Asymmetric branched F-actin networks at apical clathrin coated pits support microvillar biogenesis

Microvilli are conserved actin-based protrusions that expand surface area and absorptive capacity in epithelial tissues. In humans, the intestinal tract grows three-hundred trillion microvilli per day, yet the molecular mechanisms defining where and when microvilli grow remain unclear. Using a combination of live-cell confocal and super-resolution N-SPARC microscopy to investigate native intestinal tissues and epithelial cell culture models, we found that microvilli grow from Arp2/3-generated branched actin networks that form on the surface of clathrin coated pits. These transient networks are stabilized by cortactin and localize to the apical plasma membrane minutes before reorganizing into the linear core actin bundles that support microvilli. Moreover, subpixel precision localization of coated pit and microvilli markers revealed that F-actin asymmetrically localizes to one side of a nascent clathrin coated pit, and that microvilli grow from the side with the highest F-actin density. These findings support a model where the asymmetric accumulation of F-actin and growing barbed ends on the surface of a clathrin coated pit offer a filament source to support microvilli formation. Media: see text.