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

Schneider IC, Haugh JM - J. Cell Biol. (2005)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA.

ABSTRACT
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.

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TIRF imaging of extracellular and intracellular gradients. (A) A micropipette was coloaded with Oregon green (OG) 514–dextran and a prescribed concentration of PDGF, and PDGF gradients were presented to NIH 3T3 fibroblasts transfected with CFP-AktPH. Bar, 100 μm. (B) Although the TIRF excitation of the volume marker is partially occluded by the cells (Lanni et al., 1985), the surrounding regions allow the estimation of the PDGF concentration profile across the cell, as described in Materials and methods. The solid curve in the plot is the best polynomial fit to the fluorescence profiles on either side of the cell, along the line scan depicted in the TIRF image. Bar, 30 μm.
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fig2: TIRF imaging of extracellular and intracellular gradients. (A) A micropipette was coloaded with Oregon green (OG) 514–dextran and a prescribed concentration of PDGF, and PDGF gradients were presented to NIH 3T3 fibroblasts transfected with CFP-AktPH. Bar, 100 μm. (B) Although the TIRF excitation of the volume marker is partially occluded by the cells (Lanni et al., 1985), the surrounding regions allow the estimation of the PDGF concentration profile across the cell, as described in Materials and methods. The solid curve in the plot is the best polynomial fit to the fluorescence profiles on either side of the cell, along the line scan depicted in the TIRF image. Bar, 30 μm.

Mentions: The CFP-tagged pleckstrin homology domain of Akt (CFP-AktPH) was used as a specific biosensor for 3′ PI production at the plasma membrane. Using a micropipette coloaded with PDGF and a fluorescent volume marker (Oregon green [OG] 514–dextran), CFP-AktPH–transfected fibroblasts were presented with gradients of PDGF, and the local marker concentration and intracellular CFP-AktPH translocation were monitored using TIRF microscopy (Fig. 2). By varying the concentration of PDGF in the pipette across different experiments and observing cells at different distances from the source, we systematically analyzed responses to PDGF fields with varying midpoint concentration and gradient steepness. After 20 min of gradient stimulation, a high concentration of PDGF was added uniformly to normalize the response at each location. Subsequently, a high concentration of wortmannin was added to rapidly block PI 3-kinase and, thus, to assess the degradation of 3′ PI lipids and the contribution of cytosolic CFP-AktPH to the overall TIRF fluorescence.


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

Schneider IC, Haugh JM - J. Cell Biol. (2005)

TIRF imaging of extracellular and intracellular gradients. (A) A micropipette was coloaded with Oregon green (OG) 514–dextran and a prescribed concentration of PDGF, and PDGF gradients were presented to NIH 3T3 fibroblasts transfected with CFP-AktPH. Bar, 100 μm. (B) Although the TIRF excitation of the volume marker is partially occluded by the cells (Lanni et al., 1985), the surrounding regions allow the estimation of the PDGF concentration profile across the cell, as described in Materials and methods. The solid curve in the plot is the best polynomial fit to the fluorescence profiles on either side of the cell, along the line scan depicted in the TIRF image. Bar, 30 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171296&req=5

fig2: TIRF imaging of extracellular and intracellular gradients. (A) A micropipette was coloaded with Oregon green (OG) 514–dextran and a prescribed concentration of PDGF, and PDGF gradients were presented to NIH 3T3 fibroblasts transfected with CFP-AktPH. Bar, 100 μm. (B) Although the TIRF excitation of the volume marker is partially occluded by the cells (Lanni et al., 1985), the surrounding regions allow the estimation of the PDGF concentration profile across the cell, as described in Materials and methods. The solid curve in the plot is the best polynomial fit to the fluorescence profiles on either side of the cell, along the line scan depicted in the TIRF image. Bar, 30 μm.
Mentions: The CFP-tagged pleckstrin homology domain of Akt (CFP-AktPH) was used as a specific biosensor for 3′ PI production at the plasma membrane. Using a micropipette coloaded with PDGF and a fluorescent volume marker (Oregon green [OG] 514–dextran), CFP-AktPH–transfected fibroblasts were presented with gradients of PDGF, and the local marker concentration and intracellular CFP-AktPH translocation were monitored using TIRF microscopy (Fig. 2). By varying the concentration of PDGF in the pipette across different experiments and observing cells at different distances from the source, we systematically analyzed responses to PDGF fields with varying midpoint concentration and gradient steepness. After 20 min of gradient stimulation, a high concentration of PDGF was added uniformly to normalize the response at each location. Subsequently, a high concentration of wortmannin was added to rapidly block PI 3-kinase and, thus, to assess the degradation of 3′ PI lipids and the contribution of cytosolic CFP-AktPH to the overall TIRF fluorescence.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA.

ABSTRACT
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.

Show MeSH