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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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Results of an image-based spatial simulation of PH-GFP translocation after bradykinin-induced stimulation. The image of the cell on the right side of the images in Fig. 3 was used as the basis of the two-dimensional geometry. The cytosolic resting level of PH-GFP that was measured for this cell was 4.6 μM, and this was the value used in the simulation. Other parameters were similar to the compartmental simulations (Table AI and Table AII). Details of the simulation are given in the . (Top row) Selected time points for the relative changes in total cytosolic PH-GFP (free + bound to InsP3). (Second row) Percent change in PH-GFP associated with PIP2 in the plasma membrane. (Third row) Concentration of free InsP3, indicated by the color bar in unit of μM, in the cytosol showing the buffering effect of PH-GFP as described in the text. (Fourth row) Surface density of PIP2, indicated by the color scale in molecules/μm2, also showing the buffering effect of the indicator.
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fig6: Results of an image-based spatial simulation of PH-GFP translocation after bradykinin-induced stimulation. The image of the cell on the right side of the images in Fig. 3 was used as the basis of the two-dimensional geometry. The cytosolic resting level of PH-GFP that was measured for this cell was 4.6 μM, and this was the value used in the simulation. Other parameters were similar to the compartmental simulations (Table AI and Table AII). Details of the simulation are given in the . (Top row) Selected time points for the relative changes in total cytosolic PH-GFP (free + bound to InsP3). (Second row) Percent change in PH-GFP associated with PIP2 in the plasma membrane. (Third row) Concentration of free InsP3, indicated by the color bar in unit of μM, in the cytosol showing the buffering effect of PH-GFP as described in the text. (Fourth row) Surface density of PIP2, indicated by the color scale in molecules/μm2, also showing the buffering effect of the indicator.

Mentions: We elaborated the model used to analyze the biochemical data on inositide turnover to see if it could also predict the PH-GFP translocation experiments. The basal concentration of total PH-GFP in the cytosol (free plus bound to InsP3) was taken as 6 μM from our measurements. Binding constants to InsP3 and PIP2 were taken from in vitro measurements reported in the literature (Hirose et al., 1999). Taken with the basal levels of InsP3 and PIP2, these determined the basal levels of the bound forms of PH-GFP. Thus, all the additional model parameters were completely based on measured values (albeit in vitro), and no parameters were adjusted to fit the results of Fig. 4. All these parameters are provided in Table AII of the . The results of a compartmental model, elaborated from the one used in Fig. 1, are shown in Fig. 5. The results of a two-dimensional spatial simulation, based on the geometry of one of the cells of Fig. 3, are shown in Fig. 6.


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 PH-GFP translocation after bradykinin-induced stimulation. The image of the cell on the right side of the images in Fig. 3 was used as the basis of the two-dimensional geometry. The cytosolic resting level of PH-GFP that was measured for this cell was 4.6 μM, and this was the value used in the simulation. Other parameters were similar to the compartmental simulations (Table AI and Table AII). Details of the simulation are given in the . (Top row) Selected time points for the relative changes in total cytosolic PH-GFP (free + bound to InsP3). (Second row) Percent change in PH-GFP associated with PIP2 in the plasma membrane. (Third row) Concentration of free InsP3, indicated by the color bar in unit of μM, in the cytosol showing the buffering effect of PH-GFP as described in the text. (Fourth row) Surface density of PIP2, indicated by the color scale in molecules/μm2, also showing the buffering effect of the indicator.
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Related In: Results  -  Collection

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fig6: Results of an image-based spatial simulation of PH-GFP translocation after bradykinin-induced stimulation. The image of the cell on the right side of the images in Fig. 3 was used as the basis of the two-dimensional geometry. The cytosolic resting level of PH-GFP that was measured for this cell was 4.6 μM, and this was the value used in the simulation. Other parameters were similar to the compartmental simulations (Table AI and Table AII). Details of the simulation are given in the . (Top row) Selected time points for the relative changes in total cytosolic PH-GFP (free + bound to InsP3). (Second row) Percent change in PH-GFP associated with PIP2 in the plasma membrane. (Third row) Concentration of free InsP3, indicated by the color bar in unit of μM, in the cytosol showing the buffering effect of PH-GFP as described in the text. (Fourth row) Surface density of PIP2, indicated by the color scale in molecules/μm2, also showing the buffering effect of the indicator.
Mentions: We elaborated the model used to analyze the biochemical data on inositide turnover to see if it could also predict the PH-GFP translocation experiments. The basal concentration of total PH-GFP in the cytosol (free plus bound to InsP3) was taken as 6 μM from our measurements. Binding constants to InsP3 and PIP2 were taken from in vitro measurements reported in the literature (Hirose et al., 1999). Taken with the basal levels of InsP3 and PIP2, these determined the basal levels of the bound forms of PH-GFP. Thus, all the additional model parameters were completely based on measured values (albeit in vitro), and no parameters were adjusted to fit the results of Fig. 4. All these parameters are provided in Table AII of the . The results of a compartmental model, elaborated from the one used in Fig. 1, are shown in Fig. 5. The results of a two-dimensional spatial simulation, based on the geometry of one of the cells of Fig. 3, are shown in Fig. 6.

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