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Kinetic analysis of receptor-activated phosphoinositide turnover.

Xu C, Watras J, Loew LM - J. Cell Biol. (2003)

Bottom Line: Phosphatidylinositol-4,5-bisphosphate (PIP2) decreased over the first 30 s, and then recovered over the following 2-3 min.This was subsequently confirmed experimentally.Furthermore, this analysis could help to resolve a controversy over whether the translocation of PH-GFP from membrane to cytosol is due to a decrease in PIP2 on the membrane or an increase in InsP3 in cytosol; by computationally clamping the concentrations of each of these compounds, the model shows how both contribute to the dynamics of probe translocation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Connecticut Health Center, Farmington, CT 06030, USA.

ABSTRACT
We studied the bradykinin-induced changes in phosphoinositide composition of N1E-115 neuroblastoma cells using a combination of biochemistry, microscope imaging, and mathematical modeling. Phosphatidylinositol-4,5-bisphosphate (PIP2) decreased over the first 30 s, and then recovered over the following 2-3 min. However, the rate and amount of inositol-1,4,5-trisphosphate (InsP3) production were much greater than the rate or amount of PIP2 decline. A mathematical model of phosphoinositide turnover based on this data predicted that PIP2 synthesis is also stimulated by bradykinin, causing an early transient increase in its concentration. This was subsequently confirmed experimentally. Then, we used single-cell microscopy to further examine phosphoinositide turnover by following the translocation of the pleckstrin homology domain of PLCdelta1 fused to green fluorescent protein (PH-GFP). The observed time course could be simulated by incorporating binding of PIP2 and InsP3 to PH-GFP into the model that had been used to analyze the biochemistry. Furthermore, this analysis could help to resolve a controversy over whether the translocation of PH-GFP from membrane to cytosol is due to a decrease in PIP2 on the membrane or an increase in InsP3 in cytosol; by computationally clamping the concentrations of each of these compounds, the model shows how both contribute to the dynamics of probe translocation.

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Compartmental simulation of the response of the steady-state system to an instantaneous bolus of 1 μM InsP3. The same compartmental model was used, except that the bradykinin stimulus was never applied. Instead, InsP3 was stepped from the basal level of 0.16 μM to 1.16 μM at time 0. This was equivalent to a rapid injection or a pulse of photorelease. The response of PH-GFP translocation is displayed for three initial total cytosolic concentrations of the indicator. The central value of 6 μM (blue curve) corresponds to the average expression level in our cells.
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fig9: Compartmental simulation of the response of the steady-state system to an instantaneous bolus of 1 μM InsP3. The same compartmental model was used, except that the bradykinin stimulus was never applied. Instead, InsP3 was stepped from the basal level of 0.16 μM to 1.16 μM at time 0. This was equivalent to a rapid injection or a pulse of photorelease. The response of PH-GFP translocation is displayed for three initial total cytosolic concentrations of the indicator. The central value of 6 μM (blue curve) corresponds to the average expression level in our cells.

Mentions: We also used the model to examine the contrasting results that were reported when InsP3 is introduced into cells in the absence of receptor-mediated activation of PLC. Hirose et al. (1999) reported that injection of 1 μM InsP3 could produce large translocation of PH-GFP, whereas van der Wal et al. (2001) used photorelease of caged InsP3 to reach the opposite conclusion. Fig. 9 shows a series of simulations of the translocation response to a 1-μM instantaneous bolus of InsP3 at time 0 in the absence of phosphoinositide turnover. The three traces correspond to differing initial concentrations of total cytosolic PH-GFP, with the central concentration (6 μM) corresponding to the average concentration of the probe measured in our experiments. As can be seen, the translocation produced by 1 μM InsP3 in the presence of 6 μM total cytosolic PH-GFP is predicted to be much smaller than that produced in the full model of receptor-activated phosphoinositide turnover (Fig. 8, black trace). However, the effect is very sensitive to the expression level of the indicator over a 10-fold range. Hirose et al. (1999) and van der Wal et al. (2001) did not report the concentrations of indicator in their respective experiments. So, the difference in their results may be a consequence of a low PH-GFP expression level in the former and a high expression level in the latter. In conclusion, our results suggest that the relative or percent change in translocation after stimulated phosphoinositide turnover will be higher with lower concentrations of PH-GFP, when the PH-GFP will not buffer a significant fraction of either membrane PIP2 or cytosolic IP3. However, at very low concentrations, the intensity of the fluorescence may, of course, become limiting.


