Platelet-derived Growth Factor Gradient Research Articles

Microfluidic devices fitted with "flowver" paper pumps generate steady, tunable gradients for extended observation of chemotactic cell migration.

Microfluidics approaches have gained popularity in the field of directed cell migration, enabling control of the extracellular environment and integration with live-cell microscopy; however, technical hurdles remain. Among the challenges are the stability and predictability of the environment, which are especially critical for the observation of fibroblasts and other slow-moving cells. Such experiments require several hours and are typically plagued by the introduction of bubbles and other disturbances that naturally arise in standard microfluidics protocols. Here, we report on the development of a passive pumping strategy, driven by the high capillary pressure and evaporative capacity of paper, and its application to study fibroblast chemotaxis. The paper pumps-flowvers (flow + clover)-are inexpensive, compact, and scalable, and they allow nearly bubble-free operation, with a predictable volumetric flow rate on the order of μl/min, for several hours. To demonstrate the utility of this approach, we combined the flowver pumping strategy with a Y-junction microfluidic device to generate a chemoattractant gradient landscape that is both stable (6+ h) and predictable (by finite-element modeling calculations). Integrated with fluorescence microscopy, we were able to recapitulate previous, live-cell imaging studies of fibroblast chemotaxis to platelet derived growth factor (PDGF), with an order-of-magnitude gain in throughput. The increased throughput of single-cell analysis allowed us to more precisely define PDGF gradient conditions conducive for chemotaxis; we were also able to interpret how the orientation of signaling through the phosphoinositide 3-kinase pathway affects the cells' sensing of and response to conducive gradients.

Mechanistic models of PLC/PKC signaling implicate phosphatidic acid as a key amplifier of chemotactic gradient sensing.

Chemotaxis of fibroblasts and other mesenchymal cells is critical for embryonic development and wound healing. Fibroblast chemotaxis directed by a gradient of platelet-derived growth factor (PDGF) requires signaling through the phospholipase C (PLC)/protein kinase C (PKC) pathway. Diacylglycerol (DAG), the lipid product of PLC that activates conventional PKCs, is focally enriched at the up-gradient leading edge of fibroblasts responding to a shallow gradient of PDGF, signifying polarization. To explain the underlying mechanisms, we formulated reaction-diffusion models including as many as three putative feedback loops based on known biochemistry. These include the previously analyzed mechanism of substrate-buffering by myristoylated alanine-rich C kinase substrate (MARCKS) and two newly considered feedback loops involving the lipid, phosphatidic acid (PA). DAG kinases and phospholipase D, the enzymes that produce PA, are identified as key regulators in the models. Paradoxically, increasing DAG kinase activity can enhance the robustness of DAG/active PKC polarization with respect to chemoattractant concentration while decreasing their whole-cell levels. Finally, in simulations of wound invasion, efficient collective migration is achieved with thresholds for chemotaxis matching those of polarization in the reaction-diffusion models. This multi-scale modeling framework offers testable predictions to guide further study of signal transduction and cell behavior that affect mesenchymal chemotaxis.

A Reaction-Diffusion Model Explains Amplification of the PLC/PKC Pathway in Fibroblast Chemotaxis

During the proliferative phase of cutaneous wound healing, dermal fibroblasts are recruited into the clotted wound by a concentration gradient of platelet-derived growth factor (PDGF), together with other spatial cues. Despite the importance of this chemotactic process, the mechanisms controlling the directed migration of slow-moving mesenchymal cells such as fibroblasts are not well understood. Here, we develop and analyze a reaction-diffusion model of phospholipase C/protein kinase C (PKC) signaling, which was recently identified as a requisite PDGF-gradient-sensing pathway, with the goal of identifying mechanisms that can amplify its sensitivity in the shallow external gradients typical of chemotaxis experiments. We show that phosphorylation of myristoylated alanine-rich C kinase substrate by membrane-localized PKC constitutes a positive feedback that is sufficient for local pathway amplification. The release of phosphorylated myristoylated alanine-rich C kinase substrate and its subsequent diffusion and dephosphorylation in the cytosol also serves to suppress the pathway in down-gradient regions of the cell. By itself, this mechanism only weakly amplifies signaling in a shallow PDGF gradient, but it synergizes with other feedback mechanisms to enhance amplification. This model offers a framework for a mechanistic understanding of phospholipase C/PKC signaling in chemotactic gradient sensing and can guide the design of experiments to assess the roles of putative feedback loops.

Mesenchymal Chemotaxis Requires Selective Inactivation of Myosin II at the Leading Edge via a Noncanonical PLCγ/PKCα Pathway

Chemotaxis, migration toward soluble chemical cues, is critical for processes such as wound healing and immune surveillance and is exhibited by various cell types, from rapidly migrating leukocytes to slow-moving mesenchymal cells. To study mesenchymal chemotaxis, we observed cell migration in microfluidic chambers that generate stable gradients of platelet-derived growth factor (PDGF). Surprisingly, we found that pathways implicated in amoeboid chemotaxis, such as PI3K and mammalian target of rapamycin signaling, are dispensable for PDGF chemotaxis. Instead, we find that local inactivation of Myosin IIA, through a noncanonical Ser1/2 phosphorylation of the regulatory light chain, is essential. This site is phosphorylated by PKCα, which is activated by an intracellular gradient of diacylglycerol generated by PLCγ. Using a combination of live imaging and gradients of activators/inhibitors in the microfluidic chambers, we demonstrate that this signaling pathway and subsequent inhibition of Myosin II activity at the leading edge are required for mesenchymal chemotaxis.

Regulation of the ROS Response Dynamics and Organization to PDGF Motile Stimuli Revealed by Single Nanoparticle Imaging

Although reactive oxygen species (ROS) are better known for their harmful effects, more recently, H2O2, one of the ROS, was also found to act as a secondary messenger. However, details of spatiotemporal organization of specific signaling pathways that H2O2 is involved in are currently missing. Here, we use single nanoparticle imaging to measure the local H2O2 concentration and reveal regulation of the ROS response dynamics and organization to platelet-derived growth factor (PDGF) signaling. We demonstrate that H2O2 production is controlled by PDGFR kinase activity and EGFR transactivation, requires a persistent stimulation, and is regulated by membrane receptor diffusion. This temporal filtering is impaired in cancer cells, which may determine their pathological migration. H2O2 subcellular mapping reveals that an external PDGF gradient induces an amplification-free asymmetric H2O2 concentration profile. These results support a general model for the control of signal transduction based only on membrane receptor diffusion and second messenger degradation.

Dynamics of angiogenesis during murine retinal development: a coupledin vivoandin silicostudy

The manner in which the superficial retinal vascular plexus (RVP) develops in neonatal wild-type mice is relatively well documented and poses an interesting challenge to the mathematical modelling community. Prior to birth, astrocyte sprouting and proliferation begin around the edge of the optic nerve head, and subsequent astrocyte migration in response to a chemotactic gradient of platelet-derived growth factor (PDGF)-A results in the formation of a dense scaffold on the surface of the inner retina. Astrocytes express a variety of chemotactic and haptotactic proteins that subsequently induce endothelial cell sprouting and modulate growth of the RVP. An experimentally informed, two-dimensional hybrid partial differential equation-discrete model is derived to track the outward migration of individual astrocyte and endothelial tip cells in response to the appropriate biochemical cues. Blood perfusion is included throughout the development of the plexus, and the evolving retinal trees are allowed to adapt and remodel by means of several biological stimuli. The resulting wild-type in silico RVP structures are compared with corresponding experimental whole mounts taken at various stages of development, and agreement between the respective vascular morphologies is found to be excellent. Subsequent numerical predictions help elucidate some of the key biological processes underlying retinal development and demonstrate the potential of the virtual retina for the investigation of various vascular-related diseases of the eye.

In Chemotaxing Fibroblasts, Both High-Fidelity and Weakly Biased Cell Movements Track the Localization of PI3K Signaling

Cell movement biased by a chemical gradient, or chemotaxis, coordinates the recruitment of cells and collective migration of cell populations. During wound healing, chemotaxis of fibroblasts is stimulated by platelet-derived growth factor (PDGF) and certain other chemoattractants. Whereas the immediate PDGF gradient sensing response has been characterized previously at the level of phosphoinositide 3-kinase (PI3K) signaling, the sensitivity of the response at the level of cell migration bias has not yet been studied quantitatively. In this work, we used live-cell total internal reflection fluorescence microscopy to monitor PI3K signaling dynamics and cell movements for extended periods. We show that persistent and properly aligned (i.e., high-fidelity) fibroblast migration does indeed correlate with polarized PI3K signaling; accordingly, this behavior is seen only under conditions of high gradient steepness (>10% across a typical cell length of 50 μm) and a certain range of PDGF concentrations. Under suboptimal conditions, cells execute a random or biased random walk, but nonetheless move in a predictable fashion according to the changing pattern of PI3K signaling. Inhibition of PI3K during chemotaxis is accompanied by loss of both cell-substratum contact and morphological polarity, but after a recovery period, PI3K-inhibited fibroblasts often regain the ability to orient toward the PDGF gradient.

Cdc42 and Rac Family GTPases Regulate Mode and Speed but Not Direction of Primary Fibroblast Migration during Platelet-Derived Growth Factor-Dependent Chemotaxis

Cdc42 and Rac family GTPases are important regulators of morphology, motility, and polarity in a variety of mammalian cell types. However, comprehensive analysis of their roles in the morphological and behavioral aspects of chemotaxis within a single experimental system is still lacking. Here we demonstrate using a direct viewing chemotaxis assay that of all of the Cdc42/Rac1-related GTPases expressed in primary fibroblasts, Cdc42, Rac1, and RhoG are required for efficient migration towards platelet-derived growth factor (PDGF). During migration, Cdc42-, Rac1-, and RhoG-deficient cells show aberrant morphology characterized as cell elongation and cell body rounding, loss of lamellipodia, and formation of thick membrane extensions, respectively. Analysis of individual cell trajectories reveals that cell speed is significantly reduced, as well as persistence, but to a smaller degree, while the directional response to the gradient of PDGF is not affected. Combined knockdown of Cdc42, Rac1, and RhoG results in greater inhibition of cell speed than when each protein is knocked down alone, but the cells are still capable of migrating toward PDGF. We conclude that, Cdc42, Rac1, and RhoG function cooperatively during cell migration and that, while each GTPase is implicated in the control of morphology and cell speed, these and other Cdc42/Rac-related GTPases are not essential for the directional response toward PDGF.

Proliferation Versus Migration in Platelet-derived Growth Factor Signaling: THE KEY ROLE OF ENDOCYTOSIS

It is common knowledge that platelet-derived growth factor (PDGF) is a critical regulator of mesenchymal cell migration and proliferation. Nevertheless, these two cellular responses are mutually exclusive. To solve this apparent contradiction, we studied the behavior of NIH3T3 fibroblasts in response to increasing concentrations of PDGF. We found that there is strong cell proliferation induction only with PDGF concentrations >5 ng/ml, whereas the cell migration response arises starting from 1 ng/ml and is negligible at higher PDGF concentrations. According to these phenotypic evidences, our data indicate that cells display a differential activation of the main signaling pathways in response to PDGF as a function of the stimulation dose. At low PDGF concentrations, there is maximal activation of signaling pathways linked to cytoskeleton rearrangement needed for cell motility, whereas high PDGF concentrations activate pathways linked to mitogenesis induction. Our results suggest a mechanism by which cells switch from a migrating to a proliferating phenotype sensing the increasing gradient of PDGF. In addition, we propose that the cell decision to proliferate or migrate relies on different endocytotic routes of the PDGF receptor in response to different PDGF concentrations.

Constitutive diffuse activation of phosphoinositide 3-kinase at the plasma membrane by v-Src suppresses the chemotactic response to PDGF by abrogating the polarity of PDGF receptor signalling

Cancer cells depend on chemotaxis for invasion and frequently overexpress and/or activate Src. We previously reported that v-Src accelerates motility by promoting phosphoinositide 3-kinase (PI3-K) signalling but abrogates chemotaxis. We here addressed the mechanism of the loss of chemotactic response to platelet-derived growth factor (PDGF) gradients in fibroblasts harbouring a thermosensitive v-Src kinase. At non-permissive temperature, PDGF receptor (PDGFR) signalling, assessed by phosphoY 751-specific antibodies (a docking site for PI3-K), was not detected without PDGF and showed a concentration-dependent PDGF response. Both immunolabeling of PI3-K (p110) and live cell imaging of its product (phosphatidylinositol 3,4,5 tris-phosphate) showed PI3-K recruitment and activation at lamellipodia polarized towards a PDGF gradient. Centrosomes and PDGFR- and Src-bearing endosomes were also oriented towards this gradient. Upon v-Src thermoactivation, (i) Y 751 phosphorylation was moderately induced without PDGF and synergistically increased with PDGF; (ii) PI3-K was recruited and activated all along the plasma membrane without PDGF and did not polarize in response to a PDGF gradient; and (iii) polarization of centrosomes and of PDGFR-bearing endosomes were also abrogated. Thus, PDGF can further increase PDGFR auto-phosphorylation despite strong Src kinase activity, but diffuse downstream activation of PI3-K by Src abrogates cell polarization and chemotaxis: “signalling requires silence”.

Effectiveness factor for spatial gradient sensing in living cells

We consider the steady-state pattern of messenger molecules produced in the membrane of a cell perceiving and responding to an extracellular gradient of chemoattractant, which directs cell movement towards the chemoattractant source. Specifically, we analyze the undesirable effect of lateral diffusion in blurring the intracellular messenger profile. The concept of an effectiveness factor, akin to the analysis of reactions in porous catalysts, is applied to the spatial gradient sensing problem, with the distinction that slow, not fast, diffusion is required for effective gradient sensing. Analytical effectiveness factor expressions are derived for ideal geometries and then generalized to arbitrary cell shapes. In the case of mouse fibroblasts responding to gradients of platelet-derived growth factor, we conclude that the cell morphology and orientation with respect to the gradient can dictate whether messenger diffusion obliterates gradient sensing or has very little effect. The analysis outlined here allows the effect of intracellular messenger diffusion on spatial gradient sensing to be quantified for individual cells.

Deterministic Model of Dermal Wound Invasion Incorporating Receptor-Mediated Signal Transduction and Spatial Gradient Sensing

During dermal wound healing, platelet-derived growth factor (PDGF) serves as both a chemoattractant and mitogen for fibroblasts, potently stimulating their invasion of the fibrin clot over a period of several days. A mathematical model of this process is presented, which accurately accounts for the sensitivity of PDGF gradient sensing through PDGF receptor/phosphoinositide 3-kinase-mediated signal transduction. Analysis of the model suggests that PDGF receptor-mediated endocytosis and degradation of PDGF allows a constant PDGF concentration profile to be maintained at the leading front of the fibroblast density profile as it propagates, at a constant rate, into the clot. Thus, the constant PDGF gradient can span the optimal concentration range for asymmetric phosphoinositide 3-kinase signaling and fibroblast chemotaxis, with near-maximal invasion rates elicited over a relatively broad range of PDGF secretion rates. A somewhat surprising finding was that extremely sharp PDGF gradients do not necessarily stimulate faster progression through the clot, because maintaining such a gradient through PDGF consumption is a potentially rate-limiting process.

Quantitative elucidation of a distinct spatial gradient-sensing mechanism in fibroblasts

Migration of eukaryotic cells toward a chemoattractant often relies on their ability to distinguish receptor-mediated signaling at different subcellular locations, a phenomenon known as spatial sensing. A prominent example that is seen during wound healing is fibroblast migration in platelet-derived growth factor (PDGF) gradients. As in the well-characterized chemotactic cells Dictyostelium discoideum and neutrophils, signaling to the cytoskeleton via the phosphoinositide 3-kinase pathway in fibroblasts is spatially polarized by a PDGF gradient; however, the sensitivity of this process and how it is regulated are unknown. Through a quantitative analysis of mathematical models and live cell total internal reflection fluorescence microscopy experiments, we demonstrate that PDGF detection is governed by mechanisms that are fundamentally different from those in D. discoideum and neutrophils. Robust PDGF sensing requires steeper gradients and a much narrower range of absolute chemoattractant concentration, which is consistent with a simpler system lacking the feedback loops that yield signal amplification and adaptation in amoeboid cells.

