title: Single-molecule microscopy reveals membrane microdomain organization of cells in a living vertebrate.
abstract: It has been possible for several years to study the dynamics of fluorescently labeled proteins by single-molecule microscopy, but until now this technology has been applied only to individual cells in culture. In this study, it was extended to stem cells and living vertebrate organisms. As a molecule of interest we used yellow fluorescent protein fused to the human H-Ras membrane anchor, which has been shown to serve as a model for proteins anchored in the plasma membrane. We used a wide-field fluorescence microscopy setup to visualize individual molecules in a zebrafish cell line (ZF4) and in primary embryonic stem cells. A total-internal-reflection microscopy setup was used for imaging in living organisms, in particular in epidermal cells in the skin of 2-day-old zebrafish embryos. Our results demonstrate the occurrence of membrane microdomains in which the diffusion of membrane proteins in a living organism is confined. This membrane organization differed significantly from that observed in cultured cells, illustrating the relevance of performing single-molecule microscopy in living organisms.
authors.affiliation: Molecular Cell Biology, Institute of Biology, Leiden University, Leiden, The Netherlands. m.j.m.schaaf@biology.leidenuniv.nl
authors.forename: Marcel J M
authors.initials: MJ
authors.lastname: Schaaf
authors.forename: Wiepke J A
authors.initials: WJ
authors.lastname: Koopmans
authors.forename: Tobias
authors.initials: T
authors.lastname: Meckel
authors.forename: John
authors.initials: J
authors.lastname: van Noort
authors.forename: B Ewa
authors.initials: BE
authors.lastname: Snaar-Jagalska
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Schmidt
authors.forename: Herman P
authors.initials: HP
authors.lastname: Spaink
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology / Q000648:ultrastructure*; D021962:Membrane Microdomains / Q000648:ultrastructure*; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D015336:Molecular Probe Techniques; D015027:Zebrafish / Q000033:anatomy & histology* / Q000196:embryology*
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