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PMID: 23103167 Published · ppublish English Journal Article Research Support, N.I.H., Extramural

Dynamics of intracellular clathrin/AP1- and clathrin/AP3-containing carriers.

Cell reports ·Vol. 2 ·No. 5 ·2012-11-29 ·Pages 1111-9

Kural C, Tacheva-Grigorova SK, Boulant S, Cocucci E, Baust T, Duarte D, Kirchhausen T

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.

MeSH Terms
Adaptor Protein Complex 1/metabolism Adaptor Protein Complex 2/metabolism Adaptor Protein Complex 3/metabolism Animals Cell Line Cell Membrane/metabolism Chlorocebus aethiops Clathrin/metabolism Clathrin-Coated Vesicles/metabolism Dynamin II/antagonists & inhibitors,genetics,metabolism Endosomes/metabolism Microscopy, Fluorescence Mutation Transfection
Chemicals
Adaptor Protein Complex 1 Adaptor Protein Complex 2 Adaptor Protein Complex 3 Clathrin Dynamin II
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Kural Comert
Department of Cell Biology, Harvard Medical School, Boston and Program in Cellular and Molecular Medicine at Boston Children's Hospital, Boston, MA 02115, USA.
Tacheva-Grigorova Silvia K
Boulant Steeve
Cocucci Emanuele
Baust Thorsten
Duarte Delfim
Kirchhausen Tom
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Article Info
Journal
Cell reports
Abbr.
Cell Rep
ISSN
2211-1247
DOI
10.1016/j.celrep.2012.09.025
Published
2012-11-29
Epub
2012-00-25
Pages
1111-9
Language
English
Region
United States
NLM ID
101573691
PMCID
PMC3513667
Subset
IM
Grants
NIGMS NIH HHS · R01 GM075252 · United States
NIAID NIH HHS · U54 AI057159 · United States
NIGMS NIH HHS · GM-075252 · United States
External Links
PubMed source DOI: 10.1016/j.celrep.2012.09.025 PMC Full Text View journal details
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