Characterization of regulation and functions of tricellulin in vascular endothelial cells

背景：血液通过一层称为内皮细胞的特殊细胞层与组织分离。这些细胞形成一个保护屏障，并调节物质通过这一层的运输，这对健康的血液流动和器官功能至关重要。内皮细胞还控制白细胞何时以及如何到达组织，这一过程是防止感染和受损组织愈合的关键。内皮层的中心结构成分是细胞-细胞接触。在单个细胞相互接触的地方，大的组装体或蛋白质完成各种功能。例如，连接处的蛋白质通过形成屏障来防止细胞间的血液渗漏。连接的一个特殊版本位于3个细胞聚集的地方（三细胞接触点）。三胞蛋白是一种定位于三细胞接触处的蛋白质。这种蛋白质的作用开始在其他细胞类型中出现。我们发现三胞蛋白存在于内皮细胞中，但对其在这些细胞中的作用几乎一无所知。该计划的总体目标是确定三胞蛋白在内皮细胞中的功能，并确定其调节模式。方法：我们将使用源自大脑和心脏的培养内皮细胞。为了确定三胞蛋白的功能，在第一个项目中，我们将使用遗传技术从细胞中消除蛋白质或增加其水平。我们将重点关注两个可能的功能。首先，我们将探讨三胞蛋白是否调节细胞膜的渗漏。我们将使用先前建立的技术来跟踪层的紧密性，并测量有多少标记分子可以通过层。其次，我们将确定三胞蛋白是否控制白细胞穿过内皮细胞的运动。为此，我们将使用标记的白细胞，并测量有多少白细胞能够穿过内皮层。在第二个项目中，我们将询问如何控制三胞蛋白的水平，并探索由已知影响内皮功能的各种药物引起的变化。我们将使用生化方法来测量这些药物刺激后的三胞蛋白水平。我们还将讨论三胞蛋白周转控制的基本方面。接下来，我们将采用各种最先进的显微镜方法来确定蛋白质在细胞中的定位，并探索这在刺激下是如何改变的。最后，我们将使用遗传方法和药理学抑制剂来操纵拟议的调节剂。意义：三胞蛋白正逐渐成为内皮功能的重要调节因子。这个项目将使人们对它的作用和调控有了新的认识，并将为研究三胞蛋白如何影响疾病奠定基础。

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.