| dc.description.abstract | Microvilli are actin-bundle-supported surface protrusions that play essential roles in diverse epithelial functions, from nutrient absorption to mechanosensation. Despite the importance of microvilli for proper epithelial cell function, the mechanism by which cells build microvilli and the proteins involved in this process are not well understood. Classic electron micrographs indicate the presence of an electron dense protein complex at the distal tips of microvilli, which has been long hypothesized to coordinate protrusion growth due to its proximity to the fast-growing ends of actin filaments. Indeed, time-lapse data revealed that specific factors, including epidermal growth factor pathway substrate 8 (EPS8) and insulin-receptor tyrosine kinase substrate (IRTKS) (also known as BAIAP2L1), appear in diffraction-limited puncta at the cell surface and mark future sites of microvillus growth. New core actin bundles elongate from these puncta in parallel with the arrival of ezrin and subsequent plasma membrane encapsulation. In addition to de novo growth, we also observed that new microvilli emerge from pre-existing protrusions. Moreover, we found that nascent microvilli can also undergo collapse, resulting in disassembly of the core actin bundle itself. To further develop our understanding of microvilli biogenesis, we used a biotin proximity labeling approach to probe for new molecules involved in microvillus growth, using EPS8 as bait. Mass spectrometry of biotinylated hits identified a previously uncharacterized proximal protein, KIAA1671. In silico domain analysis indicates this protein is a large (~200 kDa), primarily disordered protein that contains few recognized domains. We find that KIAA1671 localizes to the base of the brush border in native intestinal tissue and polarized epithelial cell culture models, and to actin positive structures in non-polarized cell types. Furthermore, we find that KIAA1671 co-accumulates with EPS8 at sites of microvillus growth, suggesting that KIAA1671 is involved in the growth of microvilli. Thus, using biotin proximity labeling in conjunction with live cell imaging, we have identified a novel factor that targets to the apical surface of epithelial cells and may be involved in microvilli morphogenesis. These studies are the first to offer a temporally resolved microvillus growth mechanism and highlight factors that participate in this process; they also provide important insights on the growth of apical specializations that will likely apply to diverse epithelial contexts. | |
