Actin filament assembly facilitates vesicle formation in several trafficking pathways including clathrin-mediated endocytosis (CME) How actin assembly forces are harnessed has not been fully revealed.
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.