Limits...
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.

Show MeSH
PI 3-kinase signaling kinetics in response to transient PDGF stimulation. (A) A CFP-AktPH–transfected fibroblast was stimulated with a moving PDGF gradient for 21 min, after which 10 nM PDGF (uniform) and wortmannin (wort.) were added as in Fig. 3. The time course montage shows TIRF images of the CFP-AktPH translocation (top) and OG 514–dextran gradient (bottom). Bar, 30 μm. Video 1 shows this time course (available at http://www.jcb.org/cgi/content/full/jcb.200509028/DC1). (B) TIRF images of CFP-AktPH–transfected fibroblasts treated with a brief pulse of PDGF at 2 min, 10 nM PDGF (uniform) at 20 min, and wortmannin (wort.) at 30 min. Bar, 60 μm. (C) The left panels plot average normalized TIRF intensity in the CFP-AktPH (closed circles) and OG 514–dextran (open circles) channels as a function of time for the two cells indicated in B. The right panels are the corresponding kinetic model calculations (see supplemental Modeling details). The dashed curves show the PDGF concentration time courses assumed for each cell before the addition of the 10-nM PDGF bolus.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171296&req=5

fig5: PI 3-kinase signaling kinetics in response to transient PDGF stimulation. (A) A CFP-AktPH–transfected fibroblast was stimulated with a moving PDGF gradient for 21 min, after which 10 nM PDGF (uniform) and wortmannin (wort.) were added as in Fig. 3. The time course montage shows TIRF images of the CFP-AktPH translocation (top) and OG 514–dextran gradient (bottom). Bar, 30 μm. Video 1 shows this time course (available at http://www.jcb.org/cgi/content/full/jcb.200509028/DC1). (B) TIRF images of CFP-AktPH–transfected fibroblasts treated with a brief pulse of PDGF at 2 min, 10 nM PDGF (uniform) at 20 min, and wortmannin (wort.) at 30 min. Bar, 60 μm. (C) The left panels plot average normalized TIRF intensity in the CFP-AktPH (closed circles) and OG 514–dextran (open circles) channels as a function of time for the two cells indicated in B. The right panels are the corresponding kinetic model calculations (see supplemental Modeling details). The dashed curves show the PDGF concentration time courses assumed for each cell before the addition of the 10-nM PDGF bolus.

Mentions: Another test of a mathematical model is its ability to reproduce the cellular response to a transient or pulsed stimulus, an approach that can indicate the presence of feedback interactions (Bhalla et al., 2002). To determine whether our model could explain the 3′ PI responses to transient PDGF stimulation, PDGF was pulsed from the micropipette for a certain period, and CFP-AktPH translocation during and after the pulse was recorded using TIRF microscopy (Fig. 5). By adjusting the flow rate, the PDGF gradient during the pulse could be tuned to be steep (Fig. 5 A and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200509028/DC1) or essentially uniform across cellular dimensions (Fig. 5, B and C). In both situations, PI 3-kinase signaling tends to persist for several minutes after decay of the stimulus; in fact, the peak response was typically observed minutes after the PDGF concentration began to drop. Our kinetic model captures this behavior. It predicts that the 3′ PI decay will lag whenever the duration of the pulse is insufficient for establishing a quasi–steady state (∼5–10 min), with the time interval of the lag and rate of decay after the peak depending on the degree of PI 3-kinase saturation. Persistence of 3′ PI levels after PDGF withdrawal is not attributed to positive feedback but rather to the fairly slow kinetics of PI 3-kinase redistribution to the cytosol and 3′ PI turnover, and the model and experiment are in quantitative agreement when one allows for modest cell-to-cell variation in receptor and PI 3-kinase expression levels (Fig. 5 C).


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

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

PI 3-kinase signaling kinetics in response to transient PDGF stimulation. (A) A CFP-AktPH–transfected fibroblast was stimulated with a moving PDGF gradient for 21 min, after which 10 nM PDGF (uniform) and wortmannin (wort.) were added as in Fig. 3. The time course montage shows TIRF images of the CFP-AktPH translocation (top) and OG 514–dextran gradient (bottom). Bar, 30 μm. Video 1 shows this time course (available at http://www.jcb.org/cgi/content/full/jcb.200509028/DC1). (B) TIRF images of CFP-AktPH–transfected fibroblasts treated with a brief pulse of PDGF at 2 min, 10 nM PDGF (uniform) at 20 min, and wortmannin (wort.) at 30 min. Bar, 60 μm. (C) The left panels plot average normalized TIRF intensity in the CFP-AktPH (closed circles) and OG 514–dextran (open circles) channels as a function of time for the two cells indicated in B. The right panels are the corresponding kinetic model calculations (see supplemental Modeling details). The dashed curves show the PDGF concentration time courses assumed for each cell before the addition of the 10-nM PDGF bolus.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: PI 3-kinase signaling kinetics in response to transient PDGF stimulation. (A) A CFP-AktPH–transfected fibroblast was stimulated with a moving PDGF gradient for 21 min, after which 10 nM PDGF (uniform) and wortmannin (wort.) were added as in Fig. 3. The time course montage shows TIRF images of the CFP-AktPH translocation (top) and OG 514–dextran gradient (bottom). Bar, 30 μm. Video 1 shows this time course (available at http://www.jcb.org/cgi/content/full/jcb.200509028/DC1). (B) TIRF images of CFP-AktPH–transfected fibroblasts treated with a brief pulse of PDGF at 2 min, 10 nM PDGF (uniform) at 20 min, and wortmannin (wort.) at 30 min. Bar, 60 μm. (C) The left panels plot average normalized TIRF intensity in the CFP-AktPH (closed circles) and OG 514–dextran (open circles) channels as a function of time for the two cells indicated in B. The right panels are the corresponding kinetic model calculations (see supplemental Modeling details). The dashed curves show the PDGF concentration time courses assumed for each cell before the addition of the 10-nM PDGF bolus.
Mentions: Another test of a mathematical model is its ability to reproduce the cellular response to a transient or pulsed stimulus, an approach that can indicate the presence of feedback interactions (Bhalla et al., 2002). To determine whether our model could explain the 3′ PI responses to transient PDGF stimulation, PDGF was pulsed from the micropipette for a certain period, and CFP-AktPH translocation during and after the pulse was recorded using TIRF microscopy (Fig. 5). By adjusting the flow rate, the PDGF gradient during the pulse could be tuned to be steep (Fig. 5 A and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200509028/DC1) or essentially uniform across cellular dimensions (Fig. 5, B and C). In both situations, PI 3-kinase signaling tends to persist for several minutes after decay of the stimulus; in fact, the peak response was typically observed minutes after the PDGF concentration began to drop. Our kinetic model captures this behavior. It predicts that the 3′ PI decay will lag whenever the duration of the pulse is insufficient for establishing a quasi–steady state (∼5–10 min), with the time interval of the lag and rate of decay after the peak depending on the degree of PI 3-kinase saturation. Persistence of 3′ PI levels after PDGF withdrawal is not attributed to positive feedback but rather to the fairly slow kinetics of PI 3-kinase redistribution to the cytosol and 3′ PI turnover, and the model and experiment are in quantitative agreement when one allows for modest cell-to-cell variation in receptor and PI 3-kinase expression levels (Fig. 5 C).

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