Flat-to-curved transition during clathrin-mediated endocytosis correlates with a change in clathrin-adaptor ratio and is regulated by membrane tension

Abstract

Although essential for many cellular processes, the sequence of structural and molecular events during clathrin-mediated endocytosis remains elusive. While it was believed that clathrin-coated pits grow with a constant curvature, it was recently suggested that clathrin first assembles to form a flat structure and then bends while maintaining a constant surface area. Here, we combine correlative electron and light microscopy and mathematical modelling to quantify the sequence of ultrastructural rearrangements of the clathrin coat during endocytosis in mammalian cells. We confirm that clathrin-coated structures can initially grow flat and that lattice curvature does not show a direct correlation with clathrin coat assembly. We demonstrate that curvature begins when 70% of the final clathrin content is acquired. We find that this transition is marked by a change in the clathrin to clathrin-adaptor protein AP2 ratio and that membrane tension suppresses this transition. Our results support the model that mammalian cells dynamically regulate the flat-to-curved transition in clathrin-mediated endocytosis by both biochemical and mechanical factors.

The structure and spontaneous curvature of clathrin lattices at the plasma membrane

“…In the second, clathrin lattices grow flat to their terminal surface area and then bend to create a vesicle (Heuser, 1980). Mixtures of these models have been observed: clathrin sites growing flat, growing curved, or switching between behaviors, even within the same cell (Scott et al, 2018;Bucher et al, 2018). Indeed, clathrin imaging in live cells exhibits varied lifetime dynamics, consistent with this mixed model of endocytosis (Sochacki and Taraska, 2019;Loerke et al, 2011).…”

“…These data support a modified version of the constant area model of clathrin curvature. Previous work with electron, live cell fluorescence, and correlative microscopy has shown that in some cell lines (SK-MEL-2 and BS-C-1) (Avinoam et al, 2015;Bucher et al, 2018), flat clathrin curves. Further, in these cells, curvature can occur at the onset, during, or after the complete recruitment of clathrin to the membrane (Scott et al, 2018).…”

“…Flat clathrin bends into endocytic vesicles in both human skin melanoma (SK-MEL-2) and green monkey kidney fibroblasts (BS-C-1) (Avinoam et al, 2015;Scott et al, 2018;Bucher et al, 2018). Yet, these cell lines have been noted for a lack of large flat clathrin plaques (Saffarian et al, 2009;Bucher et al, 2018). HeLa cells, however, contain many large flat clathrin plaques (Maupin and Pollard, 1983;Saffarian et al, 2009).…”

“…Although the two major models of CME are still openly debated, recent correlative and live cell fluorescence microscopies have supported the idea that clathrin can grow flat and then curve in some cells (Avinoam et al, 2015;Bucher et al, 2018;Scott et al, 2018). This is surprising considering the atomic structure of clathrin baskets observed from cryoelectron microscopy (cryo-EM) (Fotin et al, 2004;Morris et al, 2019) (legend continued on next page) ll Article seven different triskelia.…”

α-Synuclein facilitates clathrin assembly in synaptic vesicle endocytosis

“…4). Prior studies have suggested that the clathrin:adaptor ratio is a determinant of CCP curvature (Bucher et al, 2018). The clathrin:AP180 protein ratio on synaptic membranes for αβγ-synuclein KO displayed an 1.14 and 2.42 fold increase for heavy and light chain, respectively, suggestive of altered curvature of assembled clathrin lattices.…”