Building epithelial cytoarchitecture:regulation of actin dynamics by cell-cell junctions

Cell-cell adhesion and migration are key events in the life of many different cell types, and are essential for the organization of cells into higher ordered structures such as blood vessels, lung, skin, muscle, etc. Epithelial cells differentiate and form sheets of tightly packed cells: these sheets form protective layers wrapping up the body (skin) and the inside of internal cavities (i.e. stomach, intestines, etc). The choice of whether to migrate or stick to their neighbours has implications for epithelial function in health and disease. Thus, it is important to understand how a cell is kept inside an epithelial sheet or detaches and become shapeless, homeless and aimless. Epithelial cells can not function properly in the absence of tight cell-cell adhesion and a cuboidal morphology. We are interested in understanding how cells glue to their neighbours in order to regulate their shape and differentiation. We aim at identifying the molecules that participate in these processes and investigate their role in epithelia. Insights into how epithelial cell shape is established and maintained will provide clues on how to ameliorate diseases that have compromised epithelial function.

The acquisition of a cuboidal morphology (polarization) is a hallmark of epithelia and occurs after cell division, wound closure, and during morphogenesis. Extensive reorganization of epithelial actin cytoskeleton is triggered following assembly of cadherin-dependent cell-cell adhesion. This cytoskeleton remodelling is essential for the functionality of epithelial sheets, stability of adhesive receptors at junctions and for the appropriate localization of signalling complexes. Yet, in spite of the importance of epithelia in development, homeostasis and diseases, the mechanisms via which a cuboidal morphology is acquired are poorly understood. Understanding how actin structures are assembled following cell-cell adhesion will provide major insights into the relevant signalling pathways for polarization. We have found that when cell-cell contacts are induced in keratinocytes, an increase in actin polymerization and myosin function (contractility) is observed very quickly. These processes are coordinated spatially and temporally into two distinct actin populations: junctional-actin and thin bundles. Here we aim to identify specific cytoskeletal proteins that are modulated by cell-cell adhesion and participate in the formation and/or maintenance of junctional-actin and thin bundles. As strategy, we will deplete the protein levels of specific proteins using siRNA during induction of cell-cell contacts and perform a screening using a siRNA library of actin binding proteins. Once relevant proteins are identified, we will perform functional assays to determine in which step of epithelial polarization these actin binding proteins operate: stabilization of cell-cell contacts or cell height increase (which reflects the functions of junctional-actin and thin bundles). We will use a combination of dynamic studies (live imaging), biochemical, biophysical and cell biology experiments together with rescue analysis of the specific phenotypes induced by siRNA depletion.