RESUMEN
Understanding protein dynamics is crucial in order to elucidate protein function and interactions. Advances in modern microscopy facilitate the exploration of the mobility of fluorescently tagged proteins within living cells. Fluorescence recovery after photobleaching (FRAP) is an increasingly popular functional live-cell imaging technique which enables the study of the dynamic properties of proteins at a single-cell level. As an increasing number of labs generate FRAP datasets, there is a need for fast, interactive and user-friendly applications that analyze the resulting data. Here we present easyFRAP-web, a web application that simplifies the qualitative and quantitative analysis of FRAP datasets. EasyFRAP-web permits quick analysis of FRAP datasets through an intuitive web interface with interconnected analysis steps (experimental data assessment, different types of normalization and estimation of curve-derived quantitative parameters). In addition, easyFRAP-web provides dynamic and interactive data visualization and data and figure export for further analysis after every step. We test easyFRAP-web by analyzing FRAP datasets capturing the mobility of the cell cycle regulator Cdt2 in the presence and absence of DNA damage in cultured cells. We show that easyFRAP-web yields results consistent with previous studies and highlights cell-to-cell heterogeneity in the estimated kinetic parameters. EasyFRAP-web is platform-independent and is freely accessible at: https://easyfrap.vmnet.upatras.gr/.
Asunto(s)
Recuperación de Fluorescencia tras Fotoblanqueo/estadística & datos numéricos , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusión/genética , Programas Informáticos , Supervivencia Celular , Daño del ADN , Conjuntos de Datos como Asunto , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Internet , Cinética , Células MCF-7 , Proteínas Nucleares/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Análisis de la Célula Individual/métodosRESUMEN
Fluorescence recovery after photobleaching (FRAP) is used to obtain quantitative information about molecular diffusion and binding kinetics at both cell and tissue levels of organization. FRAP models have been proposed to estimate the diffusion coefficients and binding kinetic parameters of species for a variety of biological systems and experimental settings. However, it is not clear what the connection among the diverse parameter estimates from different models of the same system is, whether the assumptions made in the model are appropriate, and what the qualities of the estimates are. Here we propose a new approach to investigate the discrepancies between parameters estimated from different models. We use a theoretical model to simulate the dynamics of a FRAP experiment and generate the data that are used in various recovery models to estimate the corresponding parameters. By postulating a recovery model identical to the theoretical model, we first establish that the appropriate choice of observation time can significantly improve the quality of estimates, especially when the diffusion and binding kinetics are not well balanced, in a sense made precise later. Secondly, we find that changing the balance between diffusion and binding kinetics by changing the size of the bleaching region, which gives rise to different FRAP curves, provides a priori knowledge of diffusion and binding kinetics, which is important for model formulation. We also show that the use of the spatial information in FRAP provides better parameter estimation. By varying the recovery model from a fixed theoretical model, we show that a simplified recovery model can adequately describe the FRAP process in some circumstances and establish the relationship between parameters in the theoretical model and those in the recovery model. We then analyze an example in which the data are generated with a model of intermediate complexity and the parameters are estimated using models of greater or less complexity, and show how sensitivity analysis can be used to improve FRAP model formulation. Lastly, we show how sophisticated global sensitivity analysis can be used to detect over-fitting when using a model that is too complex.
