Jcb_201611115 9..12

9 The Rockefeller University Press $30.00 J. Cell Biol. Vol. 216 No. 1 9–11 https://doi.org/10.1083/jcb.201611115 In eukaryotic cells, the majority of internalized transmembrane protein receptors and ligands enter by clathrin-mediated endocytosis (CME). Clathrin-coated vesicles (CCVs) assemble at the plasma membrane by forming clathrin-coated pits (CCPs) that mature into fully formed coated vesicles and undergo scission via the large GTPase, dynamin. The clathrin adapter complex AP2 (a heterotetramer made up of α, β2, μ2, and σ2 subunits) is a well-studied key regulator of CME, important for the recruitment of both cargo into CCVs and clathrin itself. AP2 directly engages short amino acid motifs found in protein cargoes: the μ2 subunit binds YXXΦ motifs (Φ is a bulky hydrophobic residue; Owen and Evans, 1998), and σ2 binds acidic dileucine ([E/D]xxxL[L/I]) motifs (Kelly et al., 2008). AP2 also binds clathrin via a clathrin box motif in the β2 hinge (ter Haar et al., 2000) and a variety of accessory proteins through C-terminal domains of α and β2 subunits (Owen et al., 1999). Models based on x-ray crystal structures (Collins et al., 2002; Jackson et al., 2010) revealed how AP2 assumes at least two conformations. In the cytoplasm, AP2 adopts an autoinhibited closed state with obstructed cargo binding sites that prevents futile clathrin binding (Fig. 1 A; Collins et al., 2002). At the plasma membrane, AP2 undergoes a conformational change to an open state that is able to bind cargo (Jackson et al., 2010). Structure-based mutagenesis combined with surface plasmon resonance experiments (Höning et al., 2005; Jackson et al., 2010) suggest the conformational change is triggered when AP2 binds phosphatidylinositol-4,5-bisphosphate (PIP2) head groups in the membrane. Further structural work (Kelly et al., 2014) revealed how the conformational change expels a short motif in the unstructured β2 hinge that recruits clathrin, which then polymerizes to form a scaffold around vesicles. The coplanar arrangement of the PIP2 and cargo binding sites on AP2 directs it, and thus clathrin polymers, to cargo-enriched sites on the membrane. This model suggests AP2 allostery is sufficient to drive CCP initiation and growth. Other data indicate that CCP initiation requires nucleation sites for AP2 formed by complexes of Eps15 with FCHo (Ma et al., 2016), which is itself hypothesized to serve as an AP2 allosteric activator (Hollopeter et al., 2014). Although these structural models are appealing, they have not been directly tested in real time in living cells. In this issue, Kadlecova et al. have now conducted a technically excellent and important study that demonstrates functional hierarchy in binding events. The authors first generated stable cell lines with highly specific structure-based point mutations. This approach allows cells to be defective in binding phosphoinositide or cargo without affecting other functions. They used total internal reflection fluorescence microscopy to quantify in real time the extent to which PIP2 and cargo binding sites on AP2 affect the dynamics of CCP initiation, formation, and stabilization. Their methods allow quantitative detection of very transient clathrin-labeled structures, as well as both abortive and productive CCPs. This allowed them to verify structural predictions and furthermore provided key additional insights into the role of AP2 in coated pit initiation and stabilization. The authors first investigated the role of PIP2 binding by the α and β2 subunits (Fig. 1 B). Previous studies probed the role of PIP2 binding in CCV formation through methods like bulk depletion of PIP2; this study takes a less disruptive approach by specifically targeting AP2-dependent CME and allows the authors to dissect the role of each PIP2 binding site. Previous data suggested that both the α and β2 PIP2 binding sites were paramount for initiating the conformational change; both in vitro and cell-based data supported the importance of the α binding event in particular (Collins et al., 2002; Höning et al., 2005). Kadlecova et al. (2017) confirmed that the α-PIP2 binding site is required for both nucleation and downstream events; cells with AP2 lacking the α binding site also yielded many fewer productive CCPs and a slower rate of clathrin polymerization. The PIP2 binding site on β2 was tested directly here for the first time in cells. The AP2 mutant lacking the β2 binding site produced the same phenotype as the α mutant, suggesting both sites are equally important. The authors used electron microscopy to show that these mutants have an increased number of small, flat CCPs. These data suggest that PIP2 binding by α and β2 is required beyond the earliest stages of CCP formation, in contrast to recent work that suggests clathrin assembly is sufficient to drive CCP formation after nucleation (Dannhauser and Ungewickell, 2012). Full allosteric activation of AP2 is necessary for rapid clathrin polymerization and efficient CCP production, and AP2 appears to require both α and β2 phosphoinositide binding sites. The authors next tested the μ2 PIP2 binding site (Fig. 1 C). Based on structural work, this site was hypothesized to be accessible after the conformational change induced by membrane The precise sequence of events promoting clathrin-coated vesicle assembly is still debated. In this issue, Kadlecova et al. (2017. J. Cell Biol. https ://doi .org /10 .1083 /jcb .201608071) test structural models using quantitative microscopy in living cells to investigate the hierarchy and temporal importance of molecular events required for clathrin-coated pit initiation. Watching real-time endocytosis in living cells