Asymmetry of epithelial cells is necessary for proper organ function in metazoans and for impeding aberrant epithelial-mesenchymal transition. Epithelial cell membranes are divided into two domains, apical and basolateral, which are demarcated by tight junctions (TJs). These electron dense structures encircle the cell and connect the apices of opposing cells. TJs are thought to maintain cell polarity by limiting the lateral diffusion of domain-specific components, such as the apical, heavily glycosylated mucins. However, the regulation and mechanism of the TJ complex as a barrier remains obscure. This proposal will determine how physical constraints on TJ proteins can drive spatial organization essential for maintaining cell polarity. Transmembrane TJ proteins, e.g. claudin-1, are linked to the actin cytoskeleton through cytosolic TJ proteins, e.g. ZO-1. Recent work has suggested that actin networks and protein size are critical determinants for membrane asymmetry, and as such, we propose that TJs are mechanically regulated. We will examine the effect of force on TJ's ability to prevent translocation of domain-specific phosphatidylinositol phosphates using a patterned cantilever and a custom-built atomic force microscope with a side-view optical imaging system. We will also test whether TJs segregate proteins based on a size-dependent sorting mechanism. This work will provide new insight into how TJs are able to maintain cell polarity and how dysregulation of TJs leads to disease, particularly cancer.
Ninety percent of cancers originate from one particular type of cell, an epithelial cell. Epithelial cells in healthy tissue have a unique property, they are asymmetric, but this cell type is known to lose this property during tumor progression. The proposed work will determine the key molecules and physical constraints that drive asymmetry in healthy epithelial cells and that cause normal cells to turn cancerous, insights which will ultimately provide new avenues and targets for drug development.
