Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 14 (10), 1252-6

Spatial Dynamics of GFP-tagged Proteins Investigated by Local Fluorescence Enhancement

Affiliations

Spatial Dynamics of GFP-tagged Proteins Investigated by Local Fluorescence Enhancement

H Yokoe et al. Nat Biotechnol.

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.

Comment in

Similar articles

See all similar articles

Cited by 44 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback