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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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Measurement of [InsP3]cyt. a, Cells were microinjected with CG-1 and NPE-InsP3, and CG-1 fluorescence was measured on the confocal microscope. Fluorescence intensity values were converted to relative [Ca2+] and plotted against time. The cell was subjected, in turn, to increasing doses of InsP3 released by UV light flashes of 20–80 ms, 500 nM BK application to the medium, and treatment with 10 μM ionomycin. A typical experiment, in which the neurite signal is followed, is shown here. The gray line connecting the tracing after addition of ionomycin indicates a period during which the cell was refocused. b, For two cells where neurite (blue) and soma (red) are monitored, relative peak [Ca2+] for each uncaging event has been plotted against [InsP3]uncaged (solid circles). The data has been fit (solid curve) with the Hill equation (R = 0.99). A dashed horizontal line was drawn indicating the [Ca2+]cyt response produced by BK stimulation in each region. A corresponding pair of lines were dropped from the Hill equation curve, indicating the [InsP3] necessary to produce the BK-induced calcium peak. c, Mean values (± SEM) of estimated InsP3 production by BK in soma (black) and neurite (gray) are compared in a bar graph (P < 0.001). d, [InsP3]cyt was measured in a N1E-115 neuroblastoma cell population during a BK-induced calcium wave using competitive radioligand binding.
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Figure 2: Measurement of [InsP3]cyt. a, Cells were microinjected with CG-1 and NPE-InsP3, and CG-1 fluorescence was measured on the confocal microscope. Fluorescence intensity values were converted to relative [Ca2+] and plotted against time. The cell was subjected, in turn, to increasing doses of InsP3 released by UV light flashes of 20–80 ms, 500 nM BK application to the medium, and treatment with 10 μM ionomycin. A typical experiment, in which the neurite signal is followed, is shown here. The gray line connecting the tracing after addition of ionomycin indicates a period during which the cell was refocused. b, For two cells where neurite (blue) and soma (red) are monitored, relative peak [Ca2+] for each uncaging event has been plotted against [InsP3]uncaged (solid circles). The data has been fit (solid curve) with the Hill equation (R = 0.99). A dashed horizontal line was drawn indicating the [Ca2+]cyt response produced by BK stimulation in each region. A corresponding pair of lines were dropped from the Hill equation curve, indicating the [InsP3] necessary to produce the BK-induced calcium peak. c, Mean values (± SEM) of estimated InsP3 production by BK in soma (black) and neurite (gray) are compared in a bar graph (P < 0.001). d, [InsP3]cyt was measured in a N1E-115 neuroblastoma cell population during a BK-induced calcium wave using competitive radioligand binding.

Mentions: Further, to determine the dependence of the [Ca2+]cyt signal on [InsP3]cyt in each region of the cell, we performed quantitative InsP3 uncaging experiments (Khodakhah and Ogden 1993; Fink et al. 1999), followed by application of BK to the same cells. A dose-response relationship can be observed between calcium release and [InsP3]cyt (Fig. 2 a), which could be fit with the Hill equation (Fig. 2 b; mean Hill coefficient for nine cells, 2.5 ± 0.5). This is consistent with other measurements on the [InsP3]cyt dependence of calcium release (Oancea and Meyer 1996). Subsequent to the uncaging flashes, the BK-evoked calcium response in the soma and neurite was compared with the dose-response. Note that the BK-induced calcium signal in the soma corresponds to uncaged [InsP3] of 2.1 ± 0.1 μM (n = 11 cells). However, BK-induced [Ca2+]cyt signals at the initiation point in the neurite could only be matched by uncaging InsP3 in the range of 5–10 μM (7.0 ± 0.8 μM; mean ± SEM; n = 9 cells; Fig. 2 c). This result is consistent with the lower amplitude of the calcium response to a uniform pulse of uncaged InsP3 in the neurite compared with the soma (Fig. 1 b), and indicates that the neurite requires three to four times as much InsP3 as the soma to produce the calcium response evoked by the physiological stimulus of Fig. 1 a.


