New tools for “hot-wiring” clathrin-mediated endocytosis with temporal and spatial precision

Abstract

Wood et al. have developed inducible chemical and optogenetic systems for triggering clathrin-mediated endocytosis using minimal clathrin-binding domains. Bypassing regulatory steps in vesicle creation will enable a better understanding of the factors required for vesicle initiation and processing, which the authors use to define how clathrin functionally interacts with its adaptor AP2.

The Structures of Natively Assembled Clathrin Coated Vesicles

“…3A). Secondly, AP2 β2-appendage has been shown experimentally to bind and assemble clathrin cages whereas the other appendage domain-containing adaptors have not (28). Finally, our CCVs are highly enriched in synaptic vesicles (Fig.…”

“…The pool of 80k CCV particle images was 2D-classified using cryoSPARC leaving 60k particles which were then 3D-classified using Relion and cisTEM into 5 different clathrin cage geometries: 1) mini-coat (15k particles, T symmetry, 28 The C2 basket geometry was delineated from a pool of football particles that were classified into two classes using cisTEM, with the D3 map as the initial model and no symmetry applied. As a last step, to prepare cage maps and metadata (Euler angles) for subparticle analysis, the Euler angles were refined in cisTEM with no symmetry applied, then the maps were reconstructed with symmetry in Relion to enable consistent subtraction of projections from images during the subparticle analysis (refer to the next section) and also to adjust the grayscale of the maps to that of the particle images.…”

“…The α2 adaptin mediates binding to membrane mainly through interactions with phosphatidylinositol-4,5bisphosphate (PtdIns(4,5)P 2 ). α2 and β2 possess C-terminal appendage domains at their Ctermini and clathrin box motifs that have been shown to bind clathrin (16,27,28). Although clathrin box binding sites are well known, the appendage binding site has been mapped to a broad region of the N-terminal CHC β-propeller and helical zig-zags (29).…”

“…CHC β-propellers provide the grooves between their blades for binding of most adaptor/accessory proteins (26) like AP1-3, AP180, HIP1, and EPSIN which possess multiple linear motifs (clathrin box) in their C-terminal unstructured regions. AP1 and 2 also have appendages (β1 and β2, respectively) C-terminal to their clathrin boxes that have been shown to bind clathrin (16,27,28). Although clathrin box binding sites are well known, the appendage binding site has been mapped to a broad region of the N-terminal CHC coiled coils (29).…”

Unintended inhibition of protein function using GFP nanobodies in human cells

“…Data analysis. Analysis of transferrin uptake was done as described previously (Wood et al, 2017). Briefly, single cells were outlined manually in Fiji.…”