Characterization of regulation and functions of tricellulin in vascular endothelial cells

背景：血液与组织之间有一层特殊的细胞层，称为内皮细胞。这些细胞创建一种保护性屏障，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。内皮细胞还控制着白细胞何时以及如何到达组织，这一过程是防止感染和修复受损组织的关键。内皮层的一个中心结构组件是细胞-细胞接触。在单个细胞相互接触的地方，大的组装或蛋白质完成各种功能。例如，交界处的蛋白质通过产生屏障来防止血液在细胞之间的泄漏。连接的特殊版本位于3个细胞聚集的地方(三细胞接触部位)。三细胞蛋白是一种定位于三细胞接触的蛋白质。这种蛋白质的作用开始出现在其他类型的细胞中。我们发现三细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。*本项目的总体目标是确定三细胞蛋白在内皮细胞中的功能，并确定其调节模式。*方法：我们将使用来源于大脑和心脏的培养内皮细胞。为了确定毛细胞蛋白的功能，在第一个项目中，我们将使用基因技术来消除细胞中的蛋白质或提高其水平。我们将重点介绍两种可能的功能。首先，我们将探索三纤维素是否调节蛋鸡的渗漏。我们将使用以前建立的技术来跟踪这一层的紧密程度，并测量有多少标记的分子可以通过这一层。其次，我们将确定三毛细胞蛋白是否控制白细胞在内皮细胞之间的移动。为此，我们将使用标记的白细胞，并测量其中有多少能够穿越内皮层。在第二个项目中，我们将询问三纤维素水平是如何控制的，并探索已知的影响内皮功能的各种因素引起的变化。我们将使用生化方法来测量在这些试剂刺激后的三纤维素水平。我们还将讨论三纤维素周转控制的基本方面。接下来，我们将使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，并探索这种定位在刺激下是如何改变的。最后，我们将使用遗传方法和药理抑制剂来操纵拟议的调节器。*意义：毛细胞蛋白正在成为内皮功能的重要调节因子。这一计划将使人们对其作用和调控有新的认识，并为研究毛细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. *****