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Morphological control of inositol-1,4,5-trisphosphate-dependent signals.

Fink CC, Slepchenko B, Moraru II, Schaff J, Watras J, Loew LM - J. Cell Biol. (1999)

Bottom Line: We conclude that the characteristic calcium dynamics requires rapid, high-amplitude production of [InsP(3)](cyt) in the neurite.This requisite InsP(3) spatiotemporal profile is provided, in turn, as an intrinsic consequence of the cell's morphology, demonstrating how geometry can locally and dramatically intensify cytosolic signals that originate at the plasma membrane.In addition, the model predicts, and experiments confirm, that stimulation of just the neurite, but not the soma or growth cone, is sufficient to generate a calcium response throughout the cell.

View Article: PubMed Central - PubMed

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

ABSTRACT
Inositol-1,4,5-trisphosphate (InsP(3))-mediated calcium signals represent an important mechanism for transmitting external stimuli to the cell. However, information about intracellular spatial patterns of InsP(3) itself is not generally available. In particular, it has not been determined how the interplay of InsP(3) generation, diffusion, and degradation within complex cellular geometries can control the patterns of InsP(3) signaling. Here, we explore the spatial and temporal characteristics of [InsP(3)](cyt) during a bradykinin-induced calcium wave in a neuroblastoma cell. This is achieved by using a unique image-based computer modeling system, Virtual Cell, to integrate experimental data on the rates and spatial distributions of the key molecular components of the process. We conclude that the characteristic calcium dynamics requires rapid, high-amplitude production of [InsP(3)](cyt) in the neurite. This requisite InsP(3) spatiotemporal profile is provided, in turn, as an intrinsic consequence of the cell's morphology, demonstrating how geometry can locally and dramatically intensify cytosolic signals that originate at the plasma membrane. In addition, the model predicts, and experiments confirm, that stimulation of just the neurite, but not the soma or growth cone, is sufficient to generate a calcium response throughout the cell.

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Modeling of observed calcium dynamics in N1E-115 neuroblastoma cells. a, Distributions of BKR and ER (SERCA2/InsP31-R) used for modeling. Cells were stained for antibodies against BKR, SERCA2, InsP31-R, and ER. Relative fluorescence intensities were measured on the confocal microscope and adjusted for convolution artifacts (Fink et al. 1998). Intensities were scaled such that the left half soma is equal to 1.0, and plotted (mean ± SEM; n = 20 cells) for six representative regions of the cell. There was no statistical difference (ANOVA; P > 0.05) between the distributions of InsP31-R, SERCA2, and ER, so they were plotted together. b, Simulation results for [Ca2+] and [InsP3] in N1E-115 neuroblastoma cells (as modeled in a). Simulations were run for three conditions: global application of a saturating concentration of BK with the average receptor and ER distribution as shown in a; global application of a saturating concentration of BK with average ER (and InsP3-R and SERCA) distributions, but with a uniform plasma membrane BKR distribution; and a step increase of [InsP3] to 1.5 μM uniformly throughout the cytosol analogous to the uncaging experiment in Fig. 1 b. [InsP3] and [Ca2+] have been pseudocolor scaled according to the color bar at the top of each column. Time is seconds after the simulated stimulus event (either BK exposure or InsP3 uncaging). Below each column are plots of [InsP3] or [Ca2+] versus time for a point in the soma or the neurite (indicated by the yellow or green dots on the 1.0 s image of the first column). For the [InsP3] versus time plot for the average receptor distribution experiment, also plotted are the [InsP3] values indicative of the average [InsP3] for the whole cell (dotted white line) and the experimental [InsP3] values determined with radioligand binding as shown in Fig. 2 d. This plot is taken to 30 s, whereas the other plots are all to 12 s.
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Figure 3: Modeling of observed calcium dynamics in N1E-115 neuroblastoma cells. a, Distributions of BKR and ER (SERCA2/InsP31-R) used for modeling. Cells were stained for antibodies against BKR, SERCA2, InsP31-R, and ER. Relative fluorescence intensities were measured on the confocal microscope and adjusted for convolution artifacts (Fink et al. 1998). Intensities were scaled such that the left half soma is equal to 1.0, and plotted (mean ± SEM; n = 20 cells) for six representative regions of the cell. There was no statistical difference (ANOVA; P > 0.05) between the distributions of InsP31-R, SERCA2, and ER, so they were plotted together. b, Simulation results for [Ca2+] and [InsP3] in N1E-115 neuroblastoma cells (as modeled in a). Simulations were run for three conditions: global application of a saturating concentration of BK with the average receptor and ER distribution as shown in a; global application of a saturating concentration of BK with average ER (and InsP3-R and SERCA) distributions, but with a uniform plasma membrane BKR distribution; and a step increase of [InsP3] to 1.5 μM uniformly throughout the cytosol analogous to the uncaging experiment in Fig. 1 b. [InsP3] and [Ca2+] have been pseudocolor scaled according to the color bar at the top of each column. Time is seconds after the simulated stimulus event (either BK exposure or InsP3 uncaging). Below each column are plots of [InsP3] or [Ca2+] versus time for a point in the soma or the neurite (indicated by the yellow or green dots on the 1.0 s image of the first column). For the [InsP3] versus time plot for the average receptor distribution experiment, also plotted are the [InsP3] values indicative of the average [InsP3] for the whole cell (dotted white line) and the experimental [InsP3] values determined with radioligand binding as shown in Fig. 2 d. This plot is taken to 30 s, whereas the other plots are all to 12 s.

Mentions: To understand how the neurite produces a higher InsP3 signal than the soma, and why it requires this higher concentration of InsP3 to set off a calcium wave, we used a computational system for cell biological modeling (Schaff et al. 1997) to construct a model based on experimental data for geometrical, electrophysiological, and biochemical components of the system. In Fig. 3 a, the geometric distributions of critical receptors (BK, InsP3, and SERCA) are mapped onto a geometry based on the cell in Fig. 1 a. This information was compiled through analysis of confocal micrographs of immunofluorescence distribution, using the quantitative procedures developed by Fink et al. 1998. The other inputs to the model comprised the individual biochemical and electrophysiological processes contributing to the BK-induced calcium wave. These included: flux of InsP3 into the cytosol from the plasma membrane; rate of InsP3 degradation; calcium uptake rate of SERCA pumps; [InsP3]cyt and [Ca2+]cyt binding to the InsP3-receptor and the consequent activation and inactivation of calcium efflux from the ER; calcium buffering in the cytosol by mobile and fixed buffers; and diffusion coefficients for InsP3, mobile buffers, and calcium.


Morphological control of inositol-1,4,5-trisphosphate-dependent signals.

Fink CC, Slepchenko B, Moraru II, Schaff J, Watras J, Loew LM - J. Cell Biol. (1999)

Modeling of observed calcium dynamics in N1E-115 neuroblastoma cells. a, Distributions of BKR and ER (SERCA2/InsP31-R) used for modeling. Cells were stained for antibodies against BKR, SERCA2, InsP31-R, and ER. Relative fluorescence intensities were measured on the confocal microscope and adjusted for convolution artifacts (Fink et al. 1998). Intensities were scaled such that the left half soma is equal to 1.0, and plotted (mean ± SEM; n = 20 cells) for six representative regions of the cell. There was no statistical difference (ANOVA; P > 0.05) between the distributions of InsP31-R, SERCA2, and ER, so they were plotted together. b, Simulation results for [Ca2+] and [InsP3] in N1E-115 neuroblastoma cells (as modeled in a). Simulations were run for three conditions: global application of a saturating concentration of BK with the average receptor and ER distribution as shown in a; global application of a saturating concentration of BK with average ER (and InsP3-R and SERCA) distributions, but with a uniform plasma membrane BKR distribution; and a step increase of [InsP3] to 1.5 μM uniformly throughout the cytosol analogous to the uncaging experiment in Fig. 1 b. [InsP3] and [Ca2+] have been pseudocolor scaled according to the color bar at the top of each column. Time is seconds after the simulated stimulus event (either BK exposure or InsP3 uncaging). Below each column are plots of [InsP3] or [Ca2+] versus time for a point in the soma or the neurite (indicated by the yellow or green dots on the 1.0 s image of the first column). For the [InsP3] versus time plot for the average receptor distribution experiment, also plotted are the [InsP3] values indicative of the average [InsP3] for the whole cell (dotted white line) and the experimental [InsP3] values determined with radioligand binding as shown in Fig. 2 d. This plot is taken to 30 s, whereas the other plots are all to 12 s.
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Related In: Results  -  Collection

