Investigating the role of clathrin-mediated endocytosis in epithelial microvilli biogenesis

Abstract

Epithelial cells from diverse contexts assemble apical specializations to serve tissue-specific functions. In virtually all cases, these features consist of arrays of microvilli: micron-scale, actin bundle-supported protrusions that mediate biochemical and physical interactions with the external environment. Despite their importance for epithelial physiology, how microvilli grow during cellular differentiation remains poorly understood. Using genetic and small molecule perturbations, we found that an epithelial cell’s potential for growing microvilli of normal size is limited by an adjacent actin-dependent process: apical clathrin-mediated endocytosis. Unexpectedly, timelapse imaging of individual microvillar growth events revealed tight spatial and temporal coupling to sites of clathrin-mediated endocytosis. Ultrastructural characterization of undifferentiated epithelial monolayers also showed that most apical endocytic pits are in contact with a nascent microvillus. Inhibition of the Arp2/3 branched nucleation complex, which drives actin polymerization on coated pits, significantly reduced the accumulation of new microvilli on the surface of differentiating epithelial cells. Finally, super-resolution microscopy further revealed that microvilli originate from asymmetric, branched actin networks surrounding clathrin-coated pits that are marked by the F-BAR protein CIP4. Based on these discoveries, we conclude that a subset of clathrin-coated pits, and their associated Arp2/3-generated actin networks, control the timing and location of microvillar growth, as well as the dimensions of the resulting protrusions.