FRAP in NIH 3T3 β-actin-GFP cell Analysis of actin cytoskeleton structures and turnover with FRAP technique Aliaksandr Halavatyi1, Ziad al Tanoury1, Marie Catillon1, Mikalai Yatskou2, Evelyne Friederich1 1Cytoskeleton and Cell Plasticity Laboratory, Life Sciences Research Unit, Faculty of Sciences, Technology and Communication, University of Luxembourg, Luxembourg 2Department of Systems Analysis, Belarusian State University, Minsk, Republic of Belarus Introduction Actin cytoskeleton, a dynamic force-generating cell system which is composed of proteanous filaments capable of undergoing continuous cycles of assembly/disassembly. Structural changes and specific spatial organisation of actin filaments are controlled by regulatory proteins. Confocal laser scanning microscopy-based fluorescence recovery after photobleaching (FRAP) technology yields new insides into actin cytoskeleton dynamics1 by providing turnover times, mobile and immobile fractions, spatial ordering and localization characteristics of actin and actin-binding proteins. Currently available fitting models and analysis protocols do not allow estimation of structural and dynamic parameters of actin cytoskeleton, such as filament length and (dis)association rate constants. Results Simulation of FRAP curves for actin turnover3 The derived FRAP fitting equation was validated using the stochastic simulation algorithm, accounting for the structural compositions of filaments. Recoveries were modelled for a broad range of model parameters to examine their influence on FRAP response. GFP-actin in NIH 3T3 cell Our aim is the quantitative investigation of dynamics of actin and actin-binding proteins in living cells with the FRAP experiments Experimental data analysis Experimental methodology The developed analysis approach was validated on the binding activity of actin-regulatory protein Tes labelled with GFP in Hela cells. Analysis resulted in the estimation of diffusion coefficient and binding/unbinding reaction rates. FRAP in NIH 3T3 β-actin-GFP cell t=0 s Diffusing Tes variant Binding Tes variant t=tbleach t=80 s FRAP experiments are performed on the Zeiss LSM510 Meta and Zeiss LSM 710 laser scanning microscopes. The size and position of the region of interest correspond to the area of specific actin structures (e.g. - focal adhesions, stress fibers, lamellipodia). Both dynamics of fluorescently-labelled actin and actin-binding proteins are analysed for evaluation of specific system parameters Analysis methodology Data processing The integrated approach has been developed for analysing the FRAP recoveries in a standardized manner. The scheme assumes selection of normalisation or modelling procedures in accordance with the type of analysed data. Planned: Analysis of structures and dynamics of actin filaments using the developed formalism. FRAPAnalyser software Utilizes the developed integrated analysis approach Currently available options: Data import/export Graphical support Normalization Modelling and fitting: binding, diffusion, binding+diffusion, polymerization Error analysis ± Planned: Global/associated analysis for series of experiments ± ± Model development Program interface Binding activity of actin regulating proteins is analysed the fitting equations reported in2. To account for the lengths and structures of bleached filaments we developed a specific mathematical formalism for the interpretation of FRAP experiments3 with actin filaments. Conclusions The created protocol can be used to investigate quantitatively the actin dynamics and estimate the filament length and assembly/disassembly reaction rates affected by regulatory proteins. Application of the presented approach is not limited to the investigation of the actin cytoskeleton systems but can be extended for evaluation of biophysical and biochemical parameters in quantitative studies of other cellular processes. Model of bleached filament References 1. Lai, F.P., et al., Arp2/3 complex interactions and actin network turnover in lamellipodia. Embo J, 2008. 27(7): p. 982-92. 2. McNally, J.G., Quantitative FRAP in analysis of molecular binding dynamics in vivo. Methods Cell Biol, 2008. 85: p. 329-51. 3. Halavatyi, A.A., et al., A mathematical model of actin filament turnover for fitting FRAP data. Eur Biophys J, 2009 (in print). <L> – the mean length of filaments cm, cf – concentrations of free and polymerised actins vb, vp – polymerisation rates Db, Dp – diffusivity coefficients P(x,t) – probability of the bleached subunit to reside in filament after time t, function of <L>, vb, vp , Db, Dp. Grants and fellowships European Science Foundation (Grant № 1500) Human Frontier Science Program Organisation National Research Fund (FNR), Luxembourg P-CUBE