Characterizing fluorescence recovery curves for nuclear proteins undergoing binding events

Carrero, Gustavo; Crawford, Ellen; Hendzel, Michael J.; Vries, Gerda de

doi:10.1016/j.bulm.2004.02.005

Cited by 55 publications

(92 citation statements)

References 26 publications

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“…Mathematical models have been successful in the quantitative analysis of FRAP curves involving protein binding reactions (Carrero et al, 2004a;Carrero et al, 2004b;McDonald et al, 2006). These models share one important assumption: the fluorescent proteins have spatially homogeneous distributions.…”

Section: Discussionmentioning

confidence: 99%

“…This technique has been used widely to determine the diffusion constant of biomolecules in membranes or various intracellular compartments (Chen et al, 2006;Carrero et al, 2003). Recent FRAP experiments have investigated direct protein activities, such as interaction with binding partners, with the fluorescence recovery curve analyzed to differentiate fast and slow components that correspond to diffusion and binding events, or different binding states (Tardy et al, 1995;Kimura et al, 2002;Dundr et al, 2002;Carrero et al, 2003;Carrero et al, 2004a;Carrero et al, 2004b;Sprague et al, 2004;McDonald et al, 2006). One recent study combined the FRAP technique with model convolution methods and measured a gradient in microtubule dynamics in yeast spindles at ~65-nm spatial intervals (Pearson et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

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In vivo assay of presynaptic microtubule cytoskeleton dynamics in Drosophila

Yan

Broadie

2007

Journal of Neuroscience Methods

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Disrupted microtubule dynamics in neuronal synapses has been suggested as an underlying cause for several devastating neurological diseases, including Hereditary Spastic Paraplegia (HSP) and Fragile X Syndrome (FXS). However, previous studies have been restricted to indirect assays of synaptic microtubules, i.e. immunocytochemistry of microtubule-associated proteins and posttranslationally modified tubulins characteristic of microtubules with different stabilities. Very little is known about synaptic microtubule dynamics in vivo, or how microtubule dynamics may be disrupted in disease states. In this study, we develop methods to analyze microtubule dynamics directly in living synaptic boutons in situ. We use fluorescence recovery after photobleaching (FRAP) of transgenic green fluorescent protein (GFP) tagged tubulin at the well-characterized Drosophila neuromuscular junction (NMJ) synapse. FRAP measurements of tubulin-GFP demonstrate biphasic recovery kinetics. Treatment with taxol to stabilize microtubules and promote microtubule assembly reduces both recovery phases. Treatment with vinblastine to disassemble microtubules increases the fast recovery phase and decreases the slow recovery phase. These data indicate that the fast recovery phase is generated by rapid diffusion of tubulin subunits and the slow phase is generated by the relatively slow turnover of microtubules. This study demonstrates that tubulin-GFP fluorescence recovery after photobleaching can be used to assay microtubule dynamics directly in living synapses.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

In vivo assay of presynaptic microtubule cytoskeleton dynamics in Drosophila

Yan

Broadie

2007

Journal of Neuroscience Methods

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“…The existing FRAP fitting models can be used for the analysis of protein diffusion (Ellenberg et al 1997;Soumpasis 1983) and binding reactions for one and several binding states, including cases where diffusion is a limiting factor for binding processes (Carrero et al 2004;Mueller et al 2008;Sprague et al 2004). This methodology provides important quantitative information on molecular dynamics in vivo, such as diffusion constants, rate constants of protein (un)binding, (im)mobile fraction(s) of proteins, spatial localisation of the fluorescent protein analogues.…”

Section: Introductionmentioning

confidence: 99%

“…Only a few studies at present used FRAP, associated with mathematical modelling, to gain quantitative information about actin polymerisation in living cells. Some of these studies have used binding approximation (Carrero et al 2004;McDonald et al 2006) to measure actin turnover in the nucleus or in cytoplasm (Campbell and Knight 2007). The model of Tardy et al (1995) was established specifically for FRAP experiments with actin filaments and was applied for the fitting of corresponding datasets.…”

