Metalloprotease-Substrate Interactions in Endothelial Cells

在脊椎动物中，白细胞通过抵御细菌、真菌和病毒感染，在免疫系统中发挥着重要作用。白血球在体内巡逻，寻找感染的迹象。这种免疫监测涉及白细胞和血管内壁细胞之间的相互作用。白细胞通过各种信号被招募到损伤区域，特别是被称为趋化因子的分子。在趋化因子家族中，有一个成员CX3CL1被锚定在血管衬里细胞的表面，CX3CL1既能吸引白细胞，又能将它们物理地附着在血管上。*CX3CL1被ADAM17切割后从血管细胞表面释放出来，ADAM17是一种分子剪刀。然而，ADAM17与CX3CL1的接触和切割方式尚不清楚。我们的初步工作表明，CX3CL1通常被限制在沿着血管细胞表面的栅栏内，并且它与ADAM17分离。使用最先进的显微镜技术，我们将检查细胞表面的CX3CL1是如何被限制的。具体地说，我们将确定CX3CL1是否被拴在维持细胞形状的内部骨架上，或者分子是否分泌在细胞外的陷阱CX3CL1内。我们还将研究ADAM17是在细胞表面还是在细胞内的单独隔室中切割CX3CL1，以及ADAM17是如何获得CX3CL1的。最后，我们将确定CX3CL1的释放如何影响血管细胞的屏障完整性，以及它们招募白细胞的能力。通过了解分子细节，我们的研究将阐明脊椎动物中保守的免疫细胞巡逻的基本过程。我们的工作将进一步阐明ADAM17分子剪刀如何在不同的物理条件下选择性地切割不同类型的分子。

In vertebrate animals, white blood cells play an important role in the immune system by protecting against bacterial, fungal, and viral infections. White blood cells patrol the body looking for signs of infection. This immune surveillance involves interactions between white cells and the cells lining blood vessels. White cells are recruited to areas of injury by various signals, especially molecules called chemokines. Among the family of chemokines, one member, CX3CL1, is anchored to the surface of cells lining blood vessels, and CX3CL1 both attracts white cells and physically attaches them to blood vessels.*******CX3CL1 is released from the surface of blood vessel cells after being cut by ADAM17, an enzyme that acts as molecular scissors. However, the way in which ADAM17 makes contact with and cuts CX3CL1 is not well understood. Our preliminary work suggests that CX3CL1 is normally restricted within fenced corrals along the surface of blood vessel cells, and that it is segregated from ADAM17. Using state-of-the-art microscopy techniques, we will examine how CX3CL1 at the surface of the cell is confined. Specifically, we will determine whether CX3CL1 is tethered to the internal skeleton that maintains the shape of the cell, or whether molecules secreted outside the cell trap CX3CL1 within their mesh. We will also examine whether ADAM17 cuts CX3CL1 at the cell surface or in a separate compartment inside the cell, and how ADAM17 gains access to CX3CL1. Lastly, we will determine how release of CX3CL1 affects the barrier integrity of blood vessel cells, and their ability to recruit white blood cells. By understanding the molecular details, our studies will shed light on a fundamental process of immune cell patrolling conserved among vertebrate animals. Our work will further clarify the way in which the ADAM17 molecular scissors can selectively cut different types of molecules under different physical conditions.************