Dynamics of Intracellular Clathrin/AP1- and Clathrin/AP3-Containing Carriers
Comert Kural,
Silvia K. Tacheva-Grigorova,
Steeve Boulant,
Emanuele Cocucci,
Thorsten Baust,
Delfim Duarte,
Tom Kirchhausen
Affiliations
Comert Kural
Department of Cell Biology, Harvard Medical School, Boston and Program in Cellular and Molecular Medicine at Boston Children’s Hospital, Boston, MA 02115, USA
Silvia K. Tacheva-Grigorova
Department of Cell Biology, Harvard Medical School, Boston and Program in Cellular and Molecular Medicine at Boston Children’s Hospital, Boston, MA 02115, USA
Steeve Boulant
Department of Cell Biology, Harvard Medical School, Boston and Program in Cellular and Molecular Medicine at Boston Children’s Hospital, Boston, MA 02115, USA
Emanuele Cocucci
Department of Cell Biology, Harvard Medical School, Boston and Program in Cellular and Molecular Medicine at Boston Children’s Hospital, Boston, MA 02115, USA
Thorsten Baust
Department of Cell Biology, Harvard Medical School, Boston and Program in Cellular and Molecular Medicine at Boston Children’s Hospital, Boston, MA 02115, USA
Delfim Duarte
Department of Cell Biology, Harvard Medical School, Boston and Program in Cellular and Molecular Medicine at Boston Children’s Hospital, Boston, MA 02115, USA
Tom Kirchhausen
Department of Cell Biology, Harvard Medical School, Boston and Program in Cellular and Molecular Medicine at Boston Children’s Hospital, Boston, MA 02115, USA
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.
