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

Abstract

SUMMARY 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.

Molecular Structure, Function, and Dynamics of Clathrin-Mediated Membrane Traffic

“…Any factor that stabilizes AP2 on the PtdIns(4,5)P 2 membrane will enhance initiation, including nonclathrin components that link multiple AP2s. In cells reconstituted with AP2 complexes lacking the appendage domain and therefore defective in recruitment of endocytic accessory factors, a large fraction of clathrin-coated pits fail to invaginate and rapidly abort (Aguet et al 2013). One obvious candidate for coated-vesicle stabilization is Eps15, which has about 15 AP2-appendage-binding sites.…”

“…The molecular organization of clathrin lattices shows that such a process would involve too drastic a deconstruction/ reconstruction to be a plausible mechanism (Kirchhausen 2009), and direct observations in living cells now show that canonical coated pits form de novo (Ehrlich et al 2004;Perrais and Merrifield 2005;Saffarian et al 2009;Cocucci et al 2012;Aguet et al 2013). Association of the larger arrays with distinct cellular processes (some of which may have endocytic function) resolves the confusion (Saffarian et al 2009).…”

Endocytic Accessory Factors and Regulation of Clathrin-Mediated Endocytosis

“…Instead low overexpression levels of endocytic accessory proteins were used, although this naturally raised concerns over potential artifacts caused by protein overexpression (Doyon et al 2011; although see Aguet et al 2013). Nonetheless, the recruitment of FP tagged endocytic accessory proteins was measured relative to single productive scission events in one cell type (mouse fibroblasts) with a temporal resolution of 2 sec (Merrifield et al 2005;Taylor et al 2011Taylor et al , 2012.…”

TRAIL-death receptor endocytosis and apoptosis are selectively regulated by dynamin-1 activation

“…In addition to their role in promoting the fission of invaginated CCPs, dynamins control earlier rate-limiting steps of clathrin-coated vesicle formation (10)(11)(12). There are three dynamin isoforms in vertebrates: dynamin-1 (Dyn1) is predominantly expressed in neurons, dynamin-2 (Dyn2) is ubiquitously expressed, and dynamin-3 (Dyn3) is expressed in neurons, lung, and testis.…”