The Molecular Mechanisms of Gradient Sensing by CXCR4

The chemokine receptor CXCR4 is essential for many physiological processes, including stem cell homing, organogenesis, inflammation and tissue repair. Expression of CXCR4 in malignant cells may influence the biology of cancer, in modulating tumor survival, growth and angiogenesis; CXCR4 plays an important role in directing metastatic CXCR4-expressing tumor cells to organs that express CXCL12. Directional cell migration towards increasing concentration of the chemokine ligand CXCL12 is result of Gαi and Gαq pathways activation. These biochemical pathways are well studied, however the mechanism of gradient sensing is not yet understood.In this study we use Ewing's sarcoma derived cells to unravel the processes that are involved in CXCR4 related metastasis formation. Cells are transformed with CXCR4-eYFP enabling use of single-molecule imaging to access diffusive dynamics of CXCR4 in the plasma membrane under different conditions. We find that receptor diffusion is not homogeneous and is present as both mobile and immobile (∼20%) receptors. Mobile receptors show free diffusion with diffusion coefficient D=0.19±0.01μm2/s. Stimulation with CXCL12, does not change receptor mobility, however it leads to changes in the ratio of mobile and immobile receptor fractions in a concentration dependent manner. Further experiments show that actin cytoskeleton depolymerization causes an unexpected drop in mobile receptor fraction. Notably, this effect is stimulation independent. Endocytosis inhibition results in an extra receptor fraction, which shows confined diffusion and significantly increases upon stimulation. Interestingly, G-proteins and dimerization do not influence diffusive behavior of CXCR4 in the plasma membrane. These findings together with whole cell migration results highlight new molecular insights into chemokine gradient recognition.