Calcium Waves

Ionised calcium (Ca2+) is an important second messenger molecule involved in a number of diverse cellular processes including cell differentiation, growth and metabolism. Propagation of intracellular Ca2+ signals is thought to be mediated by a Ca2+-induced Ca2+-release mechanism where control over the amplitude and frequency of the signal may be modulated by the underlying molecular architecture (e.g. spatial patterns of calcium uptake and release channels: ATP-dependent Ca2+ pumps, inositol triphosphate and ryanodine receptors). Ca2+ signalling represents a widely studied biochemical system for which a wealth of published experimental data currently exists. We aim to use this data to build a three dimensional lattice based model to simulate the passage of calcium waves through a segment of a hypothetical cell. The model will feature a segment of membrane, Ca2+-uptake and release channels, intraluminal Ca2+ stores and a region of the cytosol in which individual lattice sites will be used to record relative Ca2+ concentrations. By introducing rules to reflect diffusion and active transport processes, the model will be used to investigate the velocity and trajectory of Ca2+ waves in response to 1) receptor concentrations; 2) altered kinetics of calcium sequestration and release; and 3) spatial distribution of the receptors. In collaboration with experiomental groups we aim to test hypotheses and predictions arising from our simulations, which are expected to provide insights into the mechanisms of wave propagation in different cell types, both in health and disease.

This animation shows a simulation of a calcium wave propagated via the mechanism of calcium-induced calcium-release. Individual pink spots represent calcium release channels on the surface of the endoplasmic reticulum. While light blue spots represent ATPase pumps responsible for transporting calcium from the cytosol to intraluminal stores. Local fluxes in calcium concentration lead to the release of intraluminal stores of calcium (represented as the bursts of red), which diffuse within the cytosol triggering the release of calcium from other channels. By exploring patterns of distribution of the release channels in addition to the kinetics of calcium uptake and release, we are aiming to gain insights into the factors responsible for mediating calcium signalling.

Kinase signalling cascades

We are exploring a simple signaling pathway comprising two receptors with three intermediary signaling molecules. In this model, cell membrane recepters activate a cascade of three different cytosolic kinases, which in turn activate proteins in the nuclear membrane. We are investigating the effect of changes in relative concentrations of these components and the effect of spatial restriction on the ability of this pathway to transfer the signal from the outside of the cell to the nucleus. Restricting two or more components to the same area of the cell is found to greatly increase signaling efficiency.

We are also experimenting on the effect of molecular crowding on signaling efficiency (see below). Creating regions of inaccessible space within the cell reduces the volume required for the signaling particles to diffuse through. In the left example, the inaccessible space is arranged in the shape of cylinders extending out from the nucleus, as microtubules radiate outward. In the example on the right, the inaccessible space is arranged as uniformly distributed small particles.

Metabolism

This animation represents the initial stages of a simulation investigating the relationship of metabolites and enzymes in a simple 3D space. The yellow diffusing species represents a single metabolite being imported into the system. The individual coloured points represent discrete enzymes in a pathway converting the metabolite via a series of steps into an end product. Again we can explore the influence of varying parameters such as relative concentrations, enzyme kinetics and spatial constraints.

In these types of simulations, we measure the turnover rate of different metabolites over time in response to different concentrations and spatial organization of enzymes associated with a hypothetical metabolic pathway (see right). We are currently experimenting with the kinetic parameters from a real metabolic pathway, glycolysis, using this model.

Lipid Rafts

Recently membrane rafts consisting of sphingolipid/cholesterol-enriched membrane domains have been identified and implicated as platforms mediating signaling cascades for a variety of immune recognition receptors. Association of receptors and signalling components within these rafts, thought to be mediated by ligand binding, ensures the proximity necessary for triggering a signalling cascade by restricting their lateral diffusion. Here at the Hospital for Sick Children, Dr Sergio Grinstein is currently developing an in vitro model of lipid raft mediated immune signalling based on the Fcg receptor. By using fluorescence techniques, Dr Grinstein and his colleagues are able to measure the diffusion coefficients of receptors in the membrane in response to different raft densities. Signal activation in response to varying concentrations of rafts, receptors and ligands and also to different binding affinities between the receptor and ligand can also be measured. In association with this work we are constructing a model of lipid raft dependent signalling in which a section of membrane is represented by a two dimensional lattice. Rafts are modelled as diffusible clusters of lattice points on which receptors can be assembled. Simulations will follow the dynamics of the receptors and their interactions (both with membrane rafts and a set of modelled ligands). By altering the concentration of components and receptor-ligand interactions within our model, we will be able to correlate our findings with output from Dr Grinstein's in vitro model. This will allow us to generate a series of predictions which can be readily tested, allowing the formulation of new hypotheses. As more data on this system and associated components become available they will be fed back into the simulations. In this iterative procedure we aim to gain new insights into the mechanisms underlying lipid-raft mediated signalling.