Characterization of regulation and functions of tricellulin in vascular endothelial cells

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Background: Blood is separated from tissues by a specialized layer of cells, called endothelial cells. These cells create a protective barrier and regulate transport of substances through the layer, which is vital for healthy blood flow and organ functions. Endothelial cells also control when and how white blood cells can reach the tissues, and this process is key for protection against infection and healing of damaged tissues. A central structural component of the endothelial layer is the cell-cell contacts. At sites where individual cells make contact with each other, large assembly or proteins fulfil a variety of functions. For example, proteins in the junctions prevent leakage of blood between the cells by generating a barrier. A specialized version of junctions is located at the places where 3 cells come together (tricellular contact sites). Tricellulin is a protein that was found to localize at the tricellular contacts. The role of this protein is starting to emerge in other cell types. We found that tricellulin is present in endothelial cells, but almost nothing is known about its role in these cells. The overall objective of this program is to define the function of tricellulin in endothelial cells, and to determine the modes of its regulation. Approach: We will use cultured endothelial cells originating form the brain and the heart. To define tricellulin functions, in the first project we will use genetic techniques to either eliminate the protein from cells or to increase its levels. We will focus on two possible functions. First, we will explore if tricellulin regulates leakiness of the layer. We will use previously established techniques to follow the tightness of the layer, and to measure how much of labelled molecules can get through the layer. Second, we will determine whether tricellulin controls the movement of white blood cells across the endothelial cells. For this, we will use labelled white blood cells and measure how many of these were able to cross the endothelial layer. In the second project, we will ask how the levels of tricellulin are controlled, and explore changes induced by various agents that are known to affect endothelial functions. We will use biochemical methods to measure levels of tricellulin following stimulation with these agents. We will also address fundamental aspects of tricellulin turnover control. Next, we will employ a variety of state-of-the-art microscopy methods to determine localization of the protein in the cells and to explore how this is altered upon stimulation. Finally, we will manipulate proposed regulators using genetic approaches and pharmacological inhibitors. Significance: Tricellulin is emerging as an important regulator of endothelial functions. This program will allow fundamental new insights into its role and regulation and will lay the groundwork for studies into how tricellulin affects disease.