Making membranes green: construction and characterization of GFP-fusion proteins targeted to discrete plasma membrane domains.
Cell membranes contain glycolipid-enriched membrane (GEM) domains, or lipid rafts. GEM domains represent a discrete assembly ofproteins and lipids within the plasma membrane thatfunctions in cell signaling. However, studies of the GEM domains often include the disruption of cells with detergent. Thus, many of the physical and biological properties of GEM domains remain unknown and even controversial. An approach to study these domains but avoid detergent lysis is to measure their properties using the fluorescence imaging of live cells. Accordingly, GFP was targeted to either the GEM or the non-GEMfraction of the plasma membrane using the minimal membrane-anchoring signals of p56lck and pp60c-Src, respectively. The targeting of the fusion proteins to the respective membrane fractions was assayed by membrane fractionation and by quantitating the enrichment in GEM caps in stimulated T cells. The results show that the GEM marker was targeted to GEM domains with similar efficiency as other GEM-associated proteins. Conversely, the non-GEM marker was completely excluded from GEM domains. These constructs represent a useful toolfor studying the discrete fractions of the plasma membrane in live cells using fluorescence imaging.
Rodgers
William
W
Molecular Immunogenetics Program, Oklahoma Medical Research Foundation, Oklahoma City 73104, USA. william-rodgers@omrf.ouhsc.edu
D002462:Cell Membrane / Q000502:physiology*; D017930:Genes, Reporter; D006017:Glycolipids / Q000502:physiology; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism*; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000235:genetics*; D021962:Membrane Microdomains / Q000502:physiology*; D008967:Molecular Biology / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014162:Transfection
D006017:Glycolipids; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins