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

AbstractFibroblast 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±0.25μm2/s and k=1.18±0.54min−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

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

Quantitative analysis of B-lymphocyte migration directed by CXCL13

B-lymphocyte migration, directed by chemokine gradients, is essential for homing to sites of antigen presentation

Directed migration of mesenchymal cells: where signaling and the cytoskeleton meet

Cell migration directed by spatial cues, or taxis, is a primary mechanism for orchestrating concerted and collective cell movements during development, wound repair, and immune responses. Compared with the classic example of amoeboid chemotaxis, in which fast-moving cells such as neutrophils are directed by gradients of soluble factors, directed migration of slow-moving mesenchymal cells such as fibroblasts is poorly understood. Mesenchymal cells possess a distinctive organization of the actin cytoskeleton and associated adhesion complexes as its primary mechanical system, generating the asymmetric forces required for locomotion without strong polarization. The emerging hypothesis is that the molecular underpinnings of mesenchymal taxis involve distinct signaling pathways and diverse requirements for regulation

PI3K-dependent cross-talk interactions converge with Ras as quantifiable inputs integrated by Erk

Although it is appreciated that canonical signal-transduction pathways represent dominant modes of regulation embedded in larger interaction networks, relatively little has been done to quantify pathway cross-talk in such networks. Through quantitative measurements that systematically canvas an array of stimulation and molecular perturbation conditions, together with computational modeling and analysis, we have elucidated cross-talk mechanisms in the platelet-derived growth factor (PDGF) receptor signaling network, in which phosphoinositide 3-kinase (PI3K) and Ras/extracellular signal-regulated kinase (Erk) pathways are prominently activated. We show that, while PI3K signaling is insulated from cross-talk, PI3K enhances Erk activation at points both upstream and downstream of Ras. The magnitudes of these effects depend strongly on the stimulation conditions, subject to saturation effects in the respective pathways and negative feedback loops. Motivated by those dynamics, a kinetic model of the network was formulated and used to precisely quantify the relative contributions of PI3K-dependent and -independent modes of Ras/Erk activation

Development of a tandem affinity phosphoproteomic method with motif selectivity and its application in analysis of signal transduction networks

Phosphorylation is an important post-translational modification that is involved in regulating many signaling pathways. Of particular interest are the growth factor mediated Ras and phosphoinositide 3-kinase (PI3K) signaling pathways which, if misregulated, can contribute to the progression of cancer. Phosphoproteomic methods have been developed to study regulation of signaling pathways; however, due to the low stoichiometry of phosphorylation, understanding these pathways is still a challenge. In this study, we have developed a multi-dimensional method incorporating electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) with tandem IMAC-TiO2 enrichment for subsequent phosphopeptide identification by LC/MS/MS. We applied this method to PDGF-stimulated NIH 3T3 cells to provide over 11,000 unique phosphopeptide identifications. Upon motif analysis, IMAC was found to enrich for basophilic kinase substrates while the subsequent TiO2 step enriched for acidophilic kinase substrates, suggesting that both enrichment methods are necessary to capture the full complement of kinase substrates. Biological functions that were over-represented at each PDGF stimulation time point, together with the phosphorylation dynamics of several phosphopeptides containing known kinase phosphorylation sites illustrate the feasibility of this approach in quantitative phosphoproteomic studies

GMF controls branched actin content and lamellipodial retraction in fibroblasts

The lamellipodium is an important structure for cell migration containing branched actin nucleated via the Arp2/3 complex. The formation of branched actin is relatively well studied, but less is known about its disassembly and how this influences migration. GMF is implicated in both Arp2/3 debranching and inhibition of Arp2/3 activation. Modulation of GMFβ, a ubiquitous GMF isoform, by depletion or overexpression resulted in changes in lamellipodial dynamics, branched actin content, and migration. Acute pharmacological inhibition of Arp2/3 by CK-666, coupled to quantitative live-cell imaging of the complex, showed that depletion of GMFβ decreased the rate of branched actin disassembly. These data, along with mutagenesis studies, suggest that debranching (not inhibition of Arp2/3 activation) is a primary activity of GMFβ in vivo. Furthermore, depletion or overexpression of GMFβ disrupted the ability of cells to directionally migrate to a gradient of fibronectin (haptotaxis). These data suggest that debranching by GMFβ plays an important role in branched actin regulation, lamellipodial dynamics, and directional migration

Feedback control architecture and the bacterial chemotaxis network.

PMCID: PMC3088647This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Bacteria move towards favourable and away from toxic environments by changing their swimming pattern. This response is regulated by the chemotaxis signalling pathway, which has an important feature: it uses feedback to 'reset' (adapt) the bacterial sensing ability, which allows the bacteria to sense a range of background environmental changes. The role of this feedback has been studied extensively in the simple chemotaxis pathway of Escherichia coli. However it has been recently found that the majority of bacteria have multiple chemotaxis homologues of the E. coli proteins, resulting in more complex pathways. In this paper we investigate the configuration and role of feedback in Rhodobacter sphaeroides, a bacterium containing multiple homologues of the chemotaxis proteins found in E. coli. Multiple proteins could produce different possible feedback configurations, each having different chemotactic performance qualities and levels of robustness to variations and uncertainties in biological parameters and to intracellular noise. We develop four models corresponding to different feedback configurations. Using a series of carefully designed experiments we discriminate between these models and invalidate three of them. When these models are examined in terms of robustness to noise and parametric uncertainties, we find that the non-invalidated model is superior to the others. Moreover, it has a 'cascade control' feedback architecture which is used extensively in engineering to improve system performance, including robustness. Given that the majority of bacteria are known to have multiple chemotaxis pathways, in this paper we show that some feedback architectures allow them to have better performance than others. In particular, cascade control may be an important feature in achieving robust functionality in more complex signalling pathways and in improving their performance