Actin assembly facilitates vesicle formation in several trafficking pathways, including clathrin-mediated endocytosis (CME). Interestingly, actin does not assemble at all CME sites in mammalian cells. How actin networks are organized with respect to mammalian CME sites and how assembly forces are harnessed, are not fully understood. Here, branched actin network geometry at CME sites was analyzed using three different advanced imaging approaches. When endocytic dynamics of unperturbed CME sites are compared, sites with actin assembly show a distinct signature, a delay between completion of coat expansion and vesicle scission, indicating that actin assembly occurs preferentially at stalled CME sites. In addition, N-WASP and the Arp2/3 complex are recruited to one side of CME sites, where they are positioned to stimulate asymmetric actin assembly and force production. We propose that actin assembles preferentially at stalled CME sites where it pulls vesicles into the cell asymmetrically, much as a bottle opener pulls off a bottle cap.

During clathrin-mediated endocytosis (CME), flat plasma membrane is remodeled to produce nanometer-scale vesicles. The mechanisms underlying this remodeling are not completely understood. The ability of clathrin to bind membranes of distinct geometries casts uncertainty on its specific role in curvature generation/stabilization. Here, we used nanopatterning to produce substrates for live-cell imaging, with U-shaped features that bend the ventral plasma membrane of a cell into shapes resembling energetically unfavorable CME intermediates. This induced membrane curvature recruits CME proteins, promoting endocytosis. Upon AP2, FCHo1/2, or clathrin knockdown, CME on flat substrates is severely diminished. However, induced membrane curvature recruits CME proteins in the absence of FCHo1/2 or clathrin and rescues CME dynamics/cargo uptake after clathrin (but not AP2 or FCHo1/2) knockdown. Induced membrane curvature enhances CME protein recruitment upon branched actin assembly inhibition under elevated membrane tension. These data establish that membrane curvature assists in CME nucleation and that the essential function of clathrin during CME is to facilitate curvature evolution, rather than scaffold protein recruitment.

Actin filament assembly facilitates vesicle formation in several trafficking pathways including clathrin-mediated endocytosis (CME). However, how actin assembly forces are harnessed has not been fully revealed for any vesicle forming process. In this study, three-dimensional (3D) super-resolution microscopy, live-cell imaging of triple-genome-edited, induced pluripotent stem cells (iPSCs), and newly developed machine-learning-based computational analysis tools, were used to comprehensively analyze assembly dynamics and geometry of proteins representing three different CME functional modules. When hundreds of CME events with and without associated Arp2/3-dependent actin network assembly were compared, sites with actin assembly showed a distinct delay between completion of endocytic coat expansion and vesicle scission, consistent with the notion that these were stalled sites requiring actin assembly forces to complete vesicle formation. Importantly, our analysis showed that N-WASP is preferentially recruited to one side of CME sites, where it stimulates actin assembly asymmetrically. These results indicate that in mammalian cells actin assembly is induced at stalled CME sites, where asymmetric forces pull the plasma membrane into the cell much like a bottle opener pulls off a bottle cap.

Lentiviruses express non-enzymatic accessory proteins whose function is to subvert cellular machinery in the infected host. The HIV-1 accessory protein Nef hijacks clathrin adaptors to degrade or mislocalize host proteins involved in antiviral defenses. Here, we investigate the interaction between Nef and clathrin-mediated endocytosis (CME), a major pathway for membrane protein internalization in mammalian cells, using quantitative live-cell microscopy in genome-edited Jurkat cells. Nef is recruited to CME sites on the plasma membrane, and this recruitment correlates with an increase in the recruitment and lifetime of CME coat protein AP-2 and late-arriving CME protein dynamin2. Furthermore, we find that CME sites that recruit Nef are more likely to recruit dynamin2, suggesting that Nef recruitment to CME sites promotes CME site maturation to ensure high efficiency in host protein downregulation.

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