Engineering Analysis of Spatial Gradient Sensing in Platelet-derived Growth Factor-stimulated Fibroblasts

Abstract

Wound healing is a well-coordinated process in which different cell types invade the wound at different times, secreting different enzymes and factors. An important event in this process is the proliferation and directed migration of dermal fibroblasts. Evidence has shown that gradients of platelet-derived growth factor (PDGF) mediate the process of directed cell migration in fibroblasts. These cells use membrane lipids, specifically 3' phosphoinositides (PIs), to spatially sense the extracellular PDGF gradient by creating an intracellular gradient of 3' PIs. PDGF receptor stimulates 3' PI production by activating phosphoinositide 3-kinase (PI 3-kinase), a lipid kinase. However, the mechanism by which the cell generates intracellular 3' PI gradients is not well known in fibroblasts.

A 3' PI specific binding domain fused to a fluorescent protein can be used as a 3' PI probe and can be imaged quantitatively using total internal reflection fluorescence (TIRF) microscopy. This technique allows for the selective illumination of the adherent portion of the cell membrane, providing a powerful technique to visualize protein translocation to the membrane in live cells. A mathematical model has been developed to quantitatively analyze the fluorescence data and we show here that these data are sufficient to set bounds on parameters that determine 3' PI production, diffusion and degradation. Under uniform PDGF stimulation we also find areas of differentially controlled 3' PI production, diffusion and degradation that can be linked to the specific morphology of the cell. This indicates that cell polarization may play an important role in PI 3-kinase signaling. Finally, we find that fibroblasts sense gradients in a manner distinct from other more common chemotactic models such as Dictyostelium discoideum and neutrophils. Fibroblasts are only sensitive to large relative PDGF gradients and intermediate PDGF concentrations. Consequently, the ability of these cells to spatially sense relies on the competition between the intrinsic bias caused by cell polarization and the external bias caused by the PDGF gradient, each of which may have different contributions depending on the extracellular gradient condition