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Nat Biotechnol. 1996 Oct;14(10):1252-6.

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

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

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 microns2/sec, compared to 4 microns2/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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PMID:
9631088
DOI:
10.1038/nbt1096-1252
[Indexed for MEDLINE]

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