Analysis of the AP-2 adaptor complex and cargo during clathrin-mediated endocytosis

Joshua Z. Rappoport, Alexandre Benmerah, Sanford M. Simon*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

38 Scopus citations

Abstract

Previously, we reported that the hetero-tetrameric adaptor complex AP-2 co-localizes with the static population of clathrin spots, whereas it is excluded from clathrin spots that disappear from the plasma membrane (forming clathrin-coated vesicles). More recently however, another group provided evidence that AP-2 markers could be observed coincident with disappearing clathrin spots. Thus, we tested several possible explanations for the apparent discrepancies in these two studies. We evaluated the potential contribution of nonred emission of clathrin-dsRed (used in both studies) in the simultaneous measurement of AP-2 and clathrin at various times. Additionally, we directly compared two different green fluorescent protein-tagged AP-2 constructs (similar to those used in the previous reports). These studies demonstrated that the duration of expression time greatly influences the subcellular localization of the AP-2 markers. Furthermore, we quantitatively evaluated the AP-2 fluorescence at the sites of numerous static and disappearing clathrin spots (at least 80 per group) and confirmed our initial observation that while AP-2 is present in nearly all static clathrin spots, it is excluded from the disappearing population of clathrin spots. Finally, in order to verify that clathrin spot disappearance represents clathrin-coated vesicle internalization, we simultaneously imaged clathrin and the cargo molecule transferrin at the cell surface.

Original languageEnglish (US)
Pages (from-to)539-547
Number of pages9
JournalTraffic
Volume6
Issue number7
DOIs
StatePublished - Jul 2005

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology

Fingerprint Dive into the research topics of 'Analysis of the AP-2 adaptor complex and cargo during clathrin-mediated endocytosis'. Together they form a unique fingerprint.

Cite this