Cytoskeletal confinement of CX(3)CL1 limits its susceptibility to proteolytic cleavage by ADAM10

CX(3)CL1 is a unique chemokine that acts both as a transmembrane endothelial adhesion molecule and, upon proteolytic cleavage, a soluble chemoattractant for circulating leukocytes. The constitutive release of soluble CX(3)CL1 requires the interaction of its transmembrane species with the integral membrane metalloprotease ADAM10, yet the mechanisms governing this process remain elusive. Using single-particle tracking and subdiffraction imaging, we studied how ADAM10 interacts with CX(3)CL1. We observed that the majority of cell surface CX(3)CL1 diffused within restricted confinement regions structured by the cortical actin cytoskeleton. These confinement regions sequestered CX(3)CL1 from ADAM10, precluding their association. Disruption of the actin cytoskeleton reduced CX(3)CL1 confinement and increased CX(3)CL1–ADAM10 interactions, promoting the release of soluble chemokine. Our results demonstrate a novel role for the cytoskeleton in limiting membrane protein proteolysis, thereby regulating both cell surface levels and the release of soluble ligand.