Peebles, K. E. | bioRxiv Science

bioRxiv2024

Apical clathrin-coated endocytic pits control the growth and size of epithelial microvilli

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 cells 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 nascent microvilli are in contact with an apical endocytic pit. Finally, 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. Based on these discoveries, we conclude that clathrin-mediated endocytosis and its associated Arp2/3-based actin nucleation activity control the timing and location of microvillar growth, as well as the dimensions of the resulting protrusions.

cell biology↗

bioRxiv2023

Analysis of Drosophila and mouse mutants reveals that Peroxidasin is required for tissue mechanics and full viability

Basement membranes are thin strong sheets of extracellular matrix. They provide mechanical and biochemical support to epithelia, muscles, nerves, and blood vessels, among other tissues. The mechanical properties of basement membranes are conferred in part by Collagen IV (Col4), an abundant protein of basement membrane that forms an extensive two-dimensional network through head-to-head and tail-to-tail interactions. After the Col4 network is assembled into a basement membrane, it is crosslinked by the matrix-resident enzyme Peroxidasin to form a large covalent polymer. Peroxidasin and Col4 crosslinking are highly conserved, indicating they are essential, but homozygous mutant mice have mild phenotypes. To explore the role of Peroxidasin, we analyzed mutants in Drosophila, including a newly generated catalytic null, and found that homozygotes were mostly lethal with 13% viable escapers. A Mendelian analysis of mouse mutants shows a similar pattern, with homozygotes displaying [~]50% lethality and [~]50% escapers. Despite the strong mutations, the homozygous escapers had low but detectable levels of Col4 crosslinking, indicating that inefficient alternative mechanisms exist and that are probably responsible for the viable escapers. Further, fly mutants have phenotypes consistent with a decrease in stiffness. Interestingly, we found that even after adult basement membranes are assembled and crosslinked, Peroxidasin is still required to maintain stiffness. These results suggest that Peroxidasin crosslinking may be more important than previously appreciated.

cell biology↗