Section: Introductionmentioning

confidence: 99%

A mathematical model of actin filament turnover for fitting FRAP data

Halavatyi

Nazarov²,

Tanoury

et al. 2009

Eur Biophys J

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A novel mathematical model of the actin dynamics in living cells under steady-state conditions has been developed for fluorescence recovery after photobleaching (FRAP) experiments. As opposed to other FRAP fitting models, which use the average lifetime of actins in filaments and the actin turnover rate as fitting parameters, our model operates with unbiased actin association/dissociation rate constants and accounts for the filament length. The mathematical formalism is based on a system of stochastic differential equations. The derived equations were validated on synthetic theoretical data generated by a stochastic simulation algorithm adapted for the simulation of FRAP experiments. Consistent with experimental findings, the results of this work showed that (1) fluorescence recovery is a function of the average filament length, (2) the F-actin turnover and the FRAP are accelerated in the presence of actin nucleating proteins, (3) the FRAP curves may exhibit both a linear and non-linear behaviour depending on the parameters of actin polymerisation, and (4) our model resulted in more accurate parameter estimations of actin dynamics as compared with other FRAP fitting models. Additionally, we provide a computational tool that integrates the model and that can be used for interpretation of FRAP data on actin cytoskeleton.

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“…Classically, FRAP measurements were performed using a focused, static laser beam to bleach molecules. In recent years, commercial laser scanning confocal microscopes (LSCMs) have become widely utilized to study of intracellular protein dynamics [2,3,4]. As such, confocal FRAP can now be used to study binding-diffusion kinetics of proteins within their native environments, an important goal in light of the universality of reaction and diffusion processes in cells.…”

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confidence: 99%

Laser Scanning Confocal Microscope-Based Fluorescence Recovery after Photobleaching (FRAP) Models for Pure Diffusion and Binding Diffusing Kinetics on Various Geometries in 1-3 Spatial Dimensions

Kang¹,

DiBenedetto²,

Kenworthy³

2011

Microsc Microanal

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Diffusion and binding-diffusion are ubiquitous processes with importance across many scales of biology. Many cellular events depend strongly on the diffusivity and binding rates of proteins and other biomolecules, which are characterized by a diffusion coefficient (D) and binding rate constants (k on , k off ). Therefore, determining the kinetic rate constants such as diffusion coefficients or binding rate constants of proteins is important to understand cell function.FRAP has been a useful technique for studying reaction-diffusion kinetics of biomolecules in cells since its development in the 1970s [1]. Classically, FRAP measurements were performed using a focused, static laser beam to bleach molecules. In recent years, commercial laser scanning confocal microscopes (LSCMs) have become widely utilized to study of intracellular protein dynamics [2,3,4]. As such, confocal FRAP can now be used to study binding-diffusion kinetics of proteins within their native environments, an important goal in light of the universality of reaction and diffusion processes in cells.In spite of the popularity of LSCM based FRAP (confocal FRAP), most FRAP models available in the literature were developed for the static laser beam based conventional FRAP approaches, which are based on two critical assumptions: 1) the bleaching time scale is much faster than protein kinetic scales so that immediate photobleaching can be assumed; and 2) the bleach region of interest (ROI) is small enough so that the compartments from which recovery occurs (for example the plasma membrane or the cytoplasm) can be considered as infinite 2D and 3D spaces.However, the 1 st assumption does not necessarily apply in confocal FRAP experiments, especially for soluble proteins, since in laser scanning confocal microscopes using a single laser for bleaching and imaging, the time required to scan the bleach region can allow for diffusion during the bleach [5]. Due to the long scanning time of confocal laser scanning microscopes, conventional FRAP analysis applied to confocal FRAP provides confounding kinetic parameter values [5]. To address this intrinsic property of confocal FRAP, we recently developed a formalism that can be used to correct for binding and/or diffusion during the photobleaching event and showed that this is a critical consideration in confocal FRAP analysis [4,5].The 2 nd assumption is also not legitimate in many cases and geometric consideration is required depending on the locations in cells where FRAP experiments are performed. Although various versions of FRAP models have been developed in the literature, to our knowledge the consequences 212

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Characterizing fluorescence recovery curves for nuclear proteins undergoing binding events

Cited by 55 publications

References 26 publications

In vivo assay of presynaptic microtubule cytoskeleton dynamics in Drosophila

In vivo assay of presynaptic microtubule cytoskeleton dynamics in Drosophila

A mathematical model of actin filament turnover for fitting FRAP data

Laser Scanning Confocal Microscope-Based Fluorescence Recovery after Photobleaching (FRAP) Models for Pure Diffusion and Binding Diffusing Kinetics on Various Geometries in 1-3 Spatial Dimensions

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