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The movie shows the calcium wave in a differentiated N1E-115 neuroblastoma cell elicited by exposure to 0.5 micromolar bradykinin (BK). BK exposure occurs at the start of the movie, and images of fura-2 fluorescence were collected at 65 ms intervals. A cooled 12 bit CCD camera was used to acquire each 30 ms exposure, with excitation at 380 nm and emission through a 510WBP40 filter. Fluorescence change relative to pre-stimulus was calculated for each point. The initial prestimulus calcium concentration was determined from a 340/380 ratio image and this was used as a starting point for calcium changes calculated from a calibration curve of the 380nm relative fluorescence change against known Ca concentrations. The calibration was accurate only to <300nM.
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Click on the neuroblastoma cell to visualize the calcium wave. (AVI Movie 1Mb)
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The movie shows the calcium wave in a differentiated N1E-115 neuroblastoma cell elicited by exposure to 0.5 micromolar bradykinin (BK). BK exposure occurs at the start of the movie, and images of fura-2 fluorescence were collected at 65 ms intervals. A cooled 12 bit CCD camera was used to acquire each 30 ms exposure, with excitation at 380 nm and emission through a 510WBP40 filter. Fluorescence change relative to pre-stimulus was calculated for each point. The initial prestimulus calcium concentration was determined from a 340/380 ratio image and this was used as a starting point for calcium changes calculated from a calibration curve of the 380nm relative fluorescence change against known Ca concentrations. The calibration was accurate only to <300nM.
Simulation of Calcium wave by the Virtual Cell
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Click on the image to view the simulation (AVI Movie 750kb)
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Neuroblastoma cells propagate calcium-induced calcium waves in response to the application of bradykinin that have been observed to initiate preferentially in the neurite. A question to be addressed is whether this phenomena is a function of cellular geometry alone or reflects spatial in homogeneities in the distribution of specific receptors.
Physiological Model
The simulation includes a bradykinin receptor induced IP3 increase followed by ER calcium release from the IP3 receptor, wave propagation by calcium induced calcium release, and uptake via the SERCA pump.
Geometry
The spatial organization of cellular structure was captured by incorporating geometry derived from a microscope image. Specific cellular components (eg. ER, cytosol, and extracellular milieu) are associated with a mathematical model that includes electro-diffusion information and a collection of models representing discrete mechanisms for chemical reactions and membrane phenomena. The model geometry is uniformly sampled into discrete simulation elements each assigned a single component type.
In this specific application the fluxes are functions of diffusion, channels, and pumps. Membranes are represented by element boundaries separating dissimilar component types. The concentrations of molecular species within each simulation element constitute the state variables of the system. Direct correlation of simulation results with experimental time series volume data sets is possible because they are spatially coincident. The simulation agrees with experiments regarding changes in the spatial distribution of free calcium, and predicts parameters such as IP3 concentration and IP3 receptor open probability, which cannot be directly observed in vivo. The results show that geometry alone is sufficient to explain wave initiation in the neurite via the increased surface to volume ratio and its effect on local IP3 concentration. The simulation method used allows a framework for encapsulating knowledge about the interaction of intracellular components, and thus allows one to simulate complex physiology. Many other applications of this simulation method are possible, for example detailed simulations of electrophysiology such as action potentials.
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