Spatial dynamics of GFP-tagged proteins investigated by local fluorescence enhancement.
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.
Yokoe
H
H
Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.
Meyer
T
T
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D007834:Lasers; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010777:Photochemistry; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014466:Ultraviolet Rays; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D008164:Luminescent Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D018631:ras Proteins