Gab1 Contributes to Cytoskeletal Reorganization and Chemotaxis in Response to Platelet-derived Growth Factor

Gab1 is a scaffolding/docking protein that has been suggested to play a role in signal transduction downstream of certain plasma membrane receptors, including platelet-derived growth factor (PDGF) receptors. We found that PDGF induced a rapid Gab1 phosphorylation, which depended on the recruitment of Grb2, indicating that Grb2 acts as a bridge between Gab1 and the PDGF beta-receptor. PDGF also enhanced the binding of Gab1 to the phosphatase SHP-2, but not to p85. To further study the role of Gab1 in PDGF signaling, we transfected porcine aortic endothelial cells with a doxycycline-inducible Gab1 construct. Increased Gab1 expression enhanced the recruitment and activation of SHP-2, as well as the phosphorylation of the mitogen-activated protein kinases Erk and p38 by PDGF. Gab1 expression also enhanced the formation of lamellipodia and cellular protrusions. In Gab1-deficient mouse embryonic fibroblasts, the same phenotype was induced by restoring the expression of wild-type Gab1, but not a mutant Gab1 that was unable to associate with SHP-2. These effects of PDGF on the actin cytoskeleton were not altered by the inhibition of p38 or Erk, but could be blocked by a dominant-negative form of Rac (Asn(17)). Finally, Gab1-deficient fibroblasts showed a decreased chemotactic response toward gradients of PDGF as compared with wild-type cells. In conclusion, Gab1 plays a selective role in the regulation of the mitogen-activated protein kinases Erk and p38 downstream of the PDGF beta-receptor, and contributes to cytoskeletal reorganization and chemotaxis in response to PDGF.

Spatial Analysis of 3′ Phosphoinositide Signaling in Living Fibroblasts: II. Parameter Estimates for Individual Cells from Experiments

Fibroblast migration is directed by gradients of platelet-derived growth factor (PDGF) during wound healing. As in other chemotactic systems, it has been shown recently that localized stimulation of intracellular phosphoinositide (PI) 3-kinase activity and production of 3′ PI lipids in the plasma membrane are important events in the signaling of spatially biased motility processes. In turn, 3′ PI localization depends on the effective diffusion coefficient, D, and turnover rate constant, k, of these lipids. Here we present a systematic and direct comparison of mathematical model calculations and experimental measurements to estimate the values of the effective 3′ PI diffusion coefficient, D, turnover rate constant, k, and other parameters in individual fibroblasts stimulated uniformly with PDGF. In the context of our uniform stimulation model, the values of D and k in each cell were typically estimated within 10–20% or less, and the mean values across all of the cells analyzed were D = 0.37 ± 0.25 μm 2/s and k = 1.18 ± 0.54 min −1. In addition, we report that 3′ PI turnover is not affected by PDGF receptor signaling in our cells, allowing us to focus our attention on the regulation of 3′ PI production as this system is studied further.

A role for the polysialic acid-neural cell adhesion molecule in PDGF-induced chemotaxis of oligodendrocyte precursor cells.

Directed migration of oligodendrocyte precursor cells (OPCs) is important for myelin formation and repair but the mechanisms of directional control are poorly understood. Here we have tested the role of polysialic acid-neural cell adhesion molecule (PSA-NCAM) in the directional migration of OPCs towards platelet-derived growth factor (PDGF). Using a Boyden microchemotaxis chamber and the Dunn direct viewing chamber, we show that in concentration gradients of PDGF, PSA-positive OPCs polarize and efficiently migrate towards the source of PDGF (chemotaxis). The loss or inactivation of the polysialic tail of NCAM leads to an altered pattern of OPC migration in response to PDGF gradients. Cells under these conditions, while being polarized and migrating, show no bias of displacement towards the source of PDGF and make random turns. By contrast, directed migration of OPCs towards basic fibroblast growth factor was not affected by the removal of PSA. Moreover, inactivation of PSA does not interfere with the random migration pattern of cells in uniform concentrations of PDGF (chemokinesis). These results suggest that PSA-NCAM is specifically involved in establishing the directionality of OPC migration in response to the concentration gradient of PDGF, but it is not essential for cell motility per se.