Asunto(s)
Tipificación del Cuerpo/fisiología , Recuperación de Fluorescencia tras Fotoblanqueo/estadística & datos numéricos , Algoritmos , Animales , Drosophila melanogaster/crecimiento & desarrollo , Conceptos Matemáticos , Modelos Biológicos , Alas de Animales/crecimiento & desarrolloRESUMEN
BACKGROUND: Fluorescence loss in photobleaching (FLIP) is a widely used imaging technique, which provides information about protein dynamics in various cellular regions. In FLIP, a small cellular region is repeatedly illuminated by an intense laser pulse, while images are taken with reduced laser power with a time lag between the bleaches. Despite its popularity, tools are lacking for quantitative analysis of FLIP experiments. Typically, the user defines regions of interest (ROIs) for further analysis which is subjective and does not allow for comparing different cells and experimental settings. RESULTS: We present two complementary methods to detect and quantify protein transport and aggregation in living cells from FLIP image series. In the first approach, a stretched exponential (StrExp) function is fitted to fluorescence loss (FL) inside and outside the bleached region. We show by reaction-diffusion simulations, that the StrExp function can describe both, binding/barrier-limited and diffusion-limited FL kinetics. By pixel-wise regression of that function to FL kinetics of enhanced green fluorescent protein (eGFP), we determined in a user-unbiased manner from which cellular regions eGFP can be replenished in the bleached area. Spatial variation in the parameters calculated from the StrExp function allow for detecting diffusion barriers for eGFP in the nucleus and cytoplasm of living cells. Polyglutamine (polyQ) disease proteins like mutant huntingtin (mtHtt) can form large aggregates called inclusion bodies (IB's). The second method combines single particle tracking with multi-compartment modelling of FL kinetics in moving IB's to determine exchange rates of eGFP-tagged mtHtt protein (eGFP-mtHtt) between aggregates and the cytoplasm. This method is self-calibrating since it relates the FL inside and outside the bleached regions. It makes it therefore possible to compare release kinetics of eGFP-mtHtt between different cells and experiments. CONCLUSIONS: We present two complementary methods for quantitative analysis of FLIP experiments in living cells. They provide spatial maps of exchange dynamics and absolute binding parameters of fluorescent molecules to moving intracellular entities, respectively. Our methods should be of great value for quantitative studies of intracellular transport.
Asunto(s)
Recuperación de Fluorescencia tras Fotoblanqueo/estadística & datos numéricos , Fotoblanqueo , Proteínas/metabolismo , Citoplasma/metabolismo , Difusión , Fluorescencia , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteína Huntingtina , Cinética , Proteínas del Tejido Nervioso/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Transporte de ProteínasRESUMEN
Most of the important types of interactions that occur in cells can be characterized as binding-diffusion type processes, and can be quantified by kinetic rate constants such as diffusion coefficients (D) and binding rate constants (k(on) and k(off)). Confocal FRAP is a potentially important tool for the quantitative analysis of intracellular binding-diffusion kinetics, but how to dependably extract accurate kinetic constants from such analyses is still an open question. To this end, in this study, we developed what we believe is a new analytical model for confocal FRAP-based measurements of intracellular binding-diffusion processes, based on a closed-form equation of the FRAP formula for a spot photobleach geometry. This approach incorporates a binding diffusion model that allows for diffusion of both the unbound and bound species, and also compensates for binding diffusion that occurs during photobleaching, a critical consideration in confocal FRAP analysis. In addition, to address the problem of parametric multiplicity, we propose a scheme to reduce the number of fitting parameters in the effective diffusion subregime when D's for the bound and unbound species are known. 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) â¼ 255/s and k(off) â¼ 31/s.
Asunto(s)
Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Animales , Fenómenos Biofísicos , Células COS , Chlorocebus aethiops , Difusión , Retículo Endoplásmico/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo/estadística & datos numéricos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Cinética , Microscopía Confocal/métodos , Modelos Biológicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fotoblanqueo , Unión Proteica , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The analysis of fluorescence recovery after photobleaching (FRAP) data is complicated by the measurement noise, inhomogeneous fluorescence distribution, and image movement during experiment. Conventionally, these issues are tackled by data preprocessing and averaging, which causes loss of quantitative properties. In this study, we present a method which automatically estimates and compensates both the movement and inhomogeneous fluorescence distribution within the data analysis. The method is based on modeling the raw FRAP data with a parametric matrix and searching for maximum likelihood parameters between the model and the data. The developed method also automatically estimates also the bleach profile, immobile fraction, and noise variance. Suitable numerical computational method was developed and implemented in a computer grid. Simulated and experimental FRAP data was created and analyzed to evaluate the method.