Relating phosphoinositide 3-kinase (PI3K) signaling and cell motility dynamics during PDGF-stimulated chemotaxis

Abstract

Cell migration is essential for wound healing, the immune response, embryogenesis, and cancer metastasis. Chemotaxis, or cell migration directed by an external gradient of chemoattractant, is encountered in various physiological and natural settings and is a means by which cellular processes are coordinated in space and time. This cyclical process of protrusion, adhesion, and retraction is characterized by an asymmetric polarization of 3' phosphoinositides at the leading edge of a migrating cell and is common across multiple cell types. Activated by the phosphoinositide 3-kinase (PI3K) pathway, these 3' PIs act as membrane-bound second messengers which recruit additional signaling proteins, such as the Rho family GTPases, to initiate actin polymerization and membrane protrusion. These protrusions are capable of both cellular translocation as well as gradient sensing. The characteristics of chemotaxis depend on cell type. Amoeboid cells such as neutrophils and the slime mold, Dictyostelium discoideum, respond to shallow gradients of chemoattractants (~1% difference in concentration across cell dimensions) and migrate rapidly (~20 µm/min). Mesenchymal cells such as fibroblasts, on the other hand, require much steeper chemoattractant gradients to evoke similar polarization of PI3K signaling, but the sensitivity of chemotactic movement has yet to be quantitatively characterized. Using a 3' PI-specific, fluorescent biosensor and total internal reflection fluorescence (TIRF) microscopy, the spatial and temporal dynamics of PI3K signaling were monitored during chemotaxis of fibroblasts responding to gradients of the chemoattractant platelet-derived growth factor (PDGF). We show that the asymmetry of PI3K signaling positively correlates with cell directionality during chemotaxis and, consistent with previous work, that highly persistent movement aligned with the external gradient requires a steep gradient (> 10%) in PDGF receptor binding across the cell. Additionally, we present the development of novel microfluidic devices that generate linear gradients of chemoattractant and their integration with TIRF microscopy. In one particular device, fibroblasts exhibited similar characteristics of PI3K signaling asymmetry as compared with other methods. Finally, we evaluate the long-term viability of fibroblasts and methods to minimize toxicity in these devices.