title: A novel biotinylated lipid raft reporter for electron microscopic imaging of plasma membrane microdomains.
abstract: The submicroscopic spatial organization of cell surface receptors and plasma membrane signaling molecules is readily characterized by electron microscopy (EM) via immunogold labeling of plasma membrane sheets. Although various signaling molecules have been seen to segregate within plasma membrane microdomains, the biochemical identity of these microdomains and the factors affecting their formation are largely unknown. Lipid rafts are envisioned as submicron membrane subdomains of liquid ordered structure with differing lipid and protein constituents that define their specific varieties. To facilitate EM investigation of inner leaflet lipid rafts and the localization of membrane proteins therein, a unique genetically encoded reporter with the dually acylated raft-targeting motif of the Lck kinase was developed. This reporter, designated Lck-BAP-GFP, incorporates green fluorescent protein (GFP) and biotin acceptor peptide (BAP) modules, with the latter allowing its single-step labeling with streptavidin-gold. Lck-BAP-GFP was metabolically biotinylated in mammalian cells, distributed into low-density detergent-resistant membrane fractions, and was readily detected with avidin-based reagents. In EM images of plasma membrane sheets, the streptavidin-gold-labeled reporter was clustered in 20-50 nm microdomains, presumably representative of inner leaflet lipid rafts. The utility of the reporter was demonstrated in an investigation of the potential lipid raft localization of the epidermal growth factor receptor.
authors: Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, IA 52242; Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, AK 72205; and.
authors: Kimberly J
authors: KJ
authors: Krager
authors: Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, IA 52242.
authors: Mitul
authors: M
authors: Sarkar
authors: Department of Surgery, University of Iowa Carver College of Medicine, Iowa City, IA 52242.
authors: Erik C
authors: EC
authors: Twait
authors: Department of Pathology and the OSU Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210.
authors: Nancy L
authors: NL
authors: Lill
authors: Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, IA 52242. Electronic address: john-koland@uiowa.edu.
authors: John G
authors: JG
authors: Koland
mesh_terms: D000818:Animals; D019921:Biotinylation; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008055:Lipids / Q000737:chemistry; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000235:genetics* / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron
chemical_list: D008055:Lipids; D008565:Membrane Proteins; D049452:Green Fluorescent Proteins; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)