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FRAP and Other Photoperturbation Techniques

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Abstract

This chapter discusses the issues of Fluorescence recovery after photobleaching (FRAP) model selection and their applications for estimating parameter values. It highlights the important details to be considered to achieve a solid experimental basis for FRAP analysis. The chapter aims to explain the general workflow for analysing FRAP recovery curves. It provides guidelines for selecting the most suitable programs for the analysis of FRAP datasets. The chapter describes several basic pipelines to process FRAP data using freeware programs. FRAP models can be either descriptive - e.g. depend on recovery halftimes and mobile fractions - or include mechanistic description of the underlying biophysical/biochemical processes in terms of diffusion/transport coefficients, binding constants and biochemical reaction rates. FRAP can be useful for studying the rates of molecular exchange between cellular compartments. Therefore photoperturbation techniques are often still the methods of choice for measuring intracellular molecular dynamics either independently or in combination with other methods.

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Fluorescence localization after photobleaching is a new method for localized photolabeling and subsequent tracking of specific molecules within living cells. The molecular species to be located carries two different fluorophores that can be imaged independently but simultaneously by fluorescence microscopy. For the method to work, these two fluorophores should be accurately colocalized throughout the cell so that their images are closely matched. One of the fluorophores (the target fluorophore) is then rapidly photobleached at a chosen location. The unbleached (reference) fluorophore remains colocalized with the target fluorophore; thus, the subsequent fate of the photobleached molecules can be revealed by processing simultaneously acquired digital images of the two fluorophores. Here we demonstrate the simplicity and effectiveness of the FLAP method in revealing both fast and slow molecular dynamics in living cells using a Zeiss LSM 510 laser scanning confocal microscope.
Article
The formation of focal adhesions governs cell shape and function; however, there are few measurements of the binding kinetics of focal adhesion proteins in living cells. Here, we used the fluorescence recovery after photobleaching (FRAP) technique, combined with mathematical modeling and scaling analysis to quantify dissociation kinetics of focal adhesion proteins in capillary endothelial cells. Novel experimental protocols based on mathematical analysis were developed to discern the rate-limiting step during FRAP. Values for the dissociation rate constant k(OFF) ranged over an order of magnitude from 0.009+/-0.001/s for talin to 0.102+/-0.010/s for FAK, indicating that talin is bound more strongly than other proteins in focal adhesions. Comparisons with in vitro measurements reveal that multiple focal adhesion proteins form a network of bonds, rather than binding in a pair-wise manner in these anchoring structures in living cells.
FrapCalc: Github release
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