FRAP model to determine the bidirectional transport rate of GFP across the nuclear membrane and the mobile fraction in the cytoplasm and nucleus

Sylvain V. Costes; Olga Slobodskaya; Edward Cho; Maria Tsopanomihalou-nglotsu; George Pavlakis; Stephen J. Lockett

doi:10.1117/12.468359

28 May 2002 FRAP model to determine the bidirectional transport rate of GFP across the nuclear membrane and the mobile fraction in the cytoplasm and nucleus

Sylvain V. Costes, Olga Slobodskaya, Edward Cho, Maria Tsopanomihalou-nglotsu, George Pavlakis, Stephen J. Lockett

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Proceedings Volume 4622, Optical Diagnostics of Living Cells V; (2002) https://doi.org/10.1117/12.468359
Event: International Symposium on Biomedical Optics, 2002, San Jose, CA, United States

Abstract

A mathematical model was developed to predict the bi- directional transport rate of fluorescent proteins across the nuclear membrane during a fluorescence recovery after photobleaching (FRAP) experiment. The model assumes that the total amount of fluorescent protein remains the same in the cell (i.e. no production, loss or exchange with the outside of the cell) and that the cell is in a state of equilibrium; i.e. proteins are leaving and entering the nucleus at an equal rate. The latter assumption has the advantage of not needing to take into account the method of protein transport (e.g. active or passive). The model includes correction for the photobleaching that happens during image acquisition following the deliberate photobleach. In this study, the green fluorescent protein (GFP) was transfected into cells in order to study its free behavior. In the FRAP experiments, either the entire nucleus and part of the cytoplasm or only part of the cytoplasm was photobleached followed by time-series imaging of the fluorescence redistribution. The model was fitted to the curves of intensity loss or recovery after photobleaching using numerical, non-linear methods. In addition, the mobile fractions of free GFP in the cytoplasm and the nucleus could be determined.

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Sylvain V. Costes, Olga Slobodskaya, Edward Cho, Maria Tsopanomihalou-nglotsu, George Pavlakis, and Stephen J. Lockett "FRAP model to determine the bidirectional transport rate of GFP across the nuclear membrane and the mobile fraction in the cytoplasm and nucleus", Proc. SPIE 4622, Optical Diagnostics of Living Cells V, (28 May 2002); https://doi.org/10.1117/12.468359

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KEYWORDS

Proteins

Luminescence

Green fluorescent protein

Diffusion

Image acquisition

Principal component analysis

Digital image correlation

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