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

Dev Cell. 2013 Aug 12;26(3):279-91. doi: 10.1016/j.devcel.2013.06.019. Epub 2013 Jul 25.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Protein Complex 2 / chemistry
  • Adaptor Protein Complex 2 / physiology*
  • Adaptor Protein Complex mu Subunits / chemistry
  • Adaptor Protein Complex mu Subunits / physiology*
  • Algorithms
  • Clathrin Light Chains / physiology
  • Clathrin-Coated Vesicles / physiology
  • Dynamin II / chemistry
  • Dynamin II / physiology*
  • Endocytosis / physiology*
  • Fatty Acid-Binding Proteins / chemistry
  • Fatty Acid-Binding Proteins / 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*

Substances

  • 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