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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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PDGF-stimulated PI 3-kinase activation is not dependent on Rho family GTPases. (A) Bright field, TIRF, and epifluorescence images of GFP-AktPH–transfected fibroblasts that were pretreated with C. difficile toxin B responding to successive additions of 10 nM PDGF and wortmannin (wort). Inactivation of Rho family GTPases dramatically alters cell morphology but not PDGF-stimulated PI 3-kinase activation. Bar, 30 μm. (B) TIRF images of a fibroblast cotransfected with YFP-AktPH and the membrane marker Lyn-CFP treated as in A. Ratio images are YFP/CFP. The average normalized TIRF intensity is plotted as a function of time for the YFP-AktPH (closed circles) and Lyn-CFP (open circles) channels; the dotted lines indicate the additions of uniform PDGF and wortmannin. (C) PDGF gradient sensing after inactivation of Rho family GTPases. Toxin-treated cells typically showed AktPH translocation in TIRF but not a definite gradient-sensing response, which was expected given the relatively small cell dimensions. The cell shown is one of those in which a noticeable gradient response was seen. The arrow indicates the PDGF gradient orientation from high to low. (B and C) Bars, 15 μm.
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fig6: PDGF-stimulated PI 3-kinase activation is not dependent on Rho family GTPases. (A) Bright field, TIRF, and epifluorescence images of GFP-AktPH–transfected fibroblasts that were pretreated with C. difficile toxin B responding to successive additions of 10 nM PDGF and wortmannin (wort). Inactivation of Rho family GTPases dramatically alters cell morphology but not PDGF-stimulated PI 3-kinase activation. Bar, 30 μm. (B) TIRF images of a fibroblast cotransfected with YFP-AktPH and the membrane marker Lyn-CFP treated as in A. Ratio images are YFP/CFP. The average normalized TIRF intensity is plotted as a function of time for the YFP-AktPH (closed circles) and Lyn-CFP (open circles) channels; the dotted lines indicate the additions of uniform PDGF and wortmannin. (C) PDGF gradient sensing after inactivation of Rho family GTPases. Toxin-treated cells typically showed AktPH translocation in TIRF but not a definite gradient-sensing response, which was expected given the relatively small cell dimensions. The cell shown is one of those in which a noticeable gradient response was seen. The arrow indicates the PDGF gradient orientation from high to low. (B and C) Bars, 15 μm.

Mentions: Our mathematical description of PDGF gradient sensing does not invoke feedback loops, so we sought to rule out the need for Rho family GTPases in amplifying PI 3-kinase activation in our system (Fig. 6). Such feedback is plausible given that PI 3-kinase regulatory subunits can interact directly with Rac-GTP (Tolias et al., 1995; Bokoch et al., 1996). In differentiated HL-60 cells, inactivation of Rho family GTPases by Clostridium difficile toxin B treatment ablated 3′ PI accumulation in response to chemoattractants but not to insulin (Servant et al., 2000; Weiner et al., 2002). The latter signals through insulin and insulin-like growth factor receptors, which, like PDGF receptors, are receptor tyrosine kinases.


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

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

PDGF-stimulated PI 3-kinase activation is not dependent on Rho family GTPases. (A) Bright field, TIRF, and epifluorescence images of GFP-AktPH–transfected fibroblasts that were pretreated with C. difficile toxin B responding to successive additions of 10 nM PDGF and wortmannin (wort). Inactivation of Rho family GTPases dramatically alters cell morphology but not PDGF-stimulated PI 3-kinase activation. Bar, 30 μm. (B) TIRF images of a fibroblast cotransfected with YFP-AktPH and the membrane marker Lyn-CFP treated as in A. Ratio images are YFP/CFP. The average normalized TIRF intensity is plotted as a function of time for the YFP-AktPH (closed circles) and Lyn-CFP (open circles) channels; the dotted lines indicate the additions of uniform PDGF and wortmannin. (C) PDGF gradient sensing after inactivation of Rho family GTPases. Toxin-treated cells typically showed AktPH translocation in TIRF but not a definite gradient-sensing response, which was expected given the relatively small cell dimensions. The cell shown is one of those in which a noticeable gradient response was seen. The arrow indicates the PDGF gradient orientation from high to low. (B and C) Bars, 15 μm.
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fig6: PDGF-stimulated PI 3-kinase activation is not dependent on Rho family GTPases. (A) Bright field, TIRF, and epifluorescence images of GFP-AktPH–transfected fibroblasts that were pretreated with C. difficile toxin B responding to successive additions of 10 nM PDGF and wortmannin (wort). Inactivation of Rho family GTPases dramatically alters cell morphology but not PDGF-stimulated PI 3-kinase activation. Bar, 30 μm. (B) TIRF images of a fibroblast cotransfected with YFP-AktPH and the membrane marker Lyn-CFP treated as in A. Ratio images are YFP/CFP. The average normalized TIRF intensity is plotted as a function of time for the YFP-AktPH (closed circles) and Lyn-CFP (open circles) channels; the dotted lines indicate the additions of uniform PDGF and wortmannin. (C) PDGF gradient sensing after inactivation of Rho family GTPases. Toxin-treated cells typically showed AktPH translocation in TIRF but not a definite gradient-sensing response, which was expected given the relatively small cell dimensions. The cell shown is one of those in which a noticeable gradient response was seen. The arrow indicates the PDGF gradient orientation from high to low. (B and C) Bars, 15 μm.
Mentions: Our mathematical description of PDGF gradient sensing does not invoke feedback loops, so we sought to rule out the need for Rho family GTPases in amplifying PI 3-kinase activation in our system (Fig. 6). Such feedback is plausible given that PI 3-kinase regulatory subunits can interact directly with Rac-GTP (Tolias et al., 1995; Bokoch et al., 1996). In differentiated HL-60 cells, inactivation of Rho family GTPases by Clostridium difficile toxin B treatment ablated 3′ PI accumulation in response to chemoattractants but not to insulin (Servant et al., 2000; Weiner et al., 2002). The latter signals through insulin and insulin-like growth factor receptors, which, like PDGF receptors, are receptor tyrosine kinases.

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