Kinetic analysis of receptor-activated phosphoinositide turnover.

Xu C, Watras J, Loew LM - J. Cell Biol. (2003)

Compartmental simulation of the response of the steady-state system to an instantaneous bolus of 1 μM InsP3. The same compartmental model was used, except that the bradykinin stimulus was never applied. Instead, InsP3 was stepped from the basal level of 0.16 μM to 1.16 μM at time 0. This was equivalent to a rapid injection or a pulse of photorelease. The response of PH-GFP translocation is displayed for three initial total cytosolic concentrations of the indicator. The central value of 6 μM (blue curve) corresponds to the average expression level in our cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199375&req=5

fig9: Compartmental simulation of the response of the steady-state system to an instantaneous bolus of 1 μM InsP3. The same compartmental model was used, except that the bradykinin stimulus was never applied. Instead, InsP3 was stepped from the basal level of 0.16 μM to 1.16 μM at time 0. This was equivalent to a rapid injection or a pulse of photorelease. The response of PH-GFP translocation is displayed for three initial total cytosolic concentrations of the indicator. The central value of 6 μM (blue curve) corresponds to the average expression level in our cells.
Mentions: We also used the model to examine the contrasting results that were reported when InsP3 is introduced into cells in the absence of receptor-mediated activation of PLC. Hirose et al. (1999) reported that injection of 1 μM InsP3 could produce large translocation of PH-GFP, whereas van der Wal et al. (2001) used photorelease of caged InsP3 to reach the opposite conclusion. Fig. 9 shows a series of simulations of the translocation response to a 1-μM instantaneous bolus of InsP3 at time 0 in the absence of phosphoinositide turnover. The three traces correspond to differing initial concentrations of total cytosolic PH-GFP, with the central concentration (6 μM) corresponding to the average concentration of the probe measured in our experiments. As can be seen, the translocation produced by 1 μM InsP3 in the presence of 6 μM total cytosolic PH-GFP is predicted to be much smaller than that produced in the full model of receptor-activated phosphoinositide turnover (Fig. 8, black trace). However, the effect is very sensitive to the expression level of the indicator over a 10-fold range. Hirose et al. (1999) and van der Wal et al. (2001) did not report the concentrations of indicator in their respective experiments. So, the difference in their results may be a consequence of a low PH-GFP expression level in the former and a high expression level in the latter. In conclusion, our results suggest that the relative or percent change in translocation after stimulated phosphoinositide turnover will be higher with lower concentrations of PH-GFP, when the PH-GFP will not buffer a significant fraction of either membrane PIP2 or cytosolic IP3. However, at very low concentrations, the intensity of the fluorescence may, of course, become limiting.

Bottom Line: Phosphatidylinositol-4,5-bisphosphate (PIP2) decreased over the first 30 s, and then recovered over the following 2-3 min.This was subsequently confirmed experimentally.Furthermore, this analysis could help to resolve a controversy over whether the translocation of PH-GFP from membrane to cytosol is due to a decrease in PIP2 on the membrane or an increase in InsP3 in cytosol; by computationally clamping the concentrations of each of these compounds, the model shows how both contribute to the dynamics of probe translocation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Connecticut Health Center, Farmington, CT 06030, USA.

ABSTRACT
We studied the bradykinin-induced changes in phosphoinositide composition of N1E-115 neuroblastoma cells using a combination of biochemistry, microscope imaging, and mathematical modeling. Phosphatidylinositol-4,5-bisphosphate (PIP2) decreased over the first 30 s, and then recovered over the following 2-3 min. However, the rate and amount of inositol-1,4,5-trisphosphate (InsP3) production were much greater than the rate or amount of PIP2 decline. A mathematical model of phosphoinositide turnover based on this data predicted that PIP2 synthesis is also stimulated by bradykinin, causing an early transient increase in its concentration. This was subsequently confirmed experimentally. Then, we used single-cell microscopy to further examine phosphoinositide turnover by following the translocation of the pleckstrin homology domain of PLCdelta1 fused to green fluorescent protein (PH-GFP). The observed time course could be simulated by incorporating binding of PIP2 and InsP3 to PH-GFP into the model that had been used to analyze the biochemistry. Furthermore, this analysis could help to resolve a controversy over whether the translocation of PH-GFP from membrane to cytosol is due to a decrease in PIP2 on the membrane or an increase in InsP3 in cytosol; by computationally clamping the concentrations of each of these compounds, the model shows how both contribute to the dynamics of probe translocation.

Show MeSH
Related in: MedlinePlus