Assessment of the recycling of the membrane-bound chemokine, CX₃CL1.

Fractalkine (CX(3)CL1) is a membrane-anchored chemokine whose N-terminus contains a unique CX(3)C motif that is cleaved and released. The membrane-bound form functions as an adhesion molecule and the secreted form as a chemotactic factor. Like other chemokines, CX(3)CL1 is regulated at the levels of transcription and translation. Recent evidence points to additional functional regulation by cellular trafficking owing to the unique transmembrane structure. CX(3)CL1 is the only chemokine known to undergo constitutive internalization. To understand mechanisms governing the regulation and processing of such membrane-bound proteins, it is vital to study their subcellular distribution and transport. The methods outlined in this chapter describe (1) transfection of mammalian cells with plasmids encoding the expression of green fluorescent protein-tagged CX(3)CL1; (2) immunofluorescence antibody labeling as well as fluorescence recovery after photobleaching to study internalization of CX(3)CL1 by endocytosis; and (3) acid-stripping assays to study the recycling of internalized CX(3)CL1 back to the plasma membrane. Together, these methods allow for the examination of subcellular distribution and traffic of recycling membrane proteins.