title: CALM regulates clathrin-coated vesicle size and maturation by directly sensing and driving membrane curvature.
abstract: The size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of "open" clathrin-coated pits (CCPs) to "necked"/"closed" CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects. Depletion of either adaptor, however, significantly inhibits endocytosis of transferrin and epidermal growth factor. The phenotypic effects of CALM depletion can be rescued by re-expression of wild-type CALM, but not with CALM that lacks a functional N-terminal, membrane-inserting, curvature-sensing/driving amphipathic helix, the existence and properties of which are demonstrated. CALM is thus a major factor in controlling CCV size and maturation and hence in determining the rates of endocytic cargo uptake.
authors.affiliation: Cambridge Institute for Medical Research and Department of Clinical Biochemistry, University of Cambridge, Cambridge Biomedical Campus, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK. Electronic address: sem50@cam.ac.uk.
authors.forename: Sharon E
authors.initials: SE
authors.lastname: Miller
authors.affiliation: Bionanotechnology and Nanomedicine Laboratory, Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark.
authors.forename: Signe
authors.initials: S
authors.lastname: Mathiasen
authors.affiliation: Cambridge Institute for Medical Research and Department of Clinical Biochemistry, University of Cambridge, Cambridge Biomedical Campus, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Bright
authors.affiliation: Laboratoire d'Enzymologie et Biochimie Structurales, UPR3082 CNRS - Bat 34, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Pierre
authors.affiliation: Cambridge Institute for Medical Research and Department of Clinical Biochemistry, University of Cambridge, Cambridge Biomedical Campus, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Bernard T
authors.initials: BT
authors.lastname: Kelly
authors.affiliation: Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Joseph-Stelzmann-Str. 26, 50931 Cologne, Germany.
authors.forename: Nikolay
authors.initials: N
authors.lastname: Kladt
authors.affiliation: Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Joseph-Stelzmann-Str. 26, 50931 Cologne, Germany.
authors.forename: Astrid
authors.initials: A
authors.lastname: Schauss
authors.affiliation: Laboratoire d'Enzymologie et Biochimie Structurales, UPR3082 CNRS - Bat 34, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
authors.forename: Christien J
authors.initials: CJ
authors.lastname: Merrifield
authors.affiliation: Bionanotechnology and Nanomedicine Laboratory, Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Stamou
authors.affiliation: Institute of Biochemistry I and Center for Molecular Medicine Cologne, University of Cologne, Joseph-Stelzmann-Str. 52, 50931 Cologne, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Höning
authors.affiliation: Cambridge Institute for Medical Research and Department of Clinical Biochemistry, University of Cambridge, Cambridge Biomedical Campus, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK. Electronic address: djo30@cam.ac.uk.
authors.forename: David J
authors.initials: DJ
authors.lastname: Owen
mesh_terms: D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000502:physiology; D048430:Cell Shape / Q000235:genetics*; D022163:Clathrin-Coated Vesicles / Q000502:physiology*; D003034:Coated Pits, Cell-Membrane / Q000502:physiology*; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000378:metabolism; D050556:Fatty Acid-Binding Proteins / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D034041:Monomeric Clathrin Assembly Proteins / Q000235:genetics* / Q000502:physiology*; D017434:Protein Structure, Tertiary; D050683:R-SNARE Proteins / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering; D014168:Transferrin / Q000378:metabolism
chemical_list: C493410:FABP4 protein, human; D050556:Fatty Acid-Binding Proteins; D008081:Liposomes; D034041:Monomeric Clathrin Assembly Proteins; C439003:PICALM protein, human; D050683:R-SNARE Proteins; D034741:RNA, Small Interfering; D014168:Transferrin; C112938:VAMP8 protein, human; D004815:Epidermal Growth Factor