Keeping Cells in Their Places

STKE Vascular proliferative diseases, such as atherosclerosis and coronary restenosis, are characterized by dedifferentiation, abnormal proliferation, and migration of vascular smooth muscle cells, a pathophysiological reaction to injury that has been interpreted as a response to cytokines released by inflammatory cells. By comparing vascular smooth muscle cells from mice lacking elastin to cells from wild-type mice, Karnik et al . show that the extracellular matrix protein elastin, which is secreted by vascular smooth cells, inhibited cell proliferation and promoted the development of actin stress fibers, a marker for a mature contractile phenotype. Vascular smooth muscle cells migrated through a filter in response to an elastin concentration gradient, but elastin inhibited cellular migration in response to a platelet-derived growth factor gradient. In an in vivo porcine model of coronary artery stenosis, insertion of an elastin sheath into arteries after vascular injury reduced the pathophysiological response, suggesting the possibility that elastin's effects on vascular smooth muscle could be exploited in therapy to treat vascular proliferative diseases. — EA Development 130 , 411 (2002).

Elastin Keeps Cells in Their Place

Karnik et al. have obtained evidence suggesting that the extracellular matrix protein elastin acts as an autocrine regulator of vascular smooth muscle proliferation and migration, a finding that could have implications for the therapy of vascular proliferative diseases. Various vascular proliferative diseases, including atherosclerosis and coronary restenosis, are characterized by dedifferentiation, abnormal proliferation, and migration of vascular smooth muscle cells, a pathophysiological reaction to injury that has largely been interpreted as a response to cytokines released by inflammatory cells. By comparing vascular smooth muscle cells from mice lacking elastin to cells from wild-type sibling controls, Karnik et al. showed that that the extracellular matrix protein elastin, which is secreted by vascular smooth cells, inhibited cell proliferation and promoted the development of actin stress fibers, a marker for a mature contractile phenotype. Elastin influenced cell migration: vascular smooth muscle cells migrated through a filter in response to an elastin concentration gradient but elastin inhibited the cells' migration in response to a platelet-derived growth factor gradient. EDTA, which binds divalent cations thereby inhibiting integrin-mediated interactions, did not inhibit elastin's effects. Rather, pharmacological analysis indicated that elastin activates a guanosine triphosphate (GTP)-binding protein pathway involving a G i -mediated reduction in cellular cyclic adenosine monophosphate and the stimulation of Rho. In an in vivo porcine model of coronary artery stenosis, insertion of an elastin sheath into arteries following vascular injury reduced the pathophysiological response, suggesting the possibility that elastin's effects on vascular smooth muscle could be exploited in the therapy of vascular proliferative diseases. S. K. Karnik, B. S. Brooke, A. Bayes-Genis, L. Sorensen, J. D. Wythe, R. S. Schwartz, M. T. Keating, D.Y. Li, A critical role for elastin signaling in vascular morphogenesis and disease. Development 130 , 411-423 (2002). [Abstract] [Full Text]

Platelet-Derived Growth Factor B-Chain of Hematopoietic Origin Is Not Necessary for Granulation Tissue Formation and Its Absence Enhances Vascularization

The hypothesis that wound repair is augmented by delivery of platelet-derived growth factor (PDGF) from platelets and macrophages is an attractive extrapolation from the known activities of PDGF in cell culture and in vivo. To test this hypothesis in mice, we prepared hematopoietic chimeras, in which the hematopoietic system of a normal adult mouse was replaced by the hematopoietic system of a PDGF B-chain -/- or +/+ donor. We initiated local granulation tissue formation either by implanting small surgical sponges to elicit a foreign body granulation tissue response, or by ligating the left common carotid to form an organized thrombus. We found that the absence of hematopoietic PDGF B-chain did not decrease the extent of granulation tissue or vascular lesion formation, and that the vascularization of both lesions increased by approximately 100%. We conclude that PDGF B-chain from cells of hematopoietic origin, including platelets and macrophages, is not important for granulation tissue formation, and that it reduces vascularization of granulation issue, probably through disabling of the short-range chemotactic gradients of PDGF that are important for recruiting pericytes/smooth muscle cells to the endothelium of new vessels.

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.