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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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Related in: MedlinePlus

Results of an image-based spatial simulation of phosphoinositide turnover after bradykinin-induced stimulation. The simulations were performed as in Fig. 6, except that PH-GFP was not included in the system. (Top row) Concentration of InsP3 in the cytosol. (Second row) Selected time points for the surface density of PIP2. The same scales were used as in the bottom two rows of Fig. 6 to facilitate comparison.
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fig7: Results of an image-based spatial simulation of phosphoinositide turnover after bradykinin-induced stimulation. The simulations were performed as in Fig. 6, except that PH-GFP was not included in the system. (Top row) Concentration of InsP3 in the cytosol. (Second row) Selected time points for the surface density of PIP2. The same scales were used as in the bottom two rows of Fig. 6 to facilitate comparison.

Mentions: It is also instructive to compare the changes in PIP2 and InsP3 in the presence of PH-GFP, shown in the bottom two rows of Fig. 6, with the changes that the model calculates when the PH-GFP is removed, shown in Fig. 7. The simulation results in Fig. 7 are the result of simply setting the concentrations of all of the PH-GFP molecular species to zero and are mathematically equivalent to the application of the nonspatial model of Fig. 1 to our two-dimensional geometry. As can be seen, both the rates and the amplitudes of the changes in InsP3 and PIP2 are severely reduced in the presence of 6 μM PH-GFP, which effectively acts as a buffer for both of these molecules. These simulations suggest that caution is required in interpreting the physiology of PH-GFP–transfected cells. Indeed, the ability of PH-GFP to impede access of PLC to PIP2 was recognized in one of the original papers that described this probe (Varnai and Balla, 1998). Whether PHδ1-GFP inhibits InsP3-dependent calcium release has not been reported to our knowledge, although it has been reported for another PH domain isoform (Varnai et al., 2002).


Kinetic analysis of receptor-activated phosphoinositide turnover.

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

Results of an image-based spatial simulation of phosphoinositide turnover after bradykinin-induced stimulation. The simulations were performed as in Fig. 6, except that PH-GFP was not included in the system. (Top row) Concentration of InsP3 in the cytosol. (Second row) Selected time points for the surface density of PIP2. The same scales were used as in the bottom two rows of Fig. 6 to facilitate comparison.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Results of an image-based spatial simulation of phosphoinositide turnover after bradykinin-induced stimulation. The simulations were performed as in Fig. 6, except that PH-GFP was not included in the system. (Top row) Concentration of InsP3 in the cytosol. (Second row) Selected time points for the surface density of PIP2. The same scales were used as in the bottom two rows of Fig. 6 to facilitate comparison.
Mentions: It is also instructive to compare the changes in PIP2 and InsP3 in the presence of PH-GFP, shown in the bottom two rows of Fig. 6, with the changes that the model calculates when the PH-GFP is removed, shown in Fig. 7. The simulation results in Fig. 7 are the result of simply setting the concentrations of all of the PH-GFP molecular species to zero and are mathematically equivalent to the application of the nonspatial model of Fig. 1 to our two-dimensional geometry. As can be seen, both the rates and the amplitudes of the changes in InsP3 and PIP2 are severely reduced in the presence of 6 μM PH-GFP, which effectively acts as a buffer for both of these molecules. These simulations suggest that caution is required in interpreting the physiology of PH-GFP–transfected cells. Indeed, the ability of PH-GFP to impede access of PLC to PIP2 was recognized in one of the original papers that described this probe (Varnai and Balla, 1998). Whether PHδ1-GFP inhibits InsP3-dependent calcium release has not been reported to our knowledge, although it has been reported for another PH domain isoform (Varnai et al., 2002).

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