A burst of auxilin recruitment determines the onset of clathrin-coated vesicle uncoating.

Department of Cell Biology and CBR Institute for Biomedical Research, Harvard Medical School, 200 Longwood Ave, Boston, MA 02115, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 08/2006; 103(27):10265-70. DOI: 10.1073/pnas.0603369103
Source: PubMed

ABSTRACT Clathrin-coated pits assemble on a membrane and pinch off as coated vesicles. The released vesicles then rapidly lose their clathrin coats in a process mediated by the ATPase Hsc70, recruited by auxilin, a J-domain-containing cofactor. How is the uncoating process regulated? We find that during coat assembly small and variable amounts of auxilin are recruited transiently but that a much larger burst of association occurs after the peak of dynamin signal, during the transition between membrane constriction and vesicle budding. We show that the auxilin burst depends on domains of the protein likely to interact with lipid head groups. We conclude that the timing of auxilin recruitment determines the onset of uncoating. We propose that, when a diffusion barrier is established at the constricting neck of a fully formed coated pit and immediately after vesicle budding, accumulation of a specific lipid can recruit sufficient auxilin molecules to trigger uncoating.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Rabies virus (RABV) causes a fatal zoonotic encephalitis. Disease symptoms require replication and spread within neuronal cells; however, in infected animals as well as in cell culture, the virus replicates in a broad range of cell types. Here we use a single cycle RABV and a recombinant vesicular stomatitis virus (rVSV), in which the glycoprotein (G) was replaced with that of RABV (rVSV RABV G), to examine RABV uptake into the African green monkey kidney cell line, BS-C-1. Combining biochemical studies and real-time spinning disk confocal fluorescence microscopy, we show that the predominant entry pathway of RABV particles into BS-C-1 cells is clathrin-dependent. Viral particles enter cells in pits with elongated structures and incomplete clathrin coats which depend upon actin to complete the internalization process. By measuring the time of internalization and the abundance of the clathrin adaptor protein AP2 we further show that the pits that internalize RABV particles are similar to those that internalize VSV. Pharmacological perturbations of dynamin or of actin polymerization inhibit productive infection, linking our observations on particle uptake with viral infectivity. This work extends to RABV particles the finding that clathrin mediated endocytosis proceeds through incompletely coated pits which depend upon actin.
    Journal of Virology 08/2013; · 5.08 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: How does mitosis influence the critical process of endocytosis? Some experiments lead to the conclusion that endocytosis arrests completely during mitosis, whereas others indicate that endocytosis persists. We have resolved this apparent discrepancy by showing how conditions of the experiment influence its outcome. The dynamics of clathrin-coated pit formation and the uptake of transferrin are maintained in naturally dividing cells but are nearly absent in mitotic cells arrested chemically by treatment with nocodazole, S-Trityl-L-cysteine, or RO-3306. Moreover, sequentially incubating cells at 4°C and then shifting them to 37°C or to serum starvation artificially increases the amount of transferrin receptor at the surface of naturally dividing cells, leading to the incorrect conclusion that endocytosis has ceased during mitosis. Thus, our data show that endocytosis is unaffected during all stages of natural cell division.
    Cell Reports 08/2013; · 7.21 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Clathrin-coated vesicles are major carriers of vesicular traffic in eukaryotic cells. This endocytic pathway relies on cycles of clathrin coat assembly and Hsc70-mediated disassembly. Here we identify histidine residues as major determinants of lattice assembly and stability. They are located at the invariant interface between the proximal and distal segments of clathrin heavy chains, in triskelions centered on two adjacent vertices of the coated-vesicle lattice. Mutation of these histidine residues to glutamine alters the pH dependence of coat stability. We then describe single-particle fluorescence imaging experiments in which we follow the effect of these histidine mutations on susceptibility to Hsc70-dependent uncoating. Coats destabilized by these mutations require fewer Hsc70 molecules to initiate disassembly, as predicted by a model in which Hsc70 traps conformational distortions during the auxilin- and Hsc70:ATP-mediated uncoating reaction.
    Structure 05/2014; · 5.99 Impact Factor

Full-text (2 Sources)

Available from
May 31, 2014