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Figure 3: Modeling of observed calcium dynamics in N1E-115 neuroblastoma cells. a, Distributions of BKR and ER (SERCA2/InsP31-R) used for modeling. Cells were stained for antibodies against BKR, SERCA2, InsP31-R, and ER. Relative fluorescence intensities were measured on the confocal microscope and adjusted for convolution artifacts (Fink et al. 1998). Intensities were scaled such that the left half soma is equal to 1.0, and plotted (mean ± SEM; n = 20 cells) for six representative regions of the cell. There was no statistical difference (ANOVA; P > 0.05) between the distributions of InsP31-R, SERCA2, and ER, so they were plotted together. b, Simulation results for [Ca2+] and [InsP3] in N1E-115 neuroblastoma cells (as modeled in a). Simulations were run for three conditions: global application of a saturating concentration of BK with the average receptor and ER distribution as shown in a; global application of a saturating concentration of BK with average ER (and InsP3-R and SERCA) distributions, but with a uniform plasma membrane BKR distribution; and a step increase of [InsP3] to 1.5 μM uniformly throughout the cytosol analogous to the uncaging experiment in Fig. 1 b. [InsP3] and [Ca2+] have been pseudocolor scaled according to the color bar at the top of each column. Time is seconds after the simulated stimulus event (either BK exposure or InsP3 uncaging). Below each column are plots of [InsP3] or [Ca2+] versus time for a point in the soma or the neurite (indicated by the yellow or green dots on the 1.0 s image of the first column). For the [InsP3] versus time plot for the average receptor distribution experiment, also plotted are the [InsP3] values indicative of the average [InsP3] for the whole cell (dotted white line) and the experimental [InsP3] values determined with radioligand binding as shown in Fig. 2 d. This plot is taken to 30 s, whereas the other plots are all to 12 s.
Mentions: To understand how the neurite produces a higher InsP3 signal than the soma, and why it requires this higher concentration of InsP3 to set off a calcium wave, we used a computational system for cell biological modeling (Schaff et al. 1997) to construct a model based on experimental data for geometrical, electrophysiological, and biochemical components of the system. In Fig. 3 a, the geometric distributions of critical receptors (BK, InsP3, and SERCA) are mapped onto a geometry based on the cell in Fig. 1 a. This information was compiled through analysis of confocal micrographs of immunofluorescence distribution, using the quantitative procedures developed by Fink et al. 1998. The other inputs to the model comprised the individual biochemical and electrophysiological processes contributing to the BK-induced calcium wave. These included: flux of InsP3 into the cytosol from the plasma membrane; rate of InsP3 degradation; calcium uptake rate of SERCA pumps; [InsP3]cyt and [Ca2+]cyt binding to the InsP3-receptor and the consequent activation and inactivation of calcium efflux from the ER; calcium buffering in the cytosol by mobile and fixed buffers; and diffusion coefficients for InsP3, mobile buffers, and calcium.

Bottom Line: We conclude that the characteristic calcium dynamics requires rapid, high-amplitude production of [InsP(3)](cyt) in the neurite.This requisite InsP(3) spatiotemporal profile is provided, in turn, as an intrinsic consequence of the cell's morphology, demonstrating how geometry can locally and dramatically intensify cytosolic signals that originate at the plasma membrane.In addition, the model predicts, and experiments confirm, that stimulation of just the neurite, but not the soma or growth cone, is sufficient to generate a calcium response throughout the cell.

View Article: PubMed Central - PubMed

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

ABSTRACT
Inositol-1,4,5-trisphosphate (InsP(3))-mediated calcium signals represent an important mechanism for transmitting external stimuli to the cell. However, information about intracellular spatial patterns of InsP(3) itself is not generally available. In particular, it has not been determined how the interplay of InsP(3) generation, diffusion, and degradation within complex cellular geometries can control the patterns of InsP(3) signaling. Here, we explore the spatial and temporal characteristics of [InsP(3)](cyt) during a bradykinin-induced calcium wave in a neuroblastoma cell. This is achieved by using a unique image-based computer modeling system, Virtual Cell, to integrate experimental data on the rates and spatial distributions of the key molecular components of the process. We conclude that the characteristic calcium dynamics requires rapid, high-amplitude production of [InsP(3)](cyt) in the neurite. This requisite InsP(3) spatiotemporal profile is provided, in turn, as an intrinsic consequence of the cell's morphology, demonstrating how geometry can locally and dramatically intensify cytosolic signals that originate at the plasma membrane. In addition, the model predicts, and experiments confirm, that stimulation of just the neurite, but not the soma or growth cone, is sufficient to generate a calcium response throughout the cell.

Show MeSH
Related in: MedlinePlus