Advances in Analysis of Low Signal-to-Noise Images Link Dynamin and AP2 to the Functions of an Endocytic Checkpoint

• Published in: Developmental cell 2013-08, Vol.26 (3), p.279-291
• Main Authors: Aguet, François, Antonescu, Costin N., Mettlen, Marcel, Schmid, Sandra L., Danuser, Gaudenz
• Format: Article
• Language: English
• Subjects: 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; Red Fluorescent Protein; Retinal Pigment Epithelium - cytology; RNA, Small Interfering - genetics; Signal-To-Noise Ratio; Transport Vesicles - physiology
• ISSN: 1534-5807; 1878-1551
• DOI: 10.1016/j.devcel.2013.06.019

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. [Display omitted] •Highly sensitive detection of protein association with CCPs•CCPs undergo a multistep maturation process gated by a checkpoint•Early dynamin recruitment is required for CCP maturation•Compensatory mechanisms obscure EAP roles in CCP stabilization and maturation Aguet et al. perform a high-sensitivity analysis of clathrin-coated pits (CCPs). They establish methods that circumvent many difficulties associated with low signal-to-noise ratios, enable quantitative tracking of CCP/accessory dynamics, and reveal that dynamin and the α-adaptin appendage domain of AP2 each gate different stages in the progression of clathrin-mediated endocytosis.