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)

Measurement of [InsP3]cyt. a, Cells were microinjected with CG-1 and NPE-InsP3, and CG-1 fluorescence was measured on the confocal microscope. Fluorescence intensity values were converted to relative [Ca2+] and plotted against time. The cell was subjected, in turn, to increasing doses of InsP3 released by UV light flashes of 20–80 ms, 500 nM BK application to the medium, and treatment with 10 μM ionomycin. A typical experiment, in which the neurite signal is followed, is shown here. The gray line connecting the tracing after addition of ionomycin indicates a period during which the cell was refocused. b, For two cells where neurite (blue) and soma (red) are monitored, relative peak [Ca2+] for each uncaging event has been plotted against [InsP3]uncaged (solid circles). The data has been fit (solid curve) with the Hill equation (R = 0.99). A dashed horizontal line was drawn indicating the [Ca2+]cyt response produced by BK stimulation in each region. A corresponding pair of lines were dropped from the Hill equation curve, indicating the [InsP3] necessary to produce the BK-induced calcium peak. c, Mean values (± SEM) of estimated InsP3 production by BK in soma (black) and neurite (gray) are compared in a bar graph (P < 0.001). d, [InsP3]cyt was measured in a N1E-115 neuroblastoma cell population during a BK-induced calcium wave using competitive radioligand binding.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169350&req=5

Figure 2: Measurement of [InsP3]cyt. a, Cells were microinjected with CG-1 and NPE-InsP3, and CG-1 fluorescence was measured on the confocal microscope. Fluorescence intensity values were converted to relative [Ca2+] and plotted against time. The cell was subjected, in turn, to increasing doses of InsP3 released by UV light flashes of 20–80 ms, 500 nM BK application to the medium, and treatment with 10 μM ionomycin. A typical experiment, in which the neurite signal is followed, is shown here. The gray line connecting the tracing after addition of ionomycin indicates a period during which the cell was refocused. b, For two cells where neurite (blue) and soma (red) are monitored, relative peak [Ca2+] for each uncaging event has been plotted against [InsP3]uncaged (solid circles). The data has been fit (solid curve) with the Hill equation (R = 0.99). A dashed horizontal line was drawn indicating the [Ca2+]cyt response produced by BK stimulation in each region. A corresponding pair of lines were dropped from the Hill equation curve, indicating the [InsP3] necessary to produce the BK-induced calcium peak. c, Mean values (± SEM) of estimated InsP3 production by BK in soma (black) and neurite (gray) are compared in a bar graph (P < 0.001). d, [InsP3]cyt was measured in a N1E-115 neuroblastoma cell population during a BK-induced calcium wave using competitive radioligand binding.
Mentions: Further, to determine the dependence of the [Ca2+]cyt signal on [InsP3]cyt in each region of the cell, we performed quantitative InsP3 uncaging experiments (Khodakhah and Ogden 1993; Fink et al. 1999), followed by application of BK to the same cells. A dose-response relationship can be observed between calcium release and [InsP3]cyt (Fig. 2 a), which could be fit with the Hill equation (Fig. 2 b; mean Hill coefficient for nine cells, 2.5 ± 0.5). This is consistent with other measurements on the [InsP3]cyt dependence of calcium release (Oancea and Meyer 1996). Subsequent to the uncaging flashes, the BK-evoked calcium response in the soma and neurite was compared with the dose-response. Note that the BK-induced calcium signal in the soma corresponds to uncaged [InsP3] of 2.1 ± 0.1 μM (n = 11 cells). However, BK-induced [Ca2+]cyt signals at the initiation point in the neurite could only be matched by uncaging InsP3 in the range of 5–10 μM (7.0 ± 0.8 μM; mean ± SEM; n = 9 cells; Fig. 2 c). This result is consistent with the lower amplitude of the calcium response to a uniform pulse of uncaged InsP3 in the neurite compared with the soma (Fig. 1 b), and indicates that the neurite requires three to four times as much InsP3 as the soma to produce the calcium response evoked by the physiological stimulus of Fig. 1 a.

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