title: Cytoskeletal confinement of CX3CL1 limits its susceptibility to proteolytic cleavage by ADAM10.
abstract: CX3CL1 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 CX3CL1 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 CX3CL1. We observed that the majority of cell surface CX3CL1 diffused within restricted confinement regions structured by the cortical actin cytoskeleton. These confinement regions sequestered CX3CL1 from ADAM10, precluding their association. Disruption of the actin cytoskeleton reduced CX3CL1 confinement and increased CX3CL1-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.
authors.affiliation: Program in Cell Biology, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada Institute of Medical Science, University of Toronto, Toronto, ON M5S 1A8, Canada.
authors.forename: Harikesh S
authors.initials: HS
authors.lastname: Wong
authors.affiliation: Program in Cell Biology, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada.
authors.forename: Valentin
authors.initials: V
authors.lastname: Jaumouillé
authors.affiliation: Department of Microbiology and Immunology, University of Western Ontario, London, ON N6A 5C1, Canada.
authors.forename: Bryan
authors.initials: B
authors.lastname: Heit
authors.affiliation: Program in Cell Biology, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada.
authors.forename: Sasha A
authors.initials: SA
authors.lastname: Doodnauth
authors.affiliation: Program in Cell Biology, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada.
authors.forename: Sajedabanu
authors.initials: S
authors.lastname: Patel
authors.affiliation: Program in Cell Biology, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada.
authors.forename: Yi-Wei
authors.initials: YW
authors.lastname: Huang
authors.affiliation: Program in Cell Biology, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, ON M5B 1W8, Canada.
authors.forename: Sergio
authors.initials: S
authors.lastname: Grinstein
authors.affiliation: Program in Cell Biology, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada Institute of Medical Science, University of Toronto, Toronto, ON M5S 1A8, Canada Department of Paediatrics, University of Toronto, Toronto, ON M5S 2J7, Canada lisa.robinson@sickkids.ca.
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Robinson
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism*; D000072197:ADAM10 Protein; D008841:Actin Cytoskeleton / Q000378:metabolism*; D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D054428:Chemokine CX3CL1 / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D059748:Proteolysis; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014743:Videotape Recording
chemical_list: D054428:Chemokine CX3CL1; D008164:Luminescent Proteins; D008565:Membrane Proteins; D014409:Tumor Necrosis Factor-alpha; D053829:Amyloid Precursor Protein Secretases; D051722:ADAM Proteins; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human