In fibroblasts responding to gradients of platelet-derived growth factor (PDGF), an important chemoattractant in development and wound healing, signaling through the phospholipase C (PLC)/protein kinase C (PKC) pathway proved necessary for chemotaxis, whereas pathways that collaborate to activate the Arp2/3 complex were found to be dispensable.  PKC is activated through its binding to the lipid second messenger diacylglycerol (DAG), which is formed from hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP2) by PLC.  Strikingly, in fibroblasts exposed to a shallow PDGF gradient, the density of DAG in the plasma membrane is focally enriched at the up-gradient leading edge, characteristic of an internal amplification mechanism.  In previous work, we developed a reaction-diffusion model of the PLC/PKC signaling pathway and implicated phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) by membrane-localized PKC as a positive feedback mechanism sufficient for local amplification of DAG and active PKC.  However, by itself, the MARCKS feedback only weakly amplifies the signal in shallow PDGF gradients.

Our new model includes phosphatidic acid (PA), a lipid intermediate in the metabolism of DAG.  It has been shown that PA binds PLC and that active PKC can enhance the activity of phospholipase D (another enzyme that produces PA), implicating additional feedback loops.  Model simulations show that the MARCKS feedback mechanism synergizes with these new feedback loops for increased amplification even at shallow PDGF gradients and over an appreciable range of midpoint PDGF concentrations.  Simulations with variations of parameter values or cell geometry further indicate that this signaling network is a highly sensitive and robust gradient sensing circuit. We are currently integrating this model with models describing the organization of the actin cytoskeleton and directionality of cell migration for a more comprehensive understanding of how fibroblast chemotaxis proceeds during physiological processes such as wound healing.