title: Diffusion and binding analyzed with combined point FRAP and FCS.
abstract: To quantify more precisely and more reliably diffusion and reaction properties of biomolecules in living cells, a novel closed description in 3D of both the bleach and the post-bleach segment of fluorescence recovery after photobleaching (FRAP) data acquired at a point, i.e., a diffraction-limited observation area, termed point FRAP, is presented. It covers a complete coupled reaction-diffusion scheme for mobile molecules undergoing transient or long-term immobilization because of binding. We assess and confirm the feasibility with numerical solutions of the differential equations. By applying this model to free EYFP expressed in HeLa cells using a customized confocal laser scanning microscope that integrates point FRAP and fluorescence correlation spectroscopy (FCS), the applicability is validated by comparison with results from FCS. We show that by taking diffusion during bleaching into consideration and/or by employing a global analysis of series of bleach times, the results can be improved significantly. As the point FRAP approach allows to obtain data with diffraction-limited positioning accuracy, diffusion and binding properties of the exon-exon junction complex (EJC) components REF2-II and Magoh are obtained at different localizations in the nucleus of MCF7 cells and refine our view on the position-dependent association of the EJC factors with a maturating mRNP complex. Our findings corroborate the concept of combining point FRAP and FCS for a better understanding of the underlying diffusion and binding processes.
authors.affiliation: Cell Biophysics Group, Institut Pasteur Korea, Seoul, Republic of Korea; Experimental Systems Division, Institute for Basic Science, Daejeon, Republic of Korea.
authors.forename: Kang-Bin
authors.initials: KB
authors.lastname: Im
authors.forename: Ute
authors.initials: U
authors.lastname: Schmidt
authors.forename: Moon-Sik
authors.initials: MS
authors.lastname: Kang
authors.forename: Ji-Young
authors.initials: JY
authors.lastname: Lee
authors.forename: Felix
authors.initials: F
authors.lastname: Bestvater
authors.forename: Malte
authors.initials: M
authors.lastname: Wachsmuth
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D045744:Cell Line, Tumor; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry; D061986:MCF-7 Cells; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D006657:Histones; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria
keywords: coupled reaction-diffusion system; exon-exon junction complex; fluorescence correlation spectroscopy; fluorescence recovery after photobleaching