The First Five Seconds in the Life of a Clathrin-Coated Pit

Abstract

Coated pits assemble by growth of a clathrin lattice, linked by adaptors to the underlying membrane. How does this process start? We used live-cell TIRF imaging, with single-molecule EGFP sensitivity and high temporal resolution, to detect arrival of the clathrin triskelions and AP2 adaptors that initiate coat assembly. Unbiased object identification and trajectory tracking, together with a statistical model, yield the arrival times and numbers of individual proteins, as well as experimentally confirmed estimates of the extent of substitution of endogenous by expressed, fluorescently tagged proteins. Pits initiate by coordinated arrival of clathrin and AP2, usually detected as two sequential steps, each of one triskelion with two adaptors. PI-4,5-P2 is essential for initiation. The accessory proteins FCHo1/2 are not; instead, they are required for sustained growth. This objective picture of coated-pit initiation also shows that methods outlined here will be broadly useful for studies of dynamic assemblies in living cells.

Identification and Dynamics of Arabidopsis Adaptor Protein-2 Complex and Its Involvement in Floral Organ Development

“…The AP2M-GFP structures exhibited a gradual increase and rapid decrease of fluorescence intensity ( Figures 2D and 2E). Similar kinetics of the fluorescent proteinlabeled CCV components have been observed in mammalian cells (Merrifield et al, 2002;Ehrlich et al, 2004;Cocucci et al, 2012). The dynamics of the AP2M-GFP fluorescence may reflect the molecular behavior of AP2M on the plasma membrane, including incorporation into CCV and subsequent scission by the activity of DRPs, as is well studied in mammalian cells (Kirchhausen, 2009;McMahon and Boucrot, 2011).…”

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

“…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

“…A simple question in the biology of CME is how CCPs start (Ehrlich et al 2004;Antonescu et al 2010;Henne et al 2010;Nunez et al 2011;Cocucci et al 2012;Merrifield 2012;Umasankar et al 2012). Much of the spatial and temporal regulation of endocytosis is probably conducted by regulating the initiation of CCP assembly.…”

“…Thus, whereas there remains some controversy, it seems likely that AP2 complexes play an essential role in the nucleation of most CCPs and AP2 may serve as a paradigm for the role played by adaptors in CCP nucleation. The distribution of PIP2 in the inner leaflet of the mammalian plasma membrane is uniform in nonexcitable cells (Antonescu et al 2010) and AP2 has a relatively low affinity for PIP2, leading to the suggestion that AP2 "samples" the plasma membrane (Cocucci et al 2012). On binding PIP2 the AP2 complex undergoes a dramatic structural rearrangement and unfurls to reveal cargo and clathrin-binding motifs (Jackson et al 2010).…”

“…Second, the role of adaptor proteins in bud nucleation in both mammalian and yeast cells is generally clear (Motley et al 2003;Antonescu et al 2010;Cocucci et al 2012), although some areas of controversy remain. In mammalian cells a plethora of adaptor proteins have been described (Fig.…”