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PMID: 21445324 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis.

PLoS biology ·Vol. 9 ·No. 3 ·2011-03-00 ·Pages e1000604

Taylor MJ, Perrais D, Merrifield CJ

Abstract

Dual colour total internal reflection fluorescence microscopy is a powerful tool for decoding the molecular dynamics of clathrin-mediated endocytosis (CME). Typically, the recruitment of a fluorescent protein-tagged endocytic protein was referenced to the disappearance of spot-like clathrin-coated structure (CCS), but the precision of spot-like CCS disappearance as a marker for canonical CME remained unknown. Here we have used an imaging assay based on total internal reflection fluorescence microscopy to detect scission events with a resolution of ∼ 2 s. We found that scission events engulfed comparable amounts of transferrin receptor cargo at CCSs of different sizes and CCS did not always disappear following scission. We measured the recruitment dynamics of 34 types of endocytic protein to scission events: Abp1, ACK1, amphiphysin1, APPL1, Arp3, BIN1, CALM, CIP4, clathrin light chain (Clc), cofilin, coronin1B, cortactin, dynamin1/2, endophilin2, Eps15, Eps8, epsin2, FBP17, FCHo1/2, GAK, Hip1R, lifeAct, mu2 subunit of the AP2 complex, myosin1E, myosin6, NECAP, N-WASP, OCRL1, Rab5, SNX9, synaptojanin2β1, and syndapin2. For each protein we aligned ∼ 1,000 recruitment profiles to their respective scission events and constructed characteristic "recruitment signatures" that were grouped, as for yeast, to reveal the modular organization of mammalian CME. A detailed analysis revealed the unanticipated recruitment dynamics of SNX9, FBP17, and CIP4 and showed that the same set of proteins was recruited, in the same order, to scission events at CCSs of different sizes and lifetimes. Collectively these data reveal the fine-grained temporal structure of CME and suggest a simplified canonical model of mammalian CME in which the same core mechanism of CME, involving actin, operates at CCSs of diverse sizes and lifetimes.

MeSH Terms
Actins/metabolism Adaptor Proteins, Signal Transducing/metabolism Animals Clathrin/metabolism Coated Pits, Cell-Membrane/metabolism Dynamins/metabolism Endocytosis Mammals/metabolism Mice Molecular Dynamics Simulation Myosins/metabolism NIH 3T3 Cells Polymerization Protein Binding Protein Structure, Tertiary Time Factors
Chemicals
Actins Adaptor Proteins, Signal Transducing Clathrin Myosins Dynamins
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Taylor Marcus J
Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
Perrais David
Merrifield Christien J
Conflict of Interest

The authors have declared that no competing interests exist.

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Article Info
Journal
PLoS biology
Abbr.
PLoS Biol
ISSN
1545-7885
DOI
10.1371/journal.pbio.1000604
Published
2011-03-00
Epub
2011-00-22
Pages
e1000604
Language
English
Region
United States
NLM ID
101183755
PMCID
PMC3062526
Subset
IM
Grants
Medical Research Council · MC_U105178789 · United Kingdom
External Links
PubMed source DOI: 10.1371/journal.pbio.1000604 PMC Full Text View journal details
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