Neurite extension and branching are important neuronal plasticity mechanisms that can lead to the addition of synaptic contacts in developing neurons and changes in the number of synapses in mature neurons. Here we show that Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates movement, extension, and branching of filopodia and fine dendrites as well as the number of synapses in hippocampal neurons. Only CaMKIIbeta, which peaks in expression early in development, but not CaMKIIalpha, has this morphogenic activity. A small insert in CaMKIIbeta, which is absent in CaMKIIalpha, confers regulated F-actin localization to the enzyme and enables selective upregulation of dendritic motility. These results show that the two main neuronal CaMKII isoforms have markedly different roles in neuronal plasticity, with CaMKIIalpha regulating synaptic strength and CaMKIIbeta controlling the dendritic morphology and number of synapses.

The "Virtual Cell" provides a general system for testing cell biological mechanisms and creates a framework for encapsulating the burgeoning knowledge base comprising the distribution and dynamics of intracellular biochemical processes. It approaches the problem by associating biochemical and electrophysiological data describing individual reactions with experimental microscopic image data describing their subcellular localizations. Individual processes are collected within a physical and computational infrastructure that accommodates any molecular mechanism expressible as rate equations or membrane fluxes. An illustration of the method is provided by a dynamic simulation of IP3-mediated Ca2+ release from endoplasmic reticulum in a neuronal cell. The results can be directly compared to experimental observations and provide insight into the role of experimentally inaccessible components of the overall mechanism.

Calcium waves produced by bradykinin-induced inositol-1,4, 5-trisphosphate (InsP(3))-mediated release from endoplasmic reticulum (ER) have been imaged in N1E-115 neuroblastoma cells. A model of this process was built using the "virtual cell," a general computational system for integrating experimental image, biochemical, and electrophysiological data. The model geometry was based on a cell for which the calcium wave had been experimentally recorded. The distributions of the relevant cellular components [InsP(3) receptor (InsP(3)R)], sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pumps, bradykinin receptors, and ER] were based on 3D confocal immunofluorescence images. Wherever possible, known biochemical and electrophysiological data were used to constrain the model. The simulation closely matched the spatial and temporal characteristics of the experimental calcium wave. Predictions on different patterns of calcium signals after InsP(3) uncaging or for different cell geometries were confirmed experimentally, thus helping to validate the model. Models in which the spatial distributions of key components are altered suggest that initiation of the wave in the center of the neurite derives from an interplay of soma-biased ER distribution and InsP(3) generation biased toward the neurite. Simulations demonstrate that mobile buffers (like the indicator fura-2) significantly delay initiation and lower the amplitude of the wave. Analysis of the role played by calcium diffusion indicated that the speed of the wave is only slightly dependent on the ability of calcium to diffuse to and activate neighboring InsP(3) receptor sites.

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A general method is described that takes advantage of the optical sectioning properties of a confocal microscope to enable measurement of both absolute and relative concentrations of fluorescent molecules inside cells. For compartments within cells that are substantially larger than the point spread function, the fluorescence intensity is simply proportional to the concentration of the fluorophore. For small compartments, the fluorescence intensity is diluted by contributions from regions outside the compartment. Corrections for this dilution can be estimated via calibrations that are based on the intensity distribution found in a computationally synthesized model for a cell or organelle that has been blurred by convolution with the microscope point spread function. The method is illustrated with four test cases: estimation of intracellular concentration of a fluorescent calcium indicator; estimation of the relative distribution between the neurite and soma of a neuronal cell of the InsP3 receptor on the endoplasmic reticulum; estimation of the distribution of the bradykinin receptor along the surface of a neuronal cell; and relative distribution of a potentiometric dye between the mitochondria and cytosol as a means of assaying mitochondrial membrane potential.

Inositol-1,4,5-trisphosphate (InsP3)-mediated calcium signals represent an important mechanism for transmitting external stimuli to the cell. However, information about intracellular spatial patterns of InsP3 itself is not generally available. In particular, it has not been determined how the interplay of InsP3 generation, diffusion, and degradation within complex cellular geometries can control the patterns of InsP3 signaling. Here, we explore the spatial and temporal characteristics of [InsP3]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 [InsP3]cyt in the neurite. This requisite InsP3 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.

Modeling and simulation of the calcium signaling events that precede long-term depression of synaptic activity in cerebellar Purkinje cells are performed using the Virtual Cell biological modeling framework. It is found that the unusually high density and low sensitivity of inositol-1,4,5-trisphosphate receptors (IP3R) are critical to the ability of the cell to generate and localize a calcium spike in a single dendritic spine. The results also demonstrate the model's capability to simulate the supralinear calcium spike observed experimentally during coincident activation of the parallel and climbing fibers. The sensitivity of the calcium spikes to certain biological and geometrical effects is investigated as well as the mechanisms that underlie the cell's ability to generate the supralinear spike. The sensitivity of calcium release rates from the IP3R to calcium concentrations, as well as IP3 concentrations, allows the calcium spike to form. The diffusion barrier caused by the small radius of the spine neck is shown to be important, as a threshold radius is observed above which a spike cannot be formed. Additionally, the calcium buffer capacity and diffusion rates from the spine are found to be important parameters in shaping the calcium spike.

The level of [InsP3]cyt required for calcium release in A7r5 cells, a smooth muscle cell line, was determined by a new set of procedures using quantitative confocal microscopy to measure release of InsP3 from cells microinjected with caged InsP3. From these experiments, the [InsP3]cyt required to evoke a half-maximal calcium response is 100 nM. Experiments with caged glycerophosphoryl-myo-inositol 4, 5-bisphosphate (GPIP2), a slowly metabolized analogue of InsP3, gave a much slower recovery and a half-maximal response of an order of magnitude greater than InsP3. Experimental data and highly constrained variables were used to construct a mathematical model of the InsP3-dependent [Ca2+]cyt changes; the resulting simulations show high fidelity to experiment. Among the elements considered in constructing this model were the mechanism of the InsP3-receptor, InsP3 degradation, calcium buffering in the cytosol, and refilling of the ER stores via sarcoplasmic endoplasmic reticulum ATPase (SERCA) pumps. The model predicts a time constant of 0.8 s for InsP3 degradation and 13 s for GPIP2. InsP3 degradation was found to be a prerequisite for [Ca2+]cyt recovery to baseline levels and is therefore critical to the pattern of the overall [Ca2+]cyt signal. Analysis of the features of this model provides insights into the individual factors controlling the amplitude and shape of the InsP3-mediated calcium signal.