..
  43. Elvenes J, Sjøttem E, Holm T, Bjørkøy G, Johansen T. Pax6 localizes to chromatin-rich territories and displays a slow nuclear mobility altered by disease mutations. Cell Mol Life Sci. 2010;67:4079-94 pubmed publisher
    ..Here, we use fluorescence recovery after photobleaching to show that Pax6 and also other Pax family proteins display a strikingly low nuclear mobility ..
  44. Slade K, Baker R, Chua M, Thompson N, Pielak G. Effects of recombinant protein expression on green fluorescent protein diffusion in Escherichia coli. Biochemistry. 2009;48:5083-9 pubmed publisher
    b>Fluorescence recovery after photobleaching was used to measure the diffusion coefficient of green fluorescent protein (GFP, 27 kDa) in Escherichia coli in the presence or absence of four coexpressed proteins: cytoplasmic maltose binding ..
  45. Owen D, Williamson D, Rentero C, Gaus K. Quantitative microscopy: protein dynamics and membrane organisation. Traffic. 2009;10:962-71 pubmed publisher
    ..we examine the hypotheses and findings of three main techniques for analysing protein dynamics: fluorescence recovery after photobleaching, single particle tracking and fluorescence correlation spectroscopy...
  46. Kang M, Day C, Drake K, Kenworthy A, Dibenedetto E. A generalization of theory for two-dimensional fluorescence recovery after photobleaching applicable to confocal laser scanning microscopes. Biophys J. 2009;97:1501-11 pubmed publisher
    b>Fluorescence recovery after photobleaching (FRAP) using confocal laser scanning microscopes (confocal FRAP) has become a valuable technique for studying the diffusion of biomolecules in cells...
  47. Giglia Mari G, Theil A, Mari P, Mourgues S, Nonnekens J, Andrieux L, et al. Differentiation driven changes in the dynamic organization of Basal transcription initiation. PLoS Biol. 2009;7:e1000220 pubmed publisher
  48. Vladimirou E, Li M, Aldridge C, Frigerio L, Kirkilionis M, Robinson C. Diffusion of a membrane protein, Tat subunit Hcf106, is highly restricted within the chloroplast thylakoid network. FEBS Lett. 2009;583:3690-6 pubmed publisher
    ..Integral membrane proteins within both the stromal and granal membranes are therefore highly constrained, possibly forming 'microdomains' that are sharply separated...
  49. Cunningham C, Mukhopadhyay A, Lushington G, Blagg B, Prisinzano T, Krise J. Uptake, distribution and diffusivity of reactive fluorophores in cells: implications toward target identification. Mol Pharm. 2010;7:1301-10 pubmed publisher
    ..The intracellular diffusion coefficients were measured using a fluorescence recovery after photobleaching (FRAP)-based method...
  50. Waichman S, Roder F, Richter C, Birkholz O, Piehler J. Diffusion and interaction dynamics of individual membrane protein complexes confined in micropatterned polymer-supported membranes. Small. 2013;9:570-7 pubmed publisher
    ..of contiguous membranes within micropatterns is confirmed by scanning force microscopy, fluorescence recovery after photobleaching (FRAP), and super-resolved single-molecule tracking and localization microscopy...
  51. Lin W, Nebhan C, Anderson B, Webb D. Vasodilator-stimulated phosphoprotein (VASP) induces actin assembly in dendritic spines to promote their development and potentiate synaptic strength. J Biol Chem. 2010;285:36010-20 pubmed publisher
    ..with this, VASP significantly enhances the retention of GluR1 in spines as determined by fluorescence recovery after photobleaching and increases AMPAR-mediated synaptic transmission...
  52. Kiskin N, Hellen N, Babich V, Hewlett L, Knipe L, Hannah M, et al. Protein mobilities and P-selectin storage in Weibel-Palade bodies. J Cell Sci. 2010;123:2964-75 pubmed publisher
    Using fluorescence recovery after photobleaching (FRAP) we measured the mobilities of EGFP-tagged soluble secretory proteins in the endoplasmic reticulum (ER) and in individual Weibel-Palade bodies (WPBs) at early (immature) and late (..
  53. Walczak C, Rizk R, Shaw S. The use of fluorescence redistribution after photobleaching for analysis of cellular microtubule dynamics. Methods Cell Biol. 2010;97:35-52 pubmed publisher
    ..We include a discussion of cell culture and imaging conditions that maintain cell viability. We also provide an extensive discussion of both data collection and analysis that are utilized to estimate the turnover dynamics of MTs. ..