Determining averaged effective diffusion constants from experimental measurements of fluorescent proteins in an inhomogeneous medium in the presence of ligand-receptor interactions poses problems of analytical tractability. Here, we introduced a nonfitting method to evaluate the averaged effective diffusion coefficient of a region of interest (which may include a whole nucleus) by mathematical processing of the entire cellular two-dimensional spatial pattern of recovered fluorescence. Spatially and temporally resolved measurements of protein transport inside cells were obtained using the fluorescence recovery after photobleaching technique. Two-dimensional images of fluorescence patterns were collected by laser-scanning confocal microscopy. The method was demonstrated by applying it to an estimation of the mobility of green fluorescent protein-tagged heterochromatin protein 1 in the nuclei of living mouse embryonic fibroblasts. This approach does not require the mathematical solution of a corresponding system of diffusion-reaction equations that is typical of conventional fluorescence recovery after photobleaching data processing, and is most useful for investigating highly inhomogeneous areas, such as cell nuclei, which contain many protein foci and chromatin domains.

Institute of Biophysics Academy of Sciences of the Czech Republic Brno Czech Republic

Zobrazit více v PubMed

Sprague B.L., McNally J.G. FRAP analysis of binding: proper and fitting. Trends Cell Biol. 2005;15:84–91. PubMed

Festenstein R., Pagakis S.N., Kioussis D. Modulation of heterochromatin protein 1 dynamics in primary mammalian cells. Science. 2003;299:719–721. PubMed

Dundr M., Hoffmann-Rohrer U., Misteli T. A kinetic framework for a mammalian RNA polymerase in vivo. Science. 2002;298:1623–1626. PubMed

Shav-Tal Y., Darzacq X., Singer R.H. Dynamics of single mRNPs in nuclei of living cells. Science. 2004;304:1797–1800. PubMed PMC

Elsner M., Hashimoto H., Weiss M. Spatiotemporal dynamics of the COPI vesicle machinery. EMBO Rep. 2003;4:1000–1004. PubMed PMC

Giese B., Au-Yeung C.K., Müller-Newen G. Long term association of the cytokine receptor gp130 and the Janus kinase Jak1 revealed by FRAP analysis. J. Biol. Chem. 2003;278:39205–39213. PubMed

Shaw S.L., Kamyar R., Ehrhardt D.W. Sustained microtubule treadmilling in Arabidopsis cortical arrays. Science. 2003;300:1715–1718. PubMed

Smith A.J., Pfeiffer J.R., Wilson B.S. Microtubule-dependent transport of secretory vesicles in RBL-2H3 cells. Traffic. 2003;4:302–312. PubMed

von Wichert G., Haimovich B., Sheetz M.P. Force-dependent integrin–cytoskeleton linkage formation requires downregulation of focal complex dynamics by Shp2. EMBO J. 2003;22:5023–5035. PubMed PMC

Howell B.J., Moree B., Salmon E. Spindle checkpoint protein dynamics at kinetochores in living cells. Curr. Biol. 2004;14:953–964. PubMed

Peters R., Peters J., Bähr W. A microfluorimetric study of translational diffusion in erythrocyte membranes. Biochim. Biophys. Acta. 1974;367:282–294. PubMed

Axelrod D., Koppel D.E., Webb W.W. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys. J. 1976;16:1055–1069. PubMed PMC

Koppel D.E., Axelrod D., Webb W.W. Dynamics of fluorescence marker concentration as a probe of mobility. Biophys. J. 1976;16:1315–1329. PubMed PMC

Cole N.B., Smith C.L., Lippincott-Schwartz J. Diffusional mobility of Golgi proteins in membranes of living cells. Science. 1996;273:797–801. PubMed

Swaminathan R., Hoang C.P., Verkman A.S. Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion. Biophys. J. 1997;72:1900–1907. PubMed PMC

Patterson G.H., Knobel S.M., Piston D.W. Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys. J. 1997;73:2782–2790. PubMed PMC

Partikian A., Olveczky B., Verkman A.S. Rapid diffusion of green fluorescent protein in the mitochondrial matrix. J. Cell. Biol. 1998;140:821–829. PubMed PMC

Adams C.L., Chen Y.-T., Nelson W.J. Mechanisms of epithelial cell-cell adhesion and cell compaction revealed by high-resolution tracking of E-cadherin-green fluorescent protein. J. Cell. Biol. 1998;142:1105–1119. PubMed PMC

Houtsmuller A.B., Vermeulen W. Macromolecular dynamics in living cell nuclei revealed by fluorescence redistribution after photobleaching. Histochem. Cell. Biol. 2001;115:13–21. PubMed

Scholz M., Gross-Johannböcke C., Peters R. Measurement of nucleocytoplasmic transport by fluorescence microphotolysis and laser scanning microscopy. Cell. Biol. Int. Rep. 1988;12 709–27. PubMed

Tsibidis G.D., Ripoll J. Investigation of binding mechanisms of nuclear proteins using confocal scanning laser microscopy and FRAP. J. Theor. Biol. 2008;253:755–768. PubMed

Kao H.P., Abney J.R., Verkman A.S. Determinants of the translational mobility of a small solute in cell cytoplasm. J. Cell Biol. 1993;120:175–184. PubMed PMC

Verkman A.S. Diffusion in cells measured by fluorescence recovery after photobleaching. In: Marriott G., Parker I., editors. Biophotonics, Part A: Methods in Enzymology. Vol. 360. Academic Press; New York: 2003. pp. 635–648. PubMed

Bjarneson D.W., Petersen N.O. Effects of second order photobleaching on recovered diffusion parameters from fluorescence photobleaching recovery. Biophys. J. 1991;60:1128–1131. PubMed PMC

Blonk J.C.G., Don A., Birmingham J.J. Fluorescence photobleaching recovery in the confocal scanning light microscope. J. Microsc. 1993;169:363–374.

