Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation

Daniel Wüstner,Frederik W Lund,Hans J Schroll,Daniel Sage,Michael A Lomholt,Lukasz M Solanko

doi:10.1186/1471-2105-13-296

Daniel Wüstner, Frederik W Lund + Show 4 more

Open Access

https://doi.org/10.1186/1471-2105-13-296

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Abstract

BackgroundFluorescence 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.ResultsWe 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.ConclusionsWe 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.

Highlights

Fluorescence loss in photobleaching (FLIP) is a widely used imaging technique, which provides information about protein dynamics in various cellular regions
We found in additional FLIP experiments with cells expressing enhanced green fluorescent protein (eGFP) that the bleach profile has no impact on the ability of the stretched exponential (StrExp) function to fit the data
The fact, that we find this in FLIP experiments of eGFP in cells suggests that the simple compartment model is not adequate to describe local heterogeneity of intracellular eGFP motion

Summary

Introduction

Fluorescence loss in photobleaching (FLIP) is a widely used imaging technique, which provides information about protein dynamics in various cellular regions. To determine intracellular transport dynamics of proteins and lipids tagged with suitable fluorophores, one often relies on perturbing the steady image analysis tools, to measure transport kinetics of the molecule of interest by multicolour fluorescence microscopy. A variant of FRAP is continuous photobleaching (CP) introduced by Peters et al in 1981 [18] In this approach, a small area of the cell is continuously illuminated, and the fluorescence decay in this region due to photobleaching and transport is monitored. Similar as in FRAP, diffusion constants, and with extensive modelling efforts, binding parameters or flow components can be estimated from CP measurements [18,19,20] Both techniques, infer the transport parameters solely from fitting a time-dependent function to experimental data. While varying the size of the bleach area allows for controlling the diffusion time, if binding and diffusion take place on different time scales, the user cannot isolate one of the processes by adjusting the time resolution of the measurement

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