Signal transduction in single cells: Digital fluorescence imaging and mathematical modeling.

The objective of this thesis is to investigate the dynamics of cellular signal transduction through G-proteins both experimentally and mathematically. Both are used to gain insight into methods of manipulation of the cell response at the level of signal transduction. Experimentally, changes in intracellular calcium are measured in single smooth muscle (BC$\sb3$H1) cells using the fluorescent probe fura-2 in a digital fluorescence imaging system. The responses of single cells to phenylephrine stimulation are heterogeneous. Both the speed of calcium mobilization as well as the fraction of cells which mobilize calcium following phenylephrine stimulation increase with increasing ligand concentration. It is found that increases in the population response are primarily the result of increases in the number of cells responding and not increases in individual cell responses. In addition, the effect of the receptor antagonist prazosin on calcium mobilization is tested. For cells with equal equilibrium numbers of receptor/ligand complexes, cells with prazosin-blocked receptors are less responsive. Two mathematical models of signal transduction through G-proteins are developed. The first model, an ordinary differential equation model based on the calcium signal transduction cascade, is used to predict single-cell calcium responses to ligand stimulation. Cell-to-cell parameter heterogeneity is systematically incorporated in the model by randomly selecting single-cell parameter values from a Gaussian distribution. Model simulations predict both the single-cell and population-averaged trends seen experimentally with phenylephrine stimulation. However, the model is unable to predict the diminished responsiveness seen in the presence of prazosin. The second model, a Monte Carlo model of G-protein activation, is developed to examine the early events of signal transduction, those occurring at the cell membrane, in more detail. The model predicts that the presence of a receptor blocker may significantly reduce G-protein activation by restricting the movement of ligand among free receptors if the lifetime of a receptor/ligand complex is short compared to the time between diffusional encounters of a receptor/ligand complex with a G-protein. In addition, the roles of two-dimensional diffusion of receptors and G-proteins and the G-protein GTPase activity in the activation of G-protein are explored. The model quantitates how the cell response may be manipulated at the level of signal transduction by alteration of protein diffusivity, GTPase activity, receptor/ligand complex/G-protein interaction, and the numbers of signaling species.PhDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/103995/1/9423257.pdfDescription of 9423257.pdf : Restricted to UM users only