Braeckmans K., Peeters L., Demeester J. Three-dimensional fluorescence recovery after photobleaching with the confocal microscope. Biophys. J. 2003;85:2240–2252. PubMed PMC

Lopez A., Dupou L., Tocanne J. Fluorescence recovery after photobleaching (FRAP) experiments under conditions of uniform disk illumination. Biophys. J. 1988;53:963–970. PubMed PMC

Wedekind P., Kubitscheck U., Peters R. Line-scanning microphotolysis for diffraction-limited measurements of lateral diffusion. Biophys. J. 1996;71:1621–1632. PubMed PMC

Kubitscheck U., Wedekind P., Peters R. Three-dimensional diffusion measurements by scanning microphotolysis. J. Microsc. 1998;192:126–138.

Periasamy N., Verkman A.S. Analysis of fluorophore diffusion by continuous distributions of diffusion coefficients: application to photobleaching measurements of multicomponent and anomalous diffusion. Biophys. J. 1998;75:557–567. PubMed PMC

Wachsmuth M., Weidemann T., Langowski J. Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching. Biophys. J. 2003;84:3353–3363. PubMed PMC

Lele T.P., Ingber D.E. A mathematical model to determine molecular kinetic rate constants under non-steady state conditions using fluorescence recovery after photobleaching (FRAP) Biophys. Chem. 2006;120:32–35. PubMed

Kang M., Kenworthy A.K. A closed-form analytic expression for FRAP formula for the binding diffusion model. Biophys. J. 2008;95:L13–L15. PubMed PMC

Tsay T., Jacobson K.A. Spatial Fourier analysis of video photobleaching measurements, principles and optimization. Biophys. J. 1991;60:360–368. PubMed PMC

Berk D.A., Yuan F., Jain R.K. Fluorescence photobleaching with spatial Fourier analysis: measurement of diffusion in light-scattering media. Biophys. J. 1993;65:2428–2436. PubMed PMC

Mueller F., Wach P., McNally J.G. Evidence for a common mode of transcription factor interaction with chromatin as revealed by improved quantitative fluorescence recovery after photobleaching. Biophys. J. 2008;94:3323–3339. PubMed PMC

Jonsson P., Jonsson M.P., Hook F. A method improving the accuracy of fluorescence recovery after photobleaching analysis. Biophys. J. 2008;95:5334–5348. PubMed PMC

Bulinski J.C., Odde D.J., Waterman-Storer C.M. Rapid dynamics of the microtubule binding of ensconsin in vivo. J. Cell Sci. 2001;114:3885–3897. PubMed

Coscoy S., Waharte F., Amblard F. Molecular analysis of microscopic ezrin dynamics by two-photon FRAP. Proc. Natl. Acad. Sci. USA. 2002;99:12813–12818. PubMed PMC

Sprague B.L., Pego R.L., McNally J.G. Analysis of binding reactions by fluorescence recovery after photobleaching. Biophys. J. 2004;86:3473–3495. PubMed PMC

Muller K.P., Erdel F., Rippe K. Multiscale analysis of dynamics and interactions of heterochromatin protein 1 by fluorescence fluctuation microscopy. Biophys. J. 2009;97:2876–2885. PubMed PMC

Saltzman W.M., Radomsky M.L., Cone R.A. Antibody diffusion in human cervical mucus. Biophys. J. 1994;66:508–515. PubMed PMC

Phiilles G.D.J. Translational diffusion coefficient of macroparticles in solvents of high viscosity. J. Phys. Chem. 1981;85:2838–2843.

Harnicarová Horáková A., Galiová G., Bártová E. Chromocenter integrity and epigenetic marks. J. Struct. Biol. 2010;169 124–33. PubMed

Bártová E., Pacherník J., Kozubek S. Nuclear levels and patterns of histone H3 modification and HP1 proteins after inhibition of histone deacetylases. J. Cell Sci. 2005;118:5035–5046. PubMed

Bártová E., Pacherník J., Kozubek S. Differentiation-specific association of HP1 α and HP1 β with chromocenters is correlated with clustering of TIF1 β at these sites. Histochem Cell Biol. 2007;127:375–388. PubMed

Cheutin T., McNairn A.J., Misteli T. Maintenance of stable heterochromatin domains by dynamic HP1 binding. Science. 2003;299:721–723. PubMed

Scaffidi P., Misteli T. Lamin A-dependent nuclear defects in human aging. Science. 2006;312:1059–1063. PubMed PMC

Shumaker D.K., Dechat T., Goldman R.D. Mutant nuclear lamin A leads to progressive alterations of epigenetic control in premature aging. Proc. Natl. Acad. Sci. USA. 2006;103:8703–8708. PubMed PMC