Date of Award
1995
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Abstract
Aggregation of macromolecular plasma membrane receptors following ligand binding is hypothesized to be the initial step in signal transduction in cellular systems. Quantitative measurement of receptor distributions on cell surfaces is integral for elucidating the complicated mechanisms involved in transduction of chemical signals across the cells membrane. Scanning fluorescence correlation spectroscopy (S-FCS) has been used successfully to perform quantitative measurements of receptor densities and aggregate sizes. However, these measurements take a great deal of time, and require specialized equipment to implement. Here, image correlation spectroscopy (ICS) is introduced as a novel extension of scanning fluorescence correlation spectroscopy that improves on its progenitor in a number of ways. ICS entails autocorrelation analysis of confocal scanning laser microscopy images of immuno-fluorescent labeled membrane receptors. The new technique yields similar quantitative information on receptor distributions as does S-FCS, however, ICS measurements can be performed much more rapidly, and are more accurate due to the inherent increase in sampling of the imaging format. The present work outlines the underlying theory and demonstrates the technical implementation of ICS for measurements of cell surface receptor distributions on fixed preparations and living cells. ICS studies of the distribution beta receptors for platelet derived growth factor (PDGFR-{dollar}\beta{dollar}) on AG1523 human foreskin fibroblasts are presented. PDGF-{dollar}\beta{dollar} receptors were found to be distributed in clusters at a surface density of 2 clusters/{dollar}\mu{dollar}m{dollar}\sp2{dollar} at 4{dollar}\sp\circ{dollar}C on paraformaldehyde fixed cells. The clusters were estimated to contain on average 7 receptor subunits. ICS measurements were able to detect a rapid reorganization of the surface receptors into larger aggregates following incubation of the cells at 37{dollar}\sp\circ{dollar}C prior to fixation. This clustering was attributed to an antibody induced cross-linking of the PDGF-{dollar}\beta{dollar} receptors. ICS measurements performed on living cells showed similar dynamic antibody-induced clustering at 37{dollar}\sp\circ{dollar}C. The addition of platelet derived growth factor (PDGF-BB) to viable cells had no measurable effect on the receptor distribution.
Recommended Citation
Wiseman, Paul William, "Image Correlation Spectroscopy: Development And Application To Studies Of Pdgf Receptor Distributions" (1995). Digitized Theses. 2583.
https://ir.lib.uwo.ca/digitizedtheses/2583