title: Flat clathrin lattices: stable features of the plasma membrane.
abstract: Clathrin-mediated endocytosis (CME) is a fundamental property of eukaryotic cells. Classical CME proceeds via the formation of clathrin-coated pits (CCPs) at the plasma membrane, which invaginate to form clathrin-coated vesicles, a process that is well understood. However, clathrin also assembles into flat clathrin lattices (FCLs); these structures remain poorly described, and their contribution to cell biology is unclear. We used quantitative imaging to provide the first comprehensive description of FCLs and explore their influence on plasma membrane organization. Ultrastructural analysis by electron and superresolution microscopy revealed two discrete populations of clathrin structures. CCPs were typified by their sphericity, small size, and homogeneity. FCLs were planar, large, and heterogeneous and present on both the dorsal and ventral surfaces of cells. Live microscopy demonstrated that CCPs are short lived and culminate in a peak of dynamin recruitment, consistent with classical CME. In contrast, FCLs were long lived, with sustained association with dynamin. We investigated the biological relevance of FCLs using the chemokine receptor CCR5 as a model system. Agonist activation leads to sustained recruitment of CCR5 to FCLs. Quantitative molecular imaging indicated that FCLs partitioned receptors at the cell surface. Our observations suggest that FCLs provide stable platforms for the recruitment of endocytic cargo.
authors.affiliation: MRC Laboratory for Molecular Cell Biology, London WC1E 6BT, United Kingdom Institute of Immunity and Transplantation, University College London, London NW3 2PF, United Kingdom j.grove@ucl.ac.uk m.marsh@ucl.ac.uk.
authors.forename: Joe
authors.initials: J
authors.lastname: Grove
authors.affiliation: Biophysics and Diagnostics, National Physical Laboratory, Teddington TW11 0LW, United Kingdom.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Metcalf
authors.affiliation: Biophysics and Diagnostics, National Physical Laboratory, Teddington TW11 0LW, United Kingdom.
authors.forename: Alex E
authors.initials: AE
authors.lastname: Knight
authors.affiliation: MRC Laboratory for Molecular Cell Biology, London WC1E 6BT, United Kingdom.
authors.forename: Silène T
authors.initials: ST
authors.lastname: Wavre-Shapton
authors.affiliation: MRC Laboratory for Molecular Cell Biology, London WC1E 6BT, United Kingdom.
authors.forename: Tony
authors.initials: T
authors.lastname: Sun
authors.affiliation: CoMPLEX, University College London, London WC1E 6BT, United Kingdom.
authors.forename: Emmanouil D
authors.initials: ED
authors.lastname: Protonotarios
authors.affiliation: CoMPLEX, University College London, London WC1E 6BT, United Kingdom.
authors.forename: Lewis D
authors.initials: LD
authors.lastname: Griffin
authors.affiliation: Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lippincott-Schwartz
authors.affiliation: MRC Laboratory for Molecular Cell Biology, London WC1E 6BT, United Kingdom j.grove@ucl.ac.uk m.marsh@ucl.ac.uk.
authors.forename: Mark
authors.initials: M
authors.lastname: Marsh
mesh_terms: D000818:Animals; D016466:CHO Cells; D018946:Chemokine CCL5 / Q000378:metabolism / Q000494:pharmacology; D002966:Clathrin / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism* / Q000648:ultrastructure; D003412:Cricetulus; D034281:Dynamins / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging; D021381:Protein Transport / Q000187:drug effects; D019713:Receptors, CCR5 / Q000819:agonists / Q000378:metabolism*
chemical_list: C483024:CCL5 protein, human; C584933:CCR5 protein, human; D018946:Chemokine CCL5; D002966:Clathrin; D019713:Receptors, CCR5; D049452:Green Fluorescent Proteins; D034281:Dynamins