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Spatial dynamics of GFP-tagged proteins investigated by local fluorescence enhancement

Nature Biotechnologyvolume 14pages12521256 (1996) | Download Citation

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Abstract

We describe a method of monitoring the spatial dynamics of proteins in intact cells by locally enhancing the blue excited fluorescence of green fluorescent protein (GFP) using a spatially focused ultraviolet-laser pulse. GFP fusion proteins were efficiently expressed by micro-electroporation of in vitro synthesized mRNA into adherent mammalian cells. We found that the diffusion coefficient of cycle 3 mutant GFP was 43 μm2/sec, compared to 4 μm2/sec for wild-type GFP, suggesting that cycle 3 GFP diffuses freely in mammalian cells and is ideally suited as a fusion tag. The local fluorescence enhancement method was used to study the membrane dissociation rate of GFP-tagged K-ras, a small GTP binding protein that localizes to plasma membranes by a farnesyl lipid group and a polybasic region. Our data suggest that K-ras exists in a dynamic equilibrium and rapidly switches between a plasma membrane bound form and a cytosolic form with a plasma membrane dissociation time constant of 1.5 sec.

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Author notes

  1. Tobias Meyer: email: tobias@cellbio.duke.edu

Affiliations

  1. Department of Cell Biology, Duke University Medical Center, Durham, NC, 27710

    • Hiroko Yokoe
    •  & Tobias Meyer

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DOI

https://doi.org/10.1038/nbt1096-1252

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