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: 9.67). 08/2006; 103(27):10265-70. DOI: 10.1073/pnas.0603369103
Source: PubMed


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.

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Available from: Ramiro Massol, Oct 10, 2015
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    • "Reagents, cell culture, and transfections U373 human glioblastoma cells stably expressing σ2-EGFP were grown in DMEM containing 10% fetal calf serum (FCS), penicillin, and streptomycin. Transient expression of rat Tomato-LCa (Massol et al., 2006; Saffarian et al., 2009) and LifeAct-mCherry was carried out by transfection with Lipofectamine 2000 (Invitrogen) in Optimem (Life Technologies, Grand Island, NY) according to the manufacturers' instructions. SUM159 human breast carcinoma cells were geneedited to replace both alleles of the σ2 subunit of AP2 with σ2 fused at its C-terminus to EGFP using a transcription activator-like effector nuclease (TALEN)-based protocol (Sanjana et al., 2012; Cocucci et al., 2014). "
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    ABSTRACT: Clathrin/AP2-coated vesicles are the principal endocytic carriers originating at the plasma membrane. In the experiments reported here, we used spinning-disk confocal and lattice light-sheet microscopy to study the assembly dynamics of coated pits on the dorsal and ventral membranes of migrating U373 glioblastoma cells stably expressing AP2 tagged with enhanced green fluorescence (AP2-EGFP) and on lateral protrusions from immobile SUM159 breast carcinoma cells, gene-edited to express AP2-EGFP. On U373 cells, coated pits initiated on the dorsal membrane at the front of the lamellipodium and at the approximate boundary between the lamellipodium and lamella and continued to grow as they were swept back toward the cell body; coated pits were absent from the corresponding ventral membrane. We observed a similar dorsal/ventral asymmetry on membrane protrusions from SUM159 cells. Stationary coated pits formed and budded on the remainder of the dorsal and ventral surfaces of both types of cells. These observations support a previously proposed model that invokes net membrane deposition at the leading edge due to an imbalance between the endocytic and exocytic membrane flow at the front of a migrating cell.
    Molecular Biology of the Cell 05/2015; 26(11). DOI:10.1091/mbc.E15-01-0055 · 4.47 Impact Factor
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    • "Monkey BSC1 cells stably expressing σ2-EGFP (Ehrlich et al., 2004; Saffarian and Kirchhausen, 2008), σ1-EGFP (Anitei et al., 2010) or σ3a-EGFP and human SK-MEL-2 cells expressing dynamin2-EGFP (hDNM2EN) or clathrin light chain A fused to RFP and dynamin2-EGFP (hCLTAEN/DNM2EN) in their native genomic loci (Doyon et al., 2011) were grown in DMEM medium containing 10% fetal calf serum, penicillin and streptomycin. Transient expression of 2µg rat Tomato-LCa (Massol et al., 2006) in BSC1 cells was carried out using Lipofectamine 2000 (Invitrogen) in Optimem (Gibco) according to the manufacturer's instructions and were analyzed 24 hrs after transfection. Transient expression of wt and the K44A dominant negative mutant of dynamin2 were achieved by adenovirus transduction and cells analyzed for AP dynamics and fluorescence transferrin uptake 16 hr post infection (Altschuler et al., 1998). "
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    Cell Reports 10/2012; 2(5). DOI:10.1016/j.celrep.2012.09.025 · 8.36 Impact Factor
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    • "The most important structural components of the coat are clathrin and its AP2 (a-b2-m2-s2) heterotetrameric adaptor complex. Coated pit formation proceeds by sequential addition of clathrin triskelions and adaptors, generating a sharply curved coat; adaptor-mediated interactions with membrane-bound proteins (and lipids) deform the underlying membrane; dynamin mediates scission when the deformation has created a suitably narrow neck; and auxilin, which arrives immediately following scission, recruits Hsc70 to direct uncoating (Lee et al., 2006; Massol et al., 2006). Hip1R, which binds clathrin light chains, recruits actin, which is required in some instances for coatedvesicle maturation and budding (Ferguson et al., 2009; Gottfried et al., 2009; Merrifield et al., 2002; Saffarian et al., 2009). "
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