Spatial analysis of 3′ phosphoinositide signaling in living fibroblasts, III:: Influence of cell morphology and morphological polarity

Activation of phosphoinositide ( PI) 3-kinase is a required signaling pathway in. broblast migration directed by platelet-derived growth factor. The pattern of 39 PI lipids in the plasma membrane, integrating local PI 3-kinase activity as well as 39 PI diffusion and turnover, influences the spatiotemporal regulation of the cytoskeleton. In fibroblasts stimulated uniformly with platelet- derived growth factor, visualized using total internal reflection fluorescence microscopy, we consistently observed localized regions with significantly higher or lower 39 PI levels than adjacent regions ( hot and cold spots, respectively). A typical cell contained multiple hot spots, coinciding with apparent leading edge structures, and at most one cold spot at the rear. Using a framework for finite-element modeling with actual cell contact area geometries, we find that although the 39 PI pattern is affected by irregular contact area shape, cell morphology alone cannot explain the presence of hot or cold spots. Our results and analysis instead suggest that these regions reflect different local 39 PI dynamics, specifically through a combination of mechanisms: enhanced PI 3-kinase activity, reduced 39 PI turnover, and possibly slow/constrained 39 PI diffusion. The morphological polarity of the cell may thus bias 39 PI signaling to promote persistent migration in fibroblasts.