Quantitative FRAP in Analysis of Molecular Binding Dynamics In Vivo

https://doi.org/10.1016/S0091-679X(08)85014-5Get rights and content

Abstract

Fluorescence recovery after photobleaching (FRAP) reveals the dynamics of fluorescently tagged molecules within live cells. These molecular dynamics are governed by diffusion of the molecule and its in vivo binding interactions. As a result, quantitative estimates of the association and dissociation rates of binding can be extracted from the FRAP. This chapter describes a systematic procedure to acquire the FRAP data, and then fit it with appropriate mathematical models to estimate in vivo association and dissociation rates of binding. Also discussed are the applicability and limitations of the models, the utility of the estimated parameters, and the prospects for increased accuracy and confidence in the estimates.

Introduction

Fluorescence recovery after photobleaching (FRAP) is now widely used to study protein mobility in living cells. FRAP is performed by photobleaching fluorescent molecules at a specified location in a cell, and then monitoring the rate at which the bleached molecules are replaced by unbleached ones. The rate of recovery reflects the rate of movement of the fluorescently tagged molecules at that location within the cell.

Molecular mobilities as obtained from FRAP are informative for several reasons. First, they can provide information about the rates of cellular diffusion in different subcellular compartments. Indeed, most of the early FRAP studies focused on the diffusion of proteins and lipids within membranes (Edidin 1994, Liebman 1974, Poo 1974).

Second, molecular mobilities often reveal that apparently static structures within cells are actually constructed from highly dynamic protein constituents. FRAP uncovers this hidden flux by selectively marking a subset of molecules, and so discloses the exchange of proteins or other molecules that occur within and between cellular compartments even when the system is at equilibrium (Misteli 2001, Webb 2003).

Third, molecular mobilities can also be used for the measurement of cellular binding interactions. FRAP of different green fluorescent protein (GFP)‐fusion proteins will sometimes reveal that their mobilities are considerably slower than expected for a purely diffusing molecule of that size, or even for a large molecular complex. This often indicates retardation of the protein's mobility by cellular binding interactions. Since stronger interactions will retard mobilities more than weaker interactions, the FRAP curve can be used to estimate the strengths of in vivo molecular binding interactions (Sprague and McNally, 2005).

This chapter focuses on how to extract quantitative information about molecular binding interactions from the FRAP data.

Section snippets

Rationale

Most estimates of protein binding affinity have been performed in vitro. This typically involves isolating the protein and its binding target, incubating the binding partners under appropriate conditions, and then measuring binding affinities by one of several established techniques, such as surface plasmon resonance, calorimetry, capillary electrophoresis, or filter binding assays (He 2004, Leavitt 2001, Riggs 1970, Schuck 1997).

How close are these in vitro affinity measurements to in vivo

Methods

There are two principal steps in using FRAP to extract in vivo estimates of binding parameters: data acquisition and data analysis.

Materials

In addition to both a confocal microscope and cells expressing GFP and the GFP‐fusion protein, software is also required for the quantitative analysis of FRAP data. Image‐analysis software is needed for averaging image intensities within the bleach spot. Often these routines are available with the confocal microscope software. If not, the same procedures can be performed in virtually any image‐analysis package, such as ImageJ. The resultant quantitative data can be imported into a spreadsheet

Utility of Parameters Estimated by the Models

Correct fitting of experimental FRAP data provides estimates for the association and dissociation rates of binding, kon* and koff. These numbers can be compared to in vitro estimates of binding as a way to judge if in vitro biochemistry has accurately captured what is transpiring in vivo. However, in comparing in vivo and in vitro estimates, it is important to realize that the association rate, kon*, measured by FRAP is actually the product of the molecular on rate times the concentration of

Summary

Described herein is a method for acquiring FRAP data, followed by a step‐by‐step procedure for fitting the data to a series of increasingly complex mathematical models. Successful application of this procedure will yield estimates of the in vivo association and dissociation binding rates for the GFP‐tagged protein under study. The protein is presumed to bind to an immobilized substrate that is uniformly distributed throughout a cellular compartment, and diffusion within the compartment is

Acknowledgments

I thank Waltraud Müller and Tim Stasevich for comments on the manuscript, and Brian Sprague for suggesting the original format for Fig. 2. I also thank Florian Müller for help with the derivations of the diffusion‐uncoupled FRAPs and for comments on the manuscript.

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