title: Dynamics of intracellular clathrin/AP1- and clathrin/AP3-containing carriers.
abstract: Clathrin/AP1- and clathrin/AP3-coated vesicular carriers originate from endosomes and the trans-Golgi network. Here, we report the real-time visualization of these structures in living cells reliably tracked by rapid, three-dimensional imaging with the use of a spinning-disk confocal microscope. We imaged relatively sparse, diffraction-limited, fluorescent objects containing chimeric fluorescent protein (clathrin light chain, σ adaptor subunits, or dynamin2) with a spatial precision of up to ~30 nm and a temporal resolution of ~1 s. The dynamic characteristics of the intracellular clathrin/AP1 and clathrin/AP3 carriers are similar to those of endocytic clathrin/AP2 pits and vesicles; the clathrin/AP1 coats are, on average, slightly shorter-lived than their AP2 and AP3 counterparts. We confirmed that although dynamin2 is recruited as a burst to clathrin/AP2 pits immediately before their budding from the plasma membrane, we found no evidence supporting a similar association of dynamin2 with clathrin/AP1 or clathrin/AP3 carriers at any stage during their lifetime. We found no effects of chemical inhibitors of dynamin function or the K44A dominant-negative mutant of dynamin on AP1 and AP3 dynamics. This observation suggests that an alternative budding mechanism, yet to be discovered, is responsible for the scission step of clathrin/AP1 and clathrin/AP3 carriers.
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston and Program in Cellular and Molecular Medicine at Boston Children's Hospital, Boston, MA 02115, USA.
authors.forename: Comert
authors.initials: C
authors.lastname: Kural
authors.forename: Silvia K
authors.initials: SK
authors.lastname: Tacheva-Grigorova
authors.forename: Steeve
authors.initials: S
authors.lastname: Boulant
authors.forename: Emanuele
authors.initials: E
authors.lastname: Cocucci
authors.forename: Thorsten
authors.initials: T
authors.lastname: Baust
authors.forename: Delfim
authors.initials: D
authors.lastname: Duarte
authors.forename: Tom
authors.initials: T
authors.lastname: Kirchhausen
mesh_terms: D033961:Adaptor Protein Complex 1 / Q000378:metabolism*; D033962:Adaptor Protein Complex 2 / Q000378:metabolism; D033963:Adaptor Protein Complex 3 / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D034285:Dynamin II / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D014162:Transfection
chemical_list: D033961:Adaptor Protein Complex 1; D033962:Adaptor Protein Complex 2; D033963:Adaptor Protein Complex 3; D002966:Clathrin; D034285:Dynamin II