Metalloprotease-Substrate Interactions in Endothelial Cells

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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.