Spatial sensing in fibroblasts mediated by 3' phosphoinositides.
The directed movement of fibroblasts towards locally released platelet-derived growth factor (PDGF) is a critical event in wound healing. Although recent studies have implicated polarized activation of phosphoinositide (PI) 3-kinase in G protein-mediated chemotaxis, the role of 3' PI lipids in tyrosine kinase-triggered chemotaxis is not well understood. Using evanescent wave microscopy and green fluorescent protein-tagged Akt pleckstrin homology domain (GFP-AktPH) as a molecular sensor, we show that application of a shallow PDGF gradient triggers a markedly steeper gradient in 3' PI lipids in the adhesion zone of fibroblasts. Polar GFP-AktPH gradients, as well as a new type of radial gradient, were measured from front to rear and from the periphery to the center of the adhesion zone, respectively. A strong spatial correlation between polarized 3' PI production and rapid membrane spreading implicates 3' PI lipids as a direct mediator of polarized migration. Analysis of the temporal changes of 3' PI gradients in the adhesion zone revealed a fast diffusion coefficient (0.5 microm(2)/s) and short lifetime of 3' PIs of <1 min. Together, this study suggests that the tyrosine kinase-coupled directional movement of fibroblasts and their radial membrane activity are controlled by local generation and rapid degradation of 3' PI second messengers.
Haugh
J M
JM
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Codazzi
F
F
Teruel
M
M
Meyer
T
T
D016475:3T3 Cells; D000818:Animals; D002448:Cell Adhesion; D002633:Chemotaxis / Q000502:physiology*; D005347:Fibroblasts / Q000502:physiology*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008962:Models, Theoretical; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000378:metabolism*; D021381:Protein Transport; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D015290:Second Messenger Systems; D014945:Wound Healing / Q000502:physiology
D008164:Luminescent Proteins; D010716:Phosphatidylinositols; D010982:Platelet-Derived Growth Factor; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019869:Phosphatidylinositol 3-Kinases; D020794:Receptor Protein-Tyrosine Kinases