CXCR4-SDF1 Mediated Chemotaxis - from Tissue to the Single-Molecule Level

Abstract

Directed cell movement in a chemical gradient, chemotaxis, is not only a prerequisite for many vital processes like e.g. the immune response, but also the basis for cancer spreading in metastasis. Chemotaxis is governed by extracellular gradients of small molecules, the chemokines. The G protein-coupled receptor CXCR4 and its chemokine SDF1α play a crucial role in directing migration of tumor cells to neighbouring tissue as well as in metastasis to distant sites in the body via newly formed blood vessels (angiogenesis).We investigated CXCR4- SDF1α mediated chemotaxis in mouse fibroblasts in an integrated approach from the tissue to the single-molecule level. First, we characterized cellular migratory potential upon stimulation with SDF1α in wound healing assays applying phase contrast microscopy. We find that transiently transfected cells expressing CXCR4 double their migration speed in comparison to wild type 3T3 cells. Second, we applied single-molecule fluorescence microscopy to study the mobility of the G protein-coupled receptor CXCR4-eYFP in resting cells and upon stimulation with SDF1α. Two fractions of receptors prior to stimulation were identified: half of the receptors were immobile while the other half exhibited free diffusion with D ∼ 0.3 µm2/s on short timescales (up to 100 ms). At longer timescales receptors showed confined diffusion within micrometer domains. Global stimulation with SDF1α switched a subset of the receptors from the immobile to the mobile fraction. We predict that the impact of a SDF1α gradient might lead to asymmetric receptor diffusion and subsequently polarized cell behaviour as seen in the wound healing assays.