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Quantitative Fluorescence Loss in Photobleaching for Analysis of Protein Transport and Aggregation

D. Wüstner, L.M. Solanko, F.W. Lund, D. Sage, H.J. Schroll, M.A. Lomholt

Proceedings of BioImage Informatics 2012 (BI'12), Dresden, Federal Republic of Germany, September 16-19, 2012, pp. 61-62.

Fluorescence loss in photobleaching (FLIP) is a widely used imaging technique, which provides information about protein dynamics in various cellular regions. Despite its popularity, tools are lacking for quantitative analysis of FLIP experiments. We present two new 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 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 fluorescence loss kinetics. By pixel-wise regression of that function to fluorescence loss kinetics of enhanced green fluorescent protein (eGFP), we could detect barriers for protein diffusion in the nucleus of living cells. Polyglutamine (polyQ) disease proteins like mutant huntingtin (mtHtt) can form large aggregates called inclusion bodies (IB's). In the second approach, we measured fluorescence loss kinetics and trajectories of moving IB's by single particle tracking and determined exchange rates of eGFP-tagged mtHtt protein between aggregates and the cytoplasm using an analytical multi-compartment model. That analysis revealed heterogeneous release kinetics of mtHtt from IB's and should be of great value for quantitative studies of intracellular protein trafficking and aggregation in neurodegeneration.

AUTHOR="W{\"{u}}stner, D. and Solanko, L.M. and Lund, F.W. and Sage, D.
        and Schroll, H.J. and Lomholt, M.A.",
TITLE="Quantitative Fluorescence Loss in Photobleaching for Analysis of
        Protein Transport and Aggregation",
BOOKTITLE="Proceedings of BioImage Informatics 2012 ({BI'12})",
address="Dresden, Federal Republic of Germany",
month="September 16-19,",

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