title: Live-cell imaging of clathrin coats.
abstract: We compare the use of two-dimensional total internal reflection fluorescence microscopy with a rapid, simple-to-implement method for three-dimensional (3D) imaging using spinning-disk confocal microscopy suitable for reliable 3D tracking of clathrin-coated endocytic and endosomal carriers. These carriers contain about 20 EGFP (enhanced green fluorescent protein) equivalents of a chimeric fluorescent protein (either clathrin light chain or one of the clathrin adaptor subunits). Under tissue culture conditions, the clathrin-containing carriers correspond to a variable number of relatively sparse, diffraction-limited, fluorescent objects that can be identified with a spatial precision of ~30 nm or better and a temporal resolution of <1 s. The applicability of these approaches to mammalian cells in culture allows investigators detailed monitoring of the composition dynamics of the clathrin-containing carriers which can then be used to study in living cells the molecular mechanisms required for the formation and traffic of clathrin-coated pits and vesicles.
authors.affiliation: Department of Cell Biology, Harvard Medical School, and Immune Disease Institute and Program in Cellular and Molecular Medicine at Children’s Hospital, Boston, Massachusetts, USA.
authors.forename: Comert
authors.initials: C
authors.lastname: Kural
authors.forename: Tom
authors.initials: T
authors.lastname: Kirchhausen
mesh_terms: D000818:Animals; D058948:Cell Tracking / Q000379:methods*; D033941:Clathrin Light Chains / Q000235:genetics / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D049452:Green Fluorescent Proteins; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008322:Mammals; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*
chemical_list: D033941:Clathrin Light Chains; D049452:Green Fluorescent Proteins