Mechanisms of Epithelial Cell Behavior and Polarity Establishment

Vanderbilt University, Nashville TN

Investigators

Abstract

Project Summary Polarized epithelial cells are the foundational cell type of many organs, and disruptions in polarity and epithelial organization contribute to diseases of kidney, liver, skin, and intestine. Furthermore, loss of apical/basal polarity is an early event in many human cancers. Therefore, a deep knowledge of epithelial morphogenesis is crucial to understanding the development both of normal tissues and of many human diseases. This proposal builds on our previous NIGMS-funded accomplishments over many years in understanding the interactions, sorting, and delivery of polarity proteins, as well as deciphering behaviors of epithelial cells during mammary gland morphogenesis, and of epithelial cell behaviors during human embryonic stem cell differentiation. The overarching vision of this research program is to enhance our understanding of epithelial cell polarization and behaviors in tissue morphogenesis. We will probe the molecular mechanisms of apical intercalation and apical membrane protein sorting, using mammary epithelial cells in vitro as a model. Intraductal injection of genetically manipulated primary cells will provide in vivo validation. To gain insight into the extensive re-organization of intercellular junctions and polarity proteins during apical intercalation, which is an understudied process, we will employ soft matter physics and computational modeling, advanced live cell imaging, and super-resolution visualization. Additionally, we will use cells in vitro plus a new mouse model we developed to analyze the role of actomyosin contractility in both the intercalating cells and the monolayer into which they integrate, to distinguish competing mechanisms. We will also use our new mouse model to suppress contractility specifically in myoepithelial cells and fibroblasts of the mammary gland, to determine whether the shaping of mammary ducts during luminal cell intercalation int the pubertal terminal end buds is driven by physical constraints on isotropic expansion. Finally, we will investigate the mechanism of apical membrane protein sorting, based on our unanticipated discovery of a size filter at the Golgi for proteins with small cytoplasmic domains. Synthetic biology approaches, endogenous tagging, and multiplexed super-resolution fluorescence imaging will be deployed to identify locations within the Golgi where membrane proteins with small cytoplasmic domains become segregated from those with large ones. We will also analyze sorting dynamics of selected apical membrane proteins with intrinsically large cytoplasmic domains and those with multiple transmembrane domains, to determine how they circumvent this filter. Our research program will provide valuable insights into fundamental processes underlying cell organization and contribute to understanding human diseases.