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PMID: 23891661 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Advances in analysis of low signal-to-noise images link dynamin and AP2 to the functions of an endocytic checkpoint.

Developmental cell ·Vol. 26 ·No. 3 ·2013-08-12 ·Pages 279-91

Aguet F, Antonescu CN, Mettlen M, Schmid SL, Danuser G

Abstract

Numerous endocytic accessory proteins (EAPs) mediate assembly and maturation of clathrin-coated pits (CCPs) into cargo-containing vesicles. Analysis of EAP function through bulk measurement of cargo uptake has been hampered due to potential redundancy among EAPs and, as we show here, the plasticity and resilience of clathrin-mediated endocytosis (CME). Instead, EAP function is best studied by uncovering the correlation between variations in EAP association to individual CCPs and the resulting variations in maturation. However, most EAPs bind to CCPs in low numbers, making the measurement of EAP association via fused fluorescent reporters highly susceptible to detection errors. Here, we present a framework for unbiased measurement of EAP recruitment to CCPs and their direct effects on CCP dynamics. We identify dynamin and the EAP-binding α-adaptin appendage domain of the AP2 adaptor as switches in a regulated, multistep maturation process and provide direct evidence for a molecular checkpoint in CME.

MeSH Terms
Adaptor Protein Complex 2/chemistry,physiology Adaptor Protein Complex mu Subunits/chemistry,physiology Algorithms Clathrin Light Chains/physiology Clathrin-Coated Vesicles/physiology Dynamin II/chemistry,physiology Endocytosis/physiology Fatty Acid-Binding Proteins/chemistry,physiology Green Fluorescent Proteins Humans Luminescent Proteins Microscopy, Fluorescence/methods Protein Structure, Tertiary/physiology RNA, Small Interfering/genetics Retinal Pigment Epithelium/cytology Signal-To-Noise Ratio Transport Vesicles/physiology
Chemicals
Adaptor Protein Complex 2 Adaptor Protein Complex mu Subunits Clathrin Light Chains FABP4 protein, human Fatty Acid-Binding Proteins Luminescent Proteins RNA, Small Interfering adaptor protein complex 2, mu 2 subunit red fluorescent protein Green Fluorescent Proteins Dynamin II
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Aguet François
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Antonescu Costin N
Mettlen Marcel
Schmid Sandra L
Danuser Gaudenz
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Article Info
Journal
Developmental cell
Abbr.
Dev Cell
ISSN
1878-1551
DOI
10.1016/j.devcel.2013.06.019
Published
2013-08-12
Epub
2013-00-25
Pages
279-91
Language
English
Region
United States
NLM ID
101120028
PMCID
PMC3939604
Subset
IM
Grants
NIMH NIH HHS · R37 MH061345 · United States
NIMH NIH HHS · MH61345 · United States
NIMH NIH HHS · R01 MH061345 · United States
NIGMS NIH HHS · R01 GM73165 · United States
NIGMS NIH HHS · R01 GM073165 · United States
External Links
PubMed source DOI: 10.1016/j.devcel.2013.06.019 PMC Full Text View journal details
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