Kinetic analysis of receptor-activated phosphoinositide turnover

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.

Figure 1.

Experimental and simulated time courses of bradykinin-induced changes of PIP₂ and PIP in N1E-115 cells. [³H]inositol-prelabeled N1E-115 cells were incubated in the presence of 1 μM bradykinin for the indicated times. Membrane lipids were extracted and analyzed as described in Materials and methods. Each data point is the mean ± SEM of three to five experiments. All experiments were performed at RT. Black diamonds represent our initial determination of PIP₂ changes; purple triangles are the PIP data. These data, along with our prior study of InsP₃ dynamics in this cell, were used to constrain a model that produced the respective black and purple solid curves. The pathways that were modeled are shown in Fig. 2, and the model equations and parameters are described in the . The prediction of the model that there was an initial increase in [PIP₂] led us to determine the change in PIP₂ at 5 s, shown as a red diamond. The model calculation of the change in [InsP₃] is shown in the inset.

Figure 2.

Reaction scheme for the synthesis and hydrolysis of PIP₂. The membrane-associated reactions are shown at the top, and the cytosol reactions on the bottom. These reaction schemes form the basis of the mathematical models that were developed to analyze the data. Details of the rate expressions for each of the reactions (shown in red) are provided in the .

Figure 3.

Bradykinin-induced translocation of PH-GFP from the plasma membrane in a single N1E-115 cell. A single N1E-115 cell was stimulated with 1 μM bradykinin. PH-GFP translocation was reflected as a decrease in the membrane GFP fluorescence and a concomitant increase in cytosolic fluorescence. The experiment was performed at RT. (A) Time series of images with the time indicated on each frame in seconds. Bradykinin was added at time 0 (after the third frame). (B) Relative change in GFP fluorescence at two locations in the cytosol of the cell in the images in A. Region 1 is indicated in the first frame of A by the rectangle just above the nucleus; region 2 is indicated by the rectangle in the larger area of cytosol below the nucleus.

Figure 4.

Kinetics of bradykinin-induced PH-GFP translocation. The average changes in membrane and cytosol fluorescence after addition of 1 μM bradykinin are plotted versus time. Each point was the mean ± SEM of 19 experiments.

Figure 5.

Simulation of PH-GFP translocation. The compartmental model used in Fig. 1 was expanded to include PH-GFP binding. Additional parameters are in Table AII, and model equations are in the . The blue curve represents the relative change in total cytosolic GFP (free PH-GFP + InsP₃–PH-GFP), and can be directly compared with the corresponding experimental results in Fig. 4. The pink curve corresponds to the relative change in PIP₂–PH-GFP surface density on the plasma membrane. Because the confocal imaging system detects both fluorescence from the membrane and the adjacent cytosol, the PIP₂–PH-GFP results were adjusted by an appropriate contribution from the total cytosolic signal in the yellow curve, as detailed in the text, to allow comparison with the experimental result in Fig. 4.

Figure 6.

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.

Figure 7.

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.

Figure 8.

Compartmental simulations with fixed InsP₃ or PIP₂. The model of Fig. 5 was used, except that InsP₃ (green) or PIP₂ (blue) are clamped at their initial values. Only the change in total cytosolic PH-GFP is shown because this is the only model output that may be directly compared with the corresponding experimental result (shown in Fig. 4) as explained in the text. Also shown for comparison is the result produced by the full model in which all molecules are allowed to vary (black curve).

Figure 9.

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.