Enterocytes optimize their morphology for solute uptake from the intestinal lumen by building an apical specialization: a dense array of actin bundle-supported microvilli, collectively referred to as the ?brush border?. By scaffolding apical membrane, brush border microvilli significantly increase the capacity for housing membrane-bound transporters and channels that drive solute transport. In the cytoplasm immediately beneath the apical surface, the rootlets of microvillar core actin bundles are anchored in a meshwork of filaments known as the ?terminal web? first visualized in classic ultrastructural studies decades ago. Rich in intermediate and actin filaments, this network is dense enough to exclude microtubules, vesicles and other large organelles. Although the terminal web is well-positioned to regulate a range of critical subcellular activities, the function and composition of this domain and its contribution to intestinal physiology remain unclear. In exciting preliminary studies, we identified non-muscle myosin-2C (MYO2C) as a component of the enterocyte terminal web. MYO2 molecules consist of an N-terminal motor domain that binds actin and generates force, and a C-terminal rod-like tail, which drives the formation of contractile filaments in cells. Among the three non-muscle myosin-2 isoforms (2A,2B,2C), MYO2C is unique in that its expression is largely specific to the intestinal epithelium. Additionally, single cell RNAseq analysis indicates that MYO2C demonstrates clear enrichment in enterocytes relative to other epithelial cell types in the gut. We found that MYO2C is highly enriched at the base of the brush border in villus enterocytes from mouse and human small intestine, and in cultured intestinal epithelial cell lines. Super- resolution microscopy revealed that MYO2C forms an extensive network of puncta in the plane of the terminal web, which spatially overlaps with the rootlets of microvillar actin bundles. Small molecule and genetic perturbation studies in cultured epithelial cells leads to dramatic elongation of microvilli, suggesting these MYO2C may promote actin disassembly from rootlet pointed ends. Finally, we found that MYO2C KO mice have defects not only in microvillar organization, but also in enterocyte- and villus-scale tissue structure. Based on these findings, we hypothesize that MYO2C forms a terminal web contractile network that controls brush border actin architecture and propagates tissue-scale mechanical forces to enable efficient collective cell migration up the crypt-villus axis. To test this hypothesis, we will employ a combination of state-of-the-art super-resolution microscopy, advanced forms of electron microscopy, and lattice light sheet live imaging to:
(Aim 1) map the organization of the terminal web MYO2C network, (Aim 2) investigate the mechanism of MYO2C-dependent microvillar length regulation, and (Aim 3) define the function of MYO2C in the terminal web in vivo. These studies will lead to new paradigms for understanding the fundamental mechanisms that control the morphology of enterocytes and the epithelial tissue they comprise.
As the most abundant cell type in the intestinal epithelium, enterocytes build specialized structures known as ?brush borders? on their surface to enable nutrient uptake from the gut lumen. Despite occupying a critical physiological interface, our understanding on how enterocytes assemble and organize brush borders, or how they coordinate their function at the tissue-scale remains unclear. This proposal will investigate mechanisms underlying the assembly and organization of apical brush borders, and on a larger scale the nutrient absorbing luminal surface of the gut; this work will generate insight on processes underlying normal gut function as well as human diseases characterized by perturbation to enterocyte structure and function.