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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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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.
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fig3: 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.

Mentions: Knowing that the kinetics of bradykinin-induced decrease in membrane PIP2 and increase in cytosolic InsP3 were very different in terms of both time-to-peak and recovery, we tried to determine whether PH-GFP translocation mimicked changes in PIP2 or InsP3 in single cells. When expressed in N1E-115 cells, PH-GFP showed a strong accumulation at the plasma membrane and a low and homogenous distribution in the cytosol (Fig. 3), consistent with the idea that PH-GFP primarily binds to membrane PIP2 at rest. Several explanations for the low resting intracellular PH-GFP fluorescence are possible: (1) intracellular pools of PIP2 are not accessible to PH-GFP (Balla et al., 2000); (2) some intracellular PIP2 phosphatases (e.g., synaptojanin) have hydrolyzed the PIP2 on the internal membranes and restricted the steady-state accumulation of PIP2 to the plasma membrane (Stefan et al., 2002); or (3) a significant amount of PIP2 is on the organelle membranes and can bind PH-GFP (Watt et al., 2002), but its lower volumetric density compared with the plasma membrane produces a lower fluorescence. In this work, we measured fluorescence from membrane GFP, segmented by a combination of threshold operations and manual editing, and cytosolic fluorescence regions of interest individually for each time point; we did not use the more common procedure of measuring the amplitude of the fluorescence signal at the plasma membrane and cytosol region along a line across the cell for all time points because we found that bradykinin could induce cell shape changes, and hence, changes of the originally assigned membrane regions. After addition of bradykinin, there was a decrease in plasma membrane PH-GFP fluorescence and a concomitant increase in cytosolic fluorescence (Fig. 3 and Fig. 4). The maximum relative change in membrane GFP fluorescence was −11.3 ± 1.7% (n = 19), whereas the relative change in cytosolic fluorescence was 41.1 ± 4.7% (n = 19). The kinetics of bradykinin-induced PH-GFP translocation is characterized by a rapid onset, with translocation peaking at ∼20 to 30 s and returning to the baseline in ∼3 min in the continued presence of bradykinin (Fig. 3). This time course is very similar to the time course of PIP2 hydrolysis detected biochemically from [3H]inositol-labeled cells, in line with the previous finding that PH-GFP translocation primarily reports changes in membrane PIP2 content (van der Wal et al., 2001). However, the bradykinin-induced PH-GFP translocation did not exhibit an initial increase in PIP2, raising the possibility that PH-GFP translocation might not be determined solely by changes in membrane PIP2.


Kinetic analysis of receptor-activated phosphoinositide turnover.

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

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.
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Related In: Results  -  Collection

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fig3: 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.
Mentions: Knowing that the kinetics of bradykinin-induced decrease in membrane PIP2 and increase in cytosolic InsP3 were very different in terms of both time-to-peak and recovery, we tried to determine whether PH-GFP translocation mimicked changes in PIP2 or InsP3 in single cells. When expressed in N1E-115 cells, PH-GFP showed a strong accumulation at the plasma membrane and a low and homogenous distribution in the cytosol (Fig. 3), consistent with the idea that PH-GFP primarily binds to membrane PIP2 at rest. Several explanations for the low resting intracellular PH-GFP fluorescence are possible: (1) intracellular pools of PIP2 are not accessible to PH-GFP (Balla et al., 2000); (2) some intracellular PIP2 phosphatases (e.g., synaptojanin) have hydrolyzed the PIP2 on the internal membranes and restricted the steady-state accumulation of PIP2 to the plasma membrane (Stefan et al., 2002); or (3) a significant amount of PIP2 is on the organelle membranes and can bind PH-GFP (Watt et al., 2002), but its lower volumetric density compared with the plasma membrane produces a lower fluorescence. In this work, we measured fluorescence from membrane GFP, segmented by a combination of threshold operations and manual editing, and cytosolic fluorescence regions of interest individually for each time point; we did not use the more common procedure of measuring the amplitude of the fluorescence signal at the plasma membrane and cytosol region along a line across the cell for all time points because we found that bradykinin could induce cell shape changes, and hence, changes of the originally assigned membrane regions. After addition of bradykinin, there was a decrease in plasma membrane PH-GFP fluorescence and a concomitant increase in cytosolic fluorescence (Fig. 3 and Fig. 4). The maximum relative change in membrane GFP fluorescence was −11.3 ± 1.7% (n = 19), whereas the relative change in cytosolic fluorescence was 41.1 ± 4.7% (n = 19). The kinetics of bradykinin-induced PH-GFP translocation is characterized by a rapid onset, with translocation peaking at ∼20 to 30 s and returning to the baseline in ∼3 min in the continued presence of bradykinin (Fig. 3). This time course is very similar to the time course of PIP2 hydrolysis detected biochemically from [3H]inositol-labeled cells, in line with the previous finding that PH-GFP translocation primarily reports changes in membrane PIP2 content (van der Wal et al., 2001). However, the bradykinin-induced PH-GFP translocation did not exhibit an initial increase in PIP2, raising the possibility that PH-GFP translocation might not be determined solely by changes in membrane